Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ENGINEERING OF LEADER PEPTIDES FOR THE SECRETION OF RECOMBINANT
PROTEINS IN BACTERIA
BACKGROUND OF THE INVENTION
This application claims the priority of U.S. Provisional Patent Application
Ser. No.
60/337,452, filed November 5, 2001, and U.S. Provisional Patent Application
Ser. No. 60/-
--,---, filed August 21, 2002, in the name of George Georgiou and Matthew
DeLisa and
entitled "Engineering of Leader Peptides for the Secretion of Recombinant
Proteins in
Bacteria." Both of the foregoing disclosures are specifically incorporated
herein by
reference in the entirety.
1. Field of the Invention
The present invention relates generally to the fields of genetic engineering
and
protein secretion. More specifically, the present invention relates to
engineering of leader
peptides for the secretion of recombinant proteins in bacteria.
2. Description of the Related Art
Proteins destined for secretion from the cytoplasm are synthesized with an N-
terminal peptide extension of generally between 15-30 amino acids known as the
leader
peptide. The leader peptide is proteolytically removed from the mature protein
either
concomitant to or immediately following export into an exocytoplasmic
location.
Recent findings have established that there are actually four protein export
pathways
in Gram-negative bacteria (Stuart and Neupert, 2000): the general secretory
(Sec) pathway
(Danese and Silhavy, 1998; Pugsley, 1993), the signal recognition particle
(SRP)-
dependent pathway (Meyer et al., 1982), the recently discovered YidC-dependent
pathway
(Samuelson et al., 2000) and the twin-arginine translocation (Tat) system
(Berks, 1996).
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With the first three of these pathways, polypeptides cross the membrane via a
'threading'
mechanism, i.e., the unfolded polypeptides insert into a pore-like structure
formed by the
proteins Sect, SecE and Sect and are pulled across the membrane via a process
that
requires the hydrolysis of ATP (Schatz and Dobberstein, 1996).
In contrast, proteins exported through the Tat-pathway transverse the membrane
in a
partially or perhaps even fully folded conformation. The bacterial Tat system
is closely
related to the '~pH-dependent' protein import pathway of the plant chloroplast
thylakoid
membrane (Settles et al., 1997). Export through the Tat pathway does not
require ATP
hydrolysis and does not involve passage through the SecY/E/G pore. In most
instances, the
natural substrates for this pathway are proteins that have to fold in the
cytoplasm in order
to acquire a range of cofactors such as FeS centers or molybdopterin. However,
proteins
that do not contain cofactors but fold too rapidly or too tightly to be
exported via any other
pathway can be secreted from the cytoplasm by fusing them to a Tat-specific
leader peptide
(Berks, 1996; Berks et al., 2000).
The membrane proteins TatA, TatB and TatC are essential components of the Tat
translocase in E. coli Sargent et al., 1998; Weiner et al., 1998). In
addition, the TatA
homologue TatE, although not essential, may also has a role in translocation
and the
involvement of other factors cannot be ruled 'out. TatA, TatB and TatE are all
integral
membrane proteins predicted to span the inner membrane once with their C-
terminal
domain facing the cytoplasm. The TatA and B proteins are predicted to be
single-span
proteins, whereas the TatC protein has six transmembrane segments and has been
proposed
to function as the translocation channel and receptor for preproteins (Berks
et al., 2000;
Bogsch et al., 1998; Chanal et al., 1998). Mutagenesis of either TatB or C
completely
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abolishes export (Bogsch et al., 1998; Sargent et al., 1998; Weiner et al.,
1998). The Tat
complex purified from solubilized E. coli membranes contained only TatABC
(Bolhuis
et al., 2001). hZ vitro reconstitution of the translocation complex
demonstrated a minimal
requirement for TatABC and an intact membrane potential (Yahr and Wickner,
2001).
The choice of the leader peptides, and thus the pathway employed in the export
of a
particular protein, can determine whether correctly folded functional protein
will be
produced (Bowden and Georgiou, 1990; Thomas et al., 2001). Feilmeier et al.
(2000) have
shown that fusion of the green fluorescent protein (GFP) to a Sec-specific
leader peptide or
to the C-terminal of the maltose binding protein (MBP which is also exported
via the Sec
pathway) resulted in export of green fluorescent protein and MBP-GFP into the
periplasm.
However, green fluorescent protein in the periplasm was non-fluorescent
indicating that the
secreted protein was misfolded and thus the chromophore of the green
fluorescent protein
could not be formed. Since proteins exported via the Sec pathway transverse
the
membrane in an unfolded form, it was concluded that the environment in the
bacterial
secretory compartment (the periplasmic space) does not favor the folding of
green
fluorescent protein Feilmeier et al., 2000). In contrast, fusion of a Tat-
specific leader
peptide to green fluorescent protein resulted in accumulation of fluorescent
green
fluorescent protein in the periplasmic space. In this case, the Tat-GFP
propeptide was first
able to fold in the cytoplasm and then be exported into the periplasmic space
as a
completely folded protein (Santini et al., 2001; Thomas et al., 2001).
However, there has
been no evidence that leader peptides other than TorA can be employed to
export
heterologous proteins into the periplasmic space of E. coli.
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The cellular compartment where protein folding fakes place can have a dramatic
effect on the yield of biological active protein. The bacterial cytoplasm
contains a large
number of protein folding accessory factors, such as chaperones whose function
and ability
to facilitate folding of newly synthesized polypeptides is controlled by ATP
hydrolysis. In
contrast, the bacterial periplasm contains relatively few chaperones and there
is no
evidence that ATP is present in that comparhnent. Thus many proteins are
unable to fold
in the periplasm and can reach their native state only within the cytoplasmic
milieu. The
only known way to enable the secretion of folded proteins from the cytoplasm
is via fusion
to a Tat-specific leader peptide. However, the protein flux through the Tat
export system is
significantly lower than that of the more widely used Sec pathway.
Consequently, the
accumulation and steady state yield of proteins exported via the Tat pathway
is low.
The prior art is thus deficient in methods of directing efficient export of
folded
proteins from the cytoplasm. The present invention fulfills this long-standing
need and
desire in the art.
SUMMARY OF THE INVENTION
The present invention provides methods for the isolation of sequences that can
serve
as leader peptides to direct the export of heterologous proteins. One aspect
of the invention
allows the isolation of leader peptides capable of directing proteins to the
Tat secretion
pathway. Further, the present invention discloses methods for identifying
leader peptide
mutants that can confer improved protein export.
In one aspect, the invention thus provides methods of identifying leader
peptides
that direct enhanced protein secretion in bacteria. In one embodiment, the
methods
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disclosed herein comprise screening libraries of mutated leader peptides for
sequences that
allow rapid export and thus can rescue a short-lived reporter protein from
degradation in
the cytoplasm. Leader peptides that mediate secretion through the Esclaerichia
coli Twin
Arginine Translocation (Tat) pathway, as well as those that direct other
secretion pathways
such as the sec pathway in bacteria can be isolated by the methods disclosed
herein.
Mutant leader peptide sequences conferring improved export are also disclosed.
The
mutant leader peptides are shown to confer significantly higher steady state
levels of export
not only for the short lived reporter protein but also for other stable, long
lived proteins.
In one aspect of the present invention, there is provided a method of
identifying
leader peptides that direct increased protein export through pathways that
include, but are
not limited to, the Twin Arginine Translocation (TAT) pathway and the sec
pathway. Such
a method may involve constructing expression cassettes that place mutated
leader peptides
upstream of a gene encoding a short-lived reporter protein. The short-lived
reporter protein
can be created by attaching a cytoplasmic degradation sequence to the gene
encoding the
reporter protein. The resulting expression cassettes may then be expressed in
bacteria, and
reporter protein expressions in these bacteria measured. Mutated leader
peptides expressed
in cells that exhibit increased reporter protein expression comprise leader
peptides that
would direct increased protein export in bacteria. Representative leader
peptides identified
from the above methods include SEQ m NOs:120-136.
In another aspect of the present invention, there is provided a method of
increasing
polypeptide export through pathways that include, but are not limited to, the
Tat pathway
and the sec pathway. This method involves expressing expression cassettes that
place
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mutated leader peptides identified in the methods disclosed herein upstream of
the gene
encoding a heterologous polypeptide of interest.
In yet another aspect of the present invention, there is provided a method of
screening for a compound that inhibits or enhances protein export through
pathways that
include, but are not limited to, the Tat pathway and the sec pathway. This
method may
comprise first constructing expression cassettes that place mutated leader
peptides
identified in the methods disclosed herein upstream of a gene encoding a short-
lived
reporter protein. The short-lived reporter protein can be created by attaching
a cytoplasmic
degradation sequence to the gene encoding the reporter protein. The resulting
expression
cassettes may then be expressed in bacteria, and reporter protein expression
in these
bacteria are measured in the presence or absence of the candidate compound.
Increased
reporter protein expression measured in the presence of the candidate compound
indicates
that the candidate compound enhances protein export, whereas decreased
reporter protein
expression measured in the presence of the candidate compound indicates that
the
candidate compound inhibits protein export.
In another aspect of the present invention, there is provided a method for
producing
soluble and biologically-active heterologous polypeptide containing multiple
disulfide
bonds in a bacterial cell. The method may involve constructing an expression
cassette that
places a leader peptide that directs protein export through the Twin Arginine
Translocation
pathway upstream of a gene encoding the heterologous polypeptide. The
heterologous
polypeptide is then expressed in bacteria that have an oxidizing cytoplasm.
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Other and further aspects, features, and advantages of the present invention
will be
apparent from the following description of the presently preferred embodiments
of the
invention. These embodiments are given for the purpose of disclosure.
S BRIEF DESCRIPTION OF THE DRAWINGS
So that the matter in which the above-recited features, advantages and objects
of the
invention as well as others which will become clear are attained and can be
understood in
detail, more particular descriptions and certain embodiments of the invention
briefly
summarized above are illustrated in the appended drawings. These drawings form
a part of
the specification. The drawings illustrate certain embodiments of the
invention and are not
to be considered limiting in their scope.
FIG. 1 shows the expression of green fluorescent protein in different plasmid
constructs. FIG. lA shows minimal green fluorescent protein fluorescence in
cells
expressing pGFPSsrA, indicating that cytoplasmic SsrA-tagged green fluorescent
protein is
degraded almost completely. FIG. 1B shows enhanced green fluorescent protein
fluorescence in cells expressing pTorAGFPSsrA, indicating improved green
fluorescent
protein export directed by the TorA leader peptide. FIG. IC shows green
fluorescent
protein fluorescence in cells expressing pTorAGFP. The green fluorescent
protein was
expressed in both the cytoplasm and the periplasm.
FIG. 2 shows green fluorescent protein fluorescence in 6 different clones that
exhibit increased Tat-dependent export due to mutated TorA leader peptides.
FIG. 3 shows periplasmic green fluorescent protein accumulation in the B6 and
E2
clones. FIG. 3A shows western blot of green fluorescent protein in the
periplasm (lanes 1-
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3) and cytoplasm (lanes 4-6) of cells expressing the wild type construct
(lanes 1 and 4), the
B6 clone (lanes 2 and 5) and the E2 clone (lanes 3 and 6). GroEL is a
cytoplasmic marker
whereas DsbA is a periplasmic marker. FIG. 3B shows periplasmic and
cytoplasrnic
distribution of green fluorescent protein in cells expressing the wild type
construct, the B6
clone and the E2 clone.
FIG. 4 shows increased green fluorescent protein fluorescence in cells
expressing
the wild type construct, the B6, B7 or E2 construct fused to untagged,
proteolytically stable
green fluorescent protein.
FIG. 5 shows western blot of green fluorescent protein in the periplasm (lanes
1-2),
cytoplasm (lanes 3-4) and whole cell lysate (lanes 5-6) of cells expressing
the wild type
construct (lanes 1,3 and 5) or the B7 clone (lanes 2, 4 and 6). GroEL is a
cytoplasmic
marker whereas DsbA is a periplasmic marker.
FIG. 6 Shows schematic of export of disulfide linked heterodimer in which only
one polypeptide chain was fused to leader peptide.
FIG. 7 Shows western blot analysis and AP activity measurements for both
periplasmic and cytoplasmic fractions in six genetic backgrounds.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods of identifying and using leader
peptides
that direct enhanced protein secretion in bacteria. Numerous proteins of
commercial
interest are produced in secreted form in bacteria. However, many proteins,
including
many antibody fragments and several enzymes of eucaryotic origin, cannot be
exported
efficiently through the main secretory pathway, the sec pathway, of bacteria.
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An alternative pathway for the translocation of proteins from the cytoplasm of
bacteria is called the t'TAT" (twin-arginine-translocation) pathway. Whether a
protein is
directed to the sec machinery or the TAT pathway depends solely on the nature
of the
leader peptide, an amino acid extension of generally 15-30 residues located at
the
beginning of the polypeptide chain. The leader peptide consists of three
distinct regions:
(1) the amino terminal n-region, (2) the hydrophobic core or h-region, and (3)
the c-
terminal region.
A hallmark of both plant and prokaryotic TAT-specific leader peptides is the
presence of the distinctive and conserved (S/T)-R-R-x-F-L-K (SEQ m NO:1)
sequence
motif. This sequence motif is located at the n-region/h-boundary within leader
peptides of
known and predicted TAT substrates (Berks, 1996). Mutation of either arginine
residue
within the signal peptide significantly reduces the efficiency of protein
translocation
(Cristobal et al., 1999).
Relative to leader peptides specific for the Sec pathway, which is by far the
most
commonly used export pathway in bacteria, TAT-specific leader peptides are on
average 14
amino acids longer due to an extended n-region and more basic residues in the
c-region
(Cristobal et al., 1999). However, the hydrophobic h-region in the TAT-
specific leader
peptides is significantly shorter due to a higher occurrence of glycine and
threonine
residues.
The twin-arginine (RR) motifs of wheat pre-23K and pre-Hcf136 are essential
for
targeting by the thylakoid TAT pathway; this motif is probably a central
feature of TAT
signals. The twin-arginine motif is not the only important determinant in TAT-
specific
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targeting signals, and a further hydrophobic residue two or three residues
after this motif
seems also to be highly important.
Bacterial twin-arginine-signal peptides are similar to thylakoid TAT signals
and can
direct TAT-dependent targeting into plant thylakoids with high efficiency.
However, the
vast majority of bacterial signal peptides contain conserved sequence elements
in addition
to the twin-arginine motif that imply special functions. There is a heavy bias
towards
phenylalanine at the second position after the twin-arginine motif, and many
of the signals
contain lysine at the fourth position. None of the known thylakoid twin-
arginine signals
contains phenylalanine at this position and only one (Arabidopsis P29)
contains lysine as
the fourth residue after the twin-arginine motif. The precise roles of these
highly
conserved features are unclear; the phenylalanine residue can be replaced by
Leu but not by
Ala without undue effects, which indicates that hydrophobicity, rather than
the
phenylalanine side-chain, might be the important determinant. Similarly,
replacement of
the Lys residue does not impede export (Robinson and Bolhuis, 2001).
Proteins exported through the TAT system first fold into their native
conformation
within the cytoplasm and are then exported across the cytoplasmic membrane.
The ability
to export proteins that have already folded in the cytoplasm is highly
desirable with regard
to commercial protein production for several reasons. First of all, proteins
that fold very
rapidly after synthesis is completed cannot be secreted by the more common sec
export
pathway. Secondly, the bacterial cytoplasm contains a full complement of
folding
accessory factors, which can assist a nascent polypeptide in reaching its
native
conformation. In contrast, the secretory compartment of bacteria contains very
few folding
accessory factors such as chaperones and foldases. Therefore, for the
production of many
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proteins, it is preferable for folding to occur first within the cytoplasm
followed by export
into the periplasmic space through the TAT system. Thirdly, the acquisition of
cofactors
has to occur within the cytoplasm concomitant with folding. Consequently,
cofactor-
containing proteins must be secreted through the TAT pathway.
A limitation in the use of the protein secretion, and specif cally of the TAT
export
pathway, for commercial protein production has been that the amount of protein
that can be
exported in this manner is low. Tn other words, the overall protein flux
through the TAT
system is substantially lower than that of the sec pathway.
Currently, there is no reliable technology that can be used to screen for
increased
IO periplasmic secretion of recombinant proteins, nor is there an optimized
TAT-specific
leader peptide. However, results obtained from the methods disclosed herein
would lead to
characterization of optimized leader peptides that can circumvent the slow
transit rates
typically observed for wild type or native twin-arginine leader sequences. The
present
invention also enables a thorough and systematic determination on minimal
leader
sequence requirements for proper and efficient export through the TAT pathway.
Moreover, the methods disclosed herein can also identify leader peptides that
mediate
enhanced protein secretion through other pathways such as the sec pathway.
The present invention thus provides, in one aspect, a method of identifying a
leader
peptide that directs increased protein export through the Twin Arginine
Translocation or
TAT pathway by constructing expression cassettes that put mutated candidate
TAT-
specific leader peptides upstream of a gene encoding a short-lived reporter
protein. Such a
short lived reporter protein exhibits a decreased half life in the cytoplasm
relative to
reporter protein molecules that have been exported from the cytoplasm. The
short-lived
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reporter protein can be created, for example, by attaching a cytoplasmic
degradation
sequence to the gene encoding the reporter protein. In general, mutated leader
peptides
may be generated by random mutagenesis, error-prone PCR and/or site-directed
mutagenesis. The resulting expression cassettes can then be expressed in
bacteria, and
expression of the reporter protein be measured. Mutated TAT-specific leader
peptides
expressed in cells that exhibit increased expression of reporter protein are
leader peptides
that would direct increased protein export through the TAT pathway.
Methods that are well known to those skilled in the art can be used to
construct
expression cassettes or vectors containing appropriate transcriptional and
translational
control signals. See for example, the techniques described in Sambrook et al.,
200,
Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor Press,
N.Y.
Vectors of the invention include, but are not limited to, plasmid vectors and
viral vectors.
In one embodiment of the screening methods described herein, green fluorescent
protein (GFP) may be used as a reporter protein. The method takes advantage of
the fact
that functional, fluorescent green fluorescent protein can only be secreted
using a TAT-
specific leader peptide. However, the export of green fluorescent protein via
a TAT
specific leader peptide is inefficient and results in the accumulation of an
appreciable
amount of precursor protein (green fluorescent protein with the TAT-specific
leader) in the
cytoplasm. The cytoplasmic green fluorescent protein precursor protein is
folded correctly
and is fluorescent. As a result, the cells exhibit high fluorescence, which in
part is
contributed by the cytoplasm'ic precursor and in part by the secreted, mature
green
fluorescent protein in the periplasm. The overall high fluorescence of these
cells
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contributes to a high background signal which complicates the isolation of
leader peptide
mutations that give rise to a higher flux of exported green fluorescent
protein.
To circumvent this problem, a short-lived version of green fluorescent
reporter
protein may be used. This short-lived version is rapidly degraded within the
bacterial
cytoplasm. Fusion of the SsrA sequence A.ANDENYALAA (SEQ ID N0:119), for
example, to the C-terminal of green fluorescent protein targets the protein
for degradation
by the CIpXAP protease system (Karzai et al., 2000). As a result, the half
life of green
fluorescent protein in the cytoplasm is reduced from several hours to less
than 10 min,
resulting in a significant decrease in whole cell fluorescence.
It was shown that, when the short lived green fluorescent protein was fused to
a
wild-type TAT-specific leader peptide, a low level of cell fluorescence was
observed
because most of the protein was degraded prior to export from the cytoplasm.
It was
contemplated that mutations in the TAT-specific leader peptide may cause
faster and more
efficient export that rescues the short lived green fluorescent protein from
degradation in
the cytoplasm. As a result, folded green fluorescent protein would be
accumulated in the
periplasm, leading to higher cell fluorescence. Therefore, libraries of mutant
TAT-specific
leader peptides were constructed by either random mutagenesis (error-prone
PCR) or
nucleotide directed mutagenesis. These mutant leader peptides were then
screened fox their
ability to mediate enhanced protein secretion and rescue the short-lived green
fluorescent
protein from degradation in the cytoplasm, thereby leading to increased
fluorescence of the
bacteria. Clones exhibiting higher fluorescence were then isolated by flow
cytometry.
One particular feature of the present invention is that the genetic screen
described
herein results in periplasmic-only accumulation of active reporter protein.
The mutated
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leader peptides direct folded green fluorescent reporter protein to the
periplasm where the
fluorescent protein remains active. However, due to the presence of the SsrA C-
terminal
degradation peptide, virtually all cytoplasmic green fluorescent protein is
degraded. The
resulting cells glow green in a halo-type fashion due to the presence of
periplasmic-only
green fluorescent protein. In contrast, TAT-dependent export of green
fluorescent protein
that lacks the SsrA sequence would lead to green fluorescent protein
accumulation in both
the cytoplasm and the periplasm, resulting in substantial background signal
that makes cell-
based screening of GFP fluorescence impossible.
In addition to green fluorescent protein, various other reporter proteins can
be used
in the methods of the present invention. A person having ordinary skill in
this art could
readily isolate mutant leader peptides that result in higher levels of
reporter protein
expression in the periplasm in a number of ways. In one example, if the
reporter is an
antibiotic resistance enzyme (e.g., (3-lactamase), then mutant leader peptides
can be
isolated by selecting on increasing concentrations of antibiotic. In another
example, if the
reporter is an immunity protein to a toxin (e.g., colicins), mutant leader
peptides can be
isolated by selecting for resistance to toxin. In another example, if the
reporter protein is a
transport protein such as maltose binding protein, export of the transport
protein is used to
complement chromosomal mutants. In another example, the chromogenic or
fluoregenic
substrate of a reporter enzyme (e.g. alkaline phosphatase) can be used to
score for colonies
that produce higher levels of the enzyme in the bacterial periplasm.
There are a number of research and industrial uses for the screening system
described herein. Examples of these research and industrial uses include, but
are not
necessarily limited to, the following:
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(1) Bio-Production of Proteins: The secretion of several proteins via the TAT
pathway has been reported to be a relatively slow and inefficient process.
Therefore, the
need for improved export must be realized in order to make the TAT pathway a
feasible
platform for high-level production of high-value recombinant protein products.
Using the
genetic screens outlined herein, optimized TAT leader peptides have been
isolated and
tested fox their ability to rapidly export recombinant proteins of interest.
The recombinant
proteins are thus secreted into the periplasmic space or the growth medium in
a functional
and soluble form, alleviating problems associated with inclusion bodies and
simplifying
recovery. Furthermore, since proteins are folded and accumulate in the
cytoplasm prior to
TAT-dependent export, this export system will likely result in higher levels
of active
product accumulation within the host cell, thus maximizing the efficiency of
the
recombinant expression system.
(2) In High-Throughput Screening Platforms: The present invention can be
applied
in the development of technologies that capitalize on TAT-dependent export for
IS combinatorial library screening and protein engineering applications. For
example,
improved cytoplasmic folding of disulfide bond containing proteins (e.g.,
antibodies,
eucaryotic enzymes) can be assayed by fusion to optimized leader peptides that
export the
folded proteins of interest to the periplasm where it can be easily accessed
by FACS-based
or phage-based screening protocols. The amount of active protein detected in
the
periplasm would be a quantitative indicator of the efficiency of folding in
the cytoplasm.
(3) In Drug Discovery Programs: Homologues of some TAT proteins have been
identified in pathogenic bacteria such as Mycobacteriufn tuberculosis and
Helicobacter
pylof-i as well as Pseudomonas sp. This indicates that some proteins belonging
to this
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translocation system may be potential new targets fox antibacterial agents.
Using the
processes outlined herein, a large number of compounds can easily be screened
for
inhibition of TAT-dependent secretion. Furthermore, the presence of certain
proteins in
multicopy derived from genomic libraries or random deletion of genes from the
genome
can be tested using this process to identify novel enhancers/suppressors of
the TAT
secretion process in bacteria, thereby providing a more general approach to
developing
antimicrobials.
The present invention of identifying and using leader peptides that direct
enhanced
protein secretion in bacteria is not limited to the TAT pathway. The methods
disclosed
herein are equally applicable for identifying leader peptides that direct
enhanced protein
secretion through other secretion pathways as described above. Signal
sequences which
promote protein translocation to the periplasmic space of Gram-negative
bacterial are well-
known to one of skill in the art. For example, the E. coli OmpA, Lpp, Lama,
MaIE, PeIB,
and StII leader peptide sequences have been successfully used in many
applications as
signal sequences to promote protein secretion in bacterial cells such as those
used herein,
and are all contemplated to be useful in the practice of the methods of the
present
invention. A person having ordinary skill in this art can readily employ
procedures well-
known in the art to construct libraries of mutated leader sequences and
expression cassettes
that incorporate these mutated leader peptides, and screen these leader
peptides according
the methods described herein.
The present invention also relates to secretion of partially or fully folded
cytoplasmic proteins with disulfide bonds. The formation of disulfide bonds is
essential
for the correct folding and stability of numerous eukaryotic proteins of
importance to the
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pharmaceutical and bioprocessing industries. Correct folding depends on the
formation of
cysteine-cysteine linkages and subsequent stabilization of the protein into an
enzymatically
active structure. However, numerous studies have demonstrated that multiple
disulf de
bond-containing proteins cannot be expressed in active form in bacteria.
Disulfide bond
formation is blocked in the reducing environment of the cytoplasm of a cell
due to the
presence of thioredoxin reductase or reduced glutathione.
Thus, the production of technologically important proteins with four or more
disulfide bonds is costly and complicated and must rely either on expression
in higher
eukaryotes that provide a favorable environment for the formation of disulfide
bonds or
refolding from inclusion bodies (~-Ia~l~rieyz. , ~ 994, Georgian. anl',
;~al~.~, ' 19~~). For
example, tissue plasminogen activator (tPA) is currently produced in bacteria
inclusion
bodies. In typical procedures, the proteins are released from inclusion bodies
using a
variety of chaotropic agents, then isolated and refolded by employing reducing
agents.
Generally, refolding results in low yields of biologically active material.
The process of secretion disclosed herein provides an efficient method of
producing
complex eukaryotic proteins with multiple disulfide bonds. These disulfide
bonds form
from specific orientations to promote correct folding of the native protein.
Multiple
disulfide bonds resulting from improper orientation of nascently formed
proteins in the cell
lead to misfolding and loss or absence of biological activity. . In contrast,
biologically-
active polypeptide-containing multiple disulfide bonds produced according to
the instant
invention will be correctly folded; disulfide bonds will form to provide a
tertiary and where
applicable, quarternary structure leading to a molecule with native functional
activity with
respect to substrates and/or catalytic properties. The proteins produced by
the method
1~
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disclosed herein are correctly folded and biologically active without the need
for
reactivation or subsequent processing once isolated from a host cell.
The most immediate problem solved by the methods disclosed herein is that
proteins with multiple disulfide bonds can now be exported to the periplasm in
a fully
folded and therefore active conformation. Complex proteins containing multiple
disulfide
bonds can be folded in the cytoplasm with the assistance of a full complement
of folding
accessory factors that facilitate nascent polypeptides in reaching their
native conformation.
The folded proteins are then secreted into the periplasmic space or the growth
medium in a
functional and soluble form, thus alleviating problems associated with
inclusion bodies and
simplifying recovery. In addition, active recombinant proteins accumulate
simultaneously
in two bacterial compartments (cytoplasm and periplasm), leading to greater
overall yields
of numerous complex proteins which previously could not actively accumulate in
both
compartments concurrently.
Thus, the present invention provides a method of producing at least one
biologically-active heterologous polypeptide in a cell. A leader peptide that
directs protein
export through the Twin Arginine Translocation pathway may be placed upstream
of a
gene encoding the heterologous polypeptide in an expression cassette. The
expression
cassette can be expressed in a cell, wherein the heterologous polypeptide is
produced in a
biologically-active form. Generally, the heterologous polypeptide is secreted
from the
bacterial cell, is isolatable from the periplasm or the culture supernatant of
the bacterial
cell, or is an integral membrane protein. The heterologous polypeptide
produced by this
method can be a mammalian polypeptide such as tissue plasminogen activator,
pancreatic
trypsin inhibitor, an antibody, an antibody fragment or a toxin immunity
protein. The
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heterologous polypeptide may be a polypeptide in native conformation, a
mutated
polypeptide or a truncated polypeptide.
Using a cell that has an oxidizing cytoplasm, the above method can produce a
heterologous polypeptide containing from about 2 to about I7 disulfide bonds.
This
method may also produce two heterologous polypeptides that are linked by at
least one
disulfide bond. Preferably, the leader peptide comprises a sequence of SEQ ID
NOs:25-46,
120-128 or a peptide homologous to SEQ ID NOs:25-46, 120-128. Representative
cells
which are useful in this method include E. colt trxB mutants, E. coli gor
mutants, or E. coli
trxB gor double mutants such as E. coli strain FA 113 or E. coli strain DR473.
The present invention also provides a series of putative TAT-specific leader
peptides, which can be identified by a bioinformatics search from E. coli,
cloned and
examined for functional activities. Thus, the present invention encompasses
isolated leader
peptides that direct protein secretion and export through the Twin Arginine
Translocation
pathway. Representative leader peptides comprise sequences of SEQ >D NOs:25-
46, 120-
128. Moreover, the present invention includes isolated TAT leader peptides
that are
homologous to SEQ ID NOs:25-46, 120-128.
The present invention also provides a method of identifying a leader peptide
that
directs increased protein export by constructing expression cassettes that put
mutated
leader peptides upstream of a gene encoding a short-lived reporter protein.
The short-lived
reporter protein can be created by attaching a cytoplasmic degradation
sequence to the gene
encoding the reporter protein. Representative cytoplasmic degradation
sequences include
SEQ ID NO:I 19, PEST, or sequences recognized by LON, cIPAP, cIPXP, Stsh and
HsIUV.
The cytoplasmic degradation sequences are attached to either the N- or C-
terminal of the
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reporter protein. In general, reporter proteins that can be used include
fluorescent proteins,
an enzyme, a transport protein, an antibiotic resistance enzyme, a toxin
immunity protein, a
bacteriophage receptor protein and an antibody.
Mutated leader peptides can be generated, for example, by random mutagenesis,
error-prone PCR or site-directed mutagenesis, as well as other methods known
to those of
skill in the art. The resulting expression cassettes can then be expressed in
bacteria, and
expression of the reporter protein measured. Mutated leader peptides expressed
in cells
that exhibit increased expression of reporter protein comprise leader peptides
that would
direct increased protein export in bacteria. This screening method is capable
of identifying
leader peptides that direct protein secretion through the general secretory
(Sec) pathway,
the signal recognition particle (SRP)-dependent pathway, the YidC-dependent
pathway or
the twin-arginine translocation (Tat) pathway.
In another aspect of the present invention, there is provided a method of
increasing
export of heterologous polypeptide in bacteria. Expression cassettes are
constructed that
put mutated leader peptides identified according to the methods of the
invention upstream
of a coding sequence encoding a heterologous polypeptide of interest. These
expression
cassettes can then be expressed in bacteria.
The present invention also provides a method of screening for a compound that
inhibits or enhances protein export in bacteria. A leader peptide that directs
protein export
in bacteria may be placed upstream of a gene encoding a short-lived reporter
protein in an
expression cassette. The expression cassette may then be expressed in bacteria
in the
presence or absence of a test compound. Increased expression of the reporter
protein
measured in the presence of the test compound indicates the compound enhances
protein
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export, whereas decreased expression of the reporter protein measured in the
presence of
the compound indicates the compound inhibits protein export. Construction and
examples
of short-lived reporter protein are described above.
The present invention also provides a method of identifying a leader peptide
that
directs increased protein export through the Twin Arginine Translocation
pathway by
constructing expression cassettes that put mutated leader peptides specific
for the Twin
Arginine Translocation pathway upstream of a coding sequence encoding a short-
lived
reporter protein. Construction and examples of short-lived reporter protein
are described
above. The mutated leader peptides can be generated by random mutagenesis,
error-prone
PCR or site-directed mutagenesis. The resulting expression cassettes can then
be
expressed in bacteria, and expression of the reporter protein measured.
Mutated leader
peptides expressed in cells that exhibit increased expression of reporter
protein comprise
leader peptides that would direct increased protein export through the Twin
Arginine
Translocation pathway. Examples of mutated leader peptides comprise sequences
of SEQ
m Nos:120-128.
In another aspect of the present invention, there is provided a method of
increasing
export of heterologous polypeptide through the Twin Arginine Translocation
pathway.
Expression cassettes may be constructed that put mutated leader peptides
identified
according to the methods disclosed herein upstream of a gene encoding a
heterologous
polypeptide of interest. These expression cassettes may then be expressed in
bacteria.
Examples of mutated leader peptides comprise sequences of SEQ ll~ N~s:120-128.
The present invention also provides a method of screening for a compound that
inhibits or enhances protein export through the Twin Arginine Translocation
pathway. A
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leader peptide specific for the Twin Arginine Translocation pathway may be
placed
upstream of a gene encoding a short-lived reporter protein in an expression
cassette. The
expression cassette may then be expressed in bacteria in the presence or
absence of a test
compound. Increased expression of the reporter protein measured in the
presence of the
test compound indicates the compound enhances protein export, whereas
decreased
expression of the reporter protein measured in the presence of the compound
indicates the
compound inhibits protein export through the Twin Arginine Translocation
pathway.
Construction and examples of short-lived reporter protein are described above.
As used herein, "polypeptide" or "polypeptide of interest" refers generally to
peptides and proteins having more than about ten amino acids. The polypeptides
are
"heterologous," meaning that they are foreign to the host cell being utilized,
such as a
human protein produced by a CHO cell, or a yeast polypeptide produced by a
mammalian
cell, or a human polypeptide produced from a human cell line that is not the
native source
of the polypeptide. Examples of a polypeptide of interest include, but are not
limited to,
molecules such as renin, a growth hormone (including human growth hormone),
bovine
growth hormone, growth hormone releasing factor, parathyroid hormone, thyroid
stimulating hormone, lipoproteins, ocl-antitrypsin, insulin A-chain, insulin
(3-chain,
proinsulin, thrombopoietin, follicle stimulating hormone, calcitonin,
luteinizing hormone,
glucagon, clotting factors (such as factor V)ZIC, factor IX, tissue factor,
and von
Willebrands factor), anti-clotting factors (such as Protein C, atrial
naturietic factor, lung
surfactant), a plasminogen activator, (such as human tPA or urokinase),
mammalian trypsin
inhibitor, brain-derived neurotrophic growth factor, kallikreins, CTNF, gp120,
anti-HER-2,
human chorionic gonadotropin, mammalian pancreatic trypsin inhibitor,
antibodies,
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antibody fragments, protease inhibitors, therapeutic enzymes, lymphokines,
cytokines,
growth factors, neurotrophic factors, insulin chains or pro-insulin,
immunotoxins,
bombesin, thrombin, tumor necrosis factor-a or (3,.enkephalinase, a serum
albumin (such
as human serum albumin), mullerian-inhibiting substance, relaxin A-chain,
relaxin B-
chain, prorelaxin, mouse gonadotropin-associated peptide, a microbial protein
(such as (3-
lactamase), Dnase, inhibin, activin, vascular endothelial growth factor
(VEGF), receptors
for hormones or growth factors, integrin, protein A or D, rheumatoid factors,
neurotrophic
factors (such as neurotrophin-3, -4, -5, or -6), or a nerve growth factor
(such as NGF-(3),
cardiotrophins (cardiac hypertrophy factor) (such as cardiotrophin-1),
platelet-derived
growth factor (PDGF), fibroblast growth factor (such as a FGF and [3 FGF),
epidermal
growth factor (EGF), transforming growth factor (TGF) (such as TGF-a, TGF-(31,
TGF-
(32, TGF-[33, TGF-[34, or TGF-(35), insulin-like growth factor-I and -II,
des(1-3)-IGF-I
(brain IGF-n, insulin-like growth factor binding proteins, CD proteins (such
as CD-3, CD-
4, CD-8, and CD-19), erythropoietin, osteoinductive factors, bone
morphogenetic proteins
(BMPs), interferons (such as interferon-a, -(3, and -y), colony stimulating
factors (CSFs)
(e.g., M-CSF, GM-CSF, and G-CSF), interleukins (Ils) (such as IL-1 to IL-10),
superoxide
dismutase, T-cell receptors, surface membrane proteins, decay accelerating
factor, viral
antigens such as a portion of the AIDS envelope, transport proteins, homing
receptors,
addressins, regulatory proteins, antigens such as gp120(IIIb), or derivatives
or active
fragments of any of the peptides listed above. The polypeptides may be native
or mutated
polypeptides, and preferred sources for such mammalian polypeptides include
human,
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bovine, equine, porcine, lupine, and rodent sources, with human proteins being
particularly
preferred.
The following examples are given for the purpose of illustrating various
embodiments of the invention and are not meant to limit the present invention
in any
fashion.
EXAMPLE 1
Bioinformatics Search for TAT-Specific Leader Peptides
Putative TAT leader peptides were found using the Protein-Protein "BLAST"
search engine available through the National Center for Biotechnology
Information
website. The following search strings were entered: SRRRFLK (SEQ ID N0:2),
SRRXFLX (SEQ ID N0:3), TRRXFLX (SEQ ID NO:4), SRRXXLK (SEQ 1D NO:S),
SRRXXLA (SEQ ID NO:6), TRRX~~L,K (SEQ ID N0:7), TRRX~~I,A (SEQ ID N0:8),
SRRXXLT (SEQ ID N0:9), SRl~~XIK (SEQ ll~ NO:10), SRRXXIA (SEQ DJ NO:11),
SRR~FIX (SEQ ID NO:12), SRRXFMK (SEQ II? NO:13), SRRXFVK (SEQ ID N0:14),
SRRXFVA (SEQ ID NO:l S), SRRQFLK (SEQ ID N0:16), RRXFLA (SEQ 117 N0:17),
and RRXFLK (SEQ ZD N0:18). Searches were done for short, nearly exact matches
and
then screened for only those matches occurring within the first 50 residues of
the protein
while still maintaining the twin-arginines. The first 100 residues of each
leader peptide
were then examined by "SignaIP", a program for detecting Sec pathway leader
peptides
and cleavage sites (N~~lsen~ ~t eel ,. ~i997). The final list of putative TAT
leader peptides is
shown in Table 1. These peptides were cloned and examined for their abilities
to direct
secretion of a reporter protein, GFP-SsrA, through the TAT pathway.
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Bacterial Strains and Growth Conditions:
Cells were always grown at 37°C, either on solid LB agar or in liquid
LB media and
with appropriate antibiotics. Chloramphenicol (Cm) was used at the
concentration of 50
p,g/mL. The strain XL1-Blue (recAl endAl gyrA96 thi-1 hsdRl7 supE44 relAl lac
[F'proAB lacfqZtlM1 S TnlO (Tet~]) (Stratagene) was used for cloning purposes.
For
expression, the high-copy pBAD 18-Cm constructs were transformed into the
strains
MC4100-P (MC4100 pcnBl) and B1LK0-P (MC4100 ~tatCpcnBl).
Plasmids and Oligonucleotides:
Each putative leader peptide DNA sequence was first subcloned into pKKGS
(~eLisa bet ah, 'Z~?02), which is based on the low-copy pBAD33 plasmid (Guzman
et al.,
1995). Standard methods were used to amplify DNA and Qiagen kits were used for
all
DNA purification steps. Each leader peptide gene was first PCR amplified from
XL1-Blue
genomic DNA using a forward primer that contained a SacI cleavage site and a
reverse
primer that contained an XbaI cleavage site. Forward primers were designed to
incorporate
at least the first 18 nucleotides of the leader peptide. All forward primers
contained the
sequence (5'-GCGATGGAGCTCTTAAAGAGGAGAAAGGTC-3', SEQ ID N0:19)
followed by the start codon and leader peptide sequence from the desired gene.
Similarly,
all reverse primers contained the sequence (5'-GCGATGTCTAGA-3', SEQ ID N0:20).
Reverse primers were designed such that exactly six amino acid residues beyond
the
predicted leader peptide cleavage site would be incorporated into the plasmid.
The
resulting 58 primers are shown in Tables 2 and 3. All PCR products were gel
purified and
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digested using SacI and XbaI and finally cloned into the SacI and XbaI sites
of pKKGS.
All plasmid constructs were confirmed by sequencing.
Similar constructs were made using the high-copy plasmid pBADlB-Cm (Guzman
et al., 1995). Briefly, signal sequence-GFP-SsrA fusion constructs were
digested from
pBAD33 using SacI and HindIll and cloned into the identical sites of pBADlB-
Cm. In the
case of the HybO leader peptide, the HybO-GFP-SsrA fusion was cut with SacI
and SphI
and cloned into pBAD 18-Cm. As before, all plasmid constructs were confirmed
by
sequencing.
Subcellular Localization of Proteins:
Cells were pelleted by centrifugation at 5000 x g, resuspended in 1 ml of cell
fractionation buffer (30 mM Tris-HCl, pH 8.0, 20% (w/v) sucrose, 1 mM
Na2EDTA), and
incubated at 25°C for 10 min. The cells were again centrifuged at 5000
x g, the supernatant
discarded, and the pellet resuspended in 133 ~.1 of ice-cold 5 mM MgS04. After
10 min on
ice, the cells were centrifuged at full speed, and the supernatant was
retained as the
periplasmic fraction. The pellet was resuspended in 250 ~.1 of PBS and
sonicated for 30
seconds. The cells were centrifuged at full speed and the supernatant was
retained as the
cytoplasmic fraction.
Western Blotting Analysis:
Western blotting was according to Cl~en, et a~. The following primary
antibodies
were used: monoclonal mouse anti-GFP (Clontech) diluted 1:5000, monoclonal
rabbit anti-
DsbC (gift from John Joly, Genentech) diluted 1:10,000 and monoclonal rabbit
anti-GroEL
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(Sigma). diluted 1:10,000. The secondary antibody was 1:10,00'0 goat anti-
mouse-HRP
conjugate and goat anti-rabbit-HRP conjugate. Membranes were first probed with
anti-
GFP and anti-DsbC antibodies and, following development, were stripped in
TBS/2%
SDS/0.7 M (3-mercaptoethanol. Stripped membranes were re-blocked and probed
with
anti-GroEL antibody.
FRCS Screening of Putative Leader Peptide:
To express the leader peptide-GFP-SsrA constructs, overnight (o/n) cultures of
MC4100-P and B1LK0-P containing each of the 30 plasmids were grown in LB media
as
described above. Single colonies were grown overnight in 2 mL of media. Five
hundred
p.l of each o/n culture were used to inoculate 10 mL of media. After 1 h
shaking at 37°C,
cells were induced with arabinose to a final concentration of 0.02%. Following
four more
hours of incubation at 37°C, 1 mL samples were harvested and
centrifuged at 2500xg for 5
min. Cell pellets were resuspended in 1 mL of PBS. Of that, 5 ~,L were added
to 1 mL
fresh PBS and analyzed by the Becton-Dickenson FACSort.
Thirty putative TAT leader peptides were screened in a genetic screen as
described
previously (DeL~sa° et, al 2000. With this genetic screen, a leader
peptide that directs GFP
through the TAT pathway would be fluorescent in tatC + cells (MC4100-P) but
non-
fluorescent in tatC - cells (B 1 LKO-P) since tatC is absolutely necessary for
TAT export.
By contrast, a leader peptide that directed GFP to the periplasm via the Sec
pathway would
be non-fluorescent in both types of cells. Of note is the use of E. coli
strains containing a
mutation in pcnBl, which lowers the copy number of those plasmids (such as
pBADl8-
Cm) that contain the pBR322 replicon. Thus pBADl8-Cm, which is normally a high
copy
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vector, is only present at approximately 5-10 copies per cell in pcnBl
mutants. This
system proved optimal for use with the TAT pathway genetic screen.
The FACS analysis for the pBAD 1 ~-Cm constructs are shown in Table 4 (a list
of
arithmetic mean fluorescence values). Importantly, the FAGS data for the
pBADl~-Crn
constructs shows that six leader peptides (BisZ, , NapA, Nape, YaeI, YgfA, and
YggJ)
gave inconclusive GFP export through the TAT pathway (low signal in both wt
and tatC
mutant celsl) while at least 17 (AmiC, DmsA, FdnG, FdoG, FhuD, HyaA, HybA,
NrfC,
Sufl, TorA, WcaM, YacK, YahJ, YdcG, YdhX, YfhG, and, YnfE) are capable of
exporting
GFP via the TAT pathway. Five constructs (YagT, YcbK, YcdB, YedY, and YnfF')
displayed very high fluorescence means in both MC4100-P and B1LK0-P. It should
also
be noted that the higher mean fluorescence signals seen for some of the
constructs in the
tatC mutant (B1LK0-P) reflected emission from only a small population of
highly
fluorescent cells while the bulk of the cell population was non-fluorescent.
In contrast, the
high mean fluorescence of the tatC+ cells (MC4100-P) was indicative of a shift
in the
fluorescence emission throughout the population.
TABLE 1: E. coli. TAT-Specific Leader Peptides
# Gene Sequence SEQ ID
NO.
1 WcaM MPFKKLSRRTFLTASSALAFLHTPFARAL 25
2 NrfC MTWSRRQFLTGVGVLAAVSGTAGRVVAK 26
28
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3 YahJ MKESNSRREFLSQSGKMVTAAALFGTSVPLAHAA 27
4 HyaA MNNEETFYQAMRRQGVTRRSFLKYCSLAATSLGLGA 28
GMAPKIAWAL
YacK MQRRDFLKYSVALGVASALPLWSR.AVFAA 29
6 YcbK MDKFDANRRKLLALGGVALGAAIL,PTPAFAT 30
7 YfhG MRHIFQRLLPRRLWLAGLPCLALLGCVQNHNK 31
8 YcdB MQYKDENGVNEPSRRRLLKVIGALALAGSCPVAHAQ 32
9 AmiA MSTFKPLKTLTSRRQVLKAGLAALTLSGMSQAIAK 33
10YedY MKKNQFLKESDVTAESVFFMKRRQVLKfIL,GISATAL 34
SLPHAAHAD
11FhuD MSGLPLISRRRLLTAMALSPLLWQMNTAHA.A 35
12HybA l~~IVRRNFIKAASCGALLTGALPSVSHAAA 36
13YdcG MDRRRFIKGSMAMAAVCGTSGIASLFSQAAFAA 37
14Sufi MSLSRRQFIQASGIALCAGAVPLKASAA 38
15YagT MSNQGEYPEDNRVGKHEPHDLSLTRRDLIKVSAATA 39
ATAVVYPHSTLAA
16 YdhX MSWIGWTVAATALGDNQMSFTRRKFVLGMGTVIFFT 40
GSASSLLAN
17 HybO MTGDNTLIHSHG11~1RRDFMKLCAALAATMGLSSKAA 41
AE
18 YnfF MMI~ITTEALNIK_AEISRRSLMKTSALGSLALASSAFT 42
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LPFS QMVR.A.A
19 DmsA MKTKIPDAVLAAEVSRRGLVKTTAIGGLAMASSALTL 43
PFSRIAHAV
20 YnfE MSKNERMVGISRRTLVKSTAIGSLALAAGGFSLPFTL 44
RNAA.A AV
21FdoG MQVSRRQFFKICAGGMAGTTAAALGFAPSVALAE 45
22AmiC MTDYASFAKVSGQISRLLVTQLRFLLLGRGMSGSNTA 46
ISRRRL,LQGAGAMWLLSVSQVSLAA
23YggJ ilvin-arginine consensus motif: RRRGFLT 47
24YgfA twin-arginine consensus motif QRRRALT 48
25BisZ twin-arginine consensus motif TRREFIK 49
26NapA twin-arginine consensus motif SRRSFMK 50
27Nape twin-arginine consensus motif GRR1ZFLR 51
28FdnG twin-arginine consensus motif-. SRRQFFK 52
29YaeI twin-arginine consensus motif SRRRFLQ 53
*Amino acids highlighted in gray constitute the twin-arginine consensus motif.
TABLE 2: Forward Primers And Their Melting Temperature For Each of The 29
TAT-Specific Leader Peptides
Name Tn., Sequence SEQ ID
(°C) NO.
WcaM fort 57.0 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 54
CCATTTAAAAAACTCTCCCGA
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NrfC 57.5 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 55
for
ACCTGGTCTCGTCGC
YahJ 57.5 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 56
fort
AAAGAAAGCAATAGC
HyaA 55.8 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 57
fort
AATAACGAGGAAACATTTTACCAG
YggJ 62.5 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCGTG 58
for
GGGAGACGACGCGGA
YacK 51.8 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 59
for
CAACGTCGTGATTTC
Nape 57.5 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 60
for
TCCCGGTCAGCGAAA
YcbK 52.9 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 61
for
GACAAATTCGACGCT
YfhG 48.9 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 62
for
CGACACATTTTTCAA
YcdB 52.9 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 63
fort
CAGTATAAAGATGAAAACGG
AmiA 47.5 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 64
for
AGCACTTTTAAACCA
B1971 51.0 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 65
fort
AAAAAGAATCAATTTTTAAAAGAATC
FhuD 54.2 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 66
for
AGCGGCTTACCTCTT
YgfA 55.6 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 67
for
ATTCGGCAACGTCGT
BisZ 50.7 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 68
fort
ATCAGGGAGGAAGTT
HybA 60.3 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCGTG 69
fort
AACAGACGTAATTTTATTAAAGCAGCCTC
YdcG 48.6 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 70
for
GATCGTAGACGATTT
Sufi 57.1 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 71
for
TCACTCAGTCGGCGT
YagT 55.8 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 72
for
AGCAACCAAGGCGAA
B1671 51.5 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 73
for
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TCATGGATAGGGTGG
B2997 48.8 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 74
for
ACTGGAGATAACACC
NapA for 51.8 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 75
AAACTCAGTCGTCGT
B1588 58.9 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 76
fort
ATGAAAATCCATACCACAGAGGCG
DmsA for 53.1 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 77
AAAACGAAAATCCCTGATG
YnfE for 56.3 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 78
TCCAAAAATGAACGAATGGTG
FdnG for 56.8 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 79
GACGTCAGTCGCAGA
FdoG for 55.5 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 80
CAGGTCAGCAGAAGG
AmiC for 60.8 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 81
ACAGATTATGCGTCTTTCGCTAAAGTT
YaeI for 58.5 GCGATGGAGCTCTTAAAGAGGAGAAAGGTCATG 82
ATTTCACGCCGCCGA
TABLE 3: Reverse Primers And Their Melting Temperature For Each of The 29
TAT-Specific Leader Peptides
Name Tm (C) Sequence SEQ ID
NO.
WcaTvI 59.7 GCGATGTCTAGAGCTTTGTCGGGCGGG 83
rev2
AAG
NrfC rev3 56.2 GCGATGTCTAGAATTGATATTCAACGTT 84
TTCGCCAC
YahJ rev3 60.1 GCGATGTCTAGATAGGGTGCCAGCTAC 85
CGC
HyaA rev2 57.4 GCGATGTCTAGAGCGCGGTTTGTTCTCC 86
AG
YggJ rev3 57.3 GCGATGTCTAGATACGCGCCCGATATG 87
GTT
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YacK rev456.5 GCGATGTCTAGATAACGTTGGGCGTTCT 88
GC
Nape rev265.1 GCGATGTCTAGAGCGCAACCGCACGCC 89
AGA
YcbK rev256.9 GCGATGTCTAGAGCGTGGGGTAGAGAG 90
TGT
YfhG rev253.4 GCGATGTCTAGACGTATCAATGGCTGG 91
CTT
YcdB rev252.1 GCGATGTCTAGACGCACTTTGCGTTTTT 92
TG
AmiA rev447.8 GCGATGTCTAGATTTTAAAAGTTCGTCT 93
TTGG
B 1971 50.2 GCGATGTCTAGAAAACCAGCTAAGCAG 94
rev2
ATC
FhuD rev453.3 GCGATGTCTAGAATTGGGATCAATAGC 95
CGC
YgfA rev250.6 GCGATGTCTAGAGAATACAGCGACCGT 96
ATG
BisZ rev252.3 GCGATGTCTAGATTTACCGCCCTTCTCT 97
TC
HybA rev262.5 GCGATGTCTAGATGGCGGGCGGTTTTC 98
AGC
YdcG rev248.6 GCGATGTCTAGAGGCAATATCAGAATC 99
TGC
Sufl rev263.2 GCGATGTCTAGACGGTTGCTGTTGCCCG 100
GC
YagT rev262.3 GCGATGTCTAGAAGCTGCGGGAACGCT 101
TGC
B1671 51.3 GCGATGTCTAGACTTTTCTTGCCTCGTG 102
rev2
TT
B2997 55.5 GCGATGTCTAGAAACCGATTCGGCCAT 103
rev2
CTC
NapA rev260.3 GCGATGTCTAGACTGACCAACAACGGC 104
GCG
B1588 56.9 GCGATGTCTAGATTCTACCGGAGCCTCT 105
rev4
GC
DmsA rev 55.5 GCGATGTCTAGATGGAATGGCGCTATC 106
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GAC
YnfE rev 57.5 GCGATGTCTAGATTTTTCGCGGGCCTGT 107
TG
FdnG rev 54.9 GCGATGTCTAGATAATTTGTAGTTTCGC 108
GCCTG
FdoG rev 54.7 GCGATGTCTAGACAGTTTATACTGCCGG 109
GTTTC
AmiC rev 61.6 GCGATGTCTAGACGCCACGACCTGGCT 110
GAC
YaeI rev 58.9 GCGATGTCTAGAGCTCGTGGCTATCGTC 111
GC
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TABLE 4: FRCS Screening of Putative Leader Peptides
Leader PeptideMC4100-P B1LK0-P (% Cells) Protein Export
AmiC 9 2 (95) +
BisZ 2 3 (100)
DmsA 287 11 (95) +++
FdnG 1 2 (100) ND
FdoG 44 2 (97.1) +
FhuD 10 2 (99.5) +
HyaA 90 3 (96.6) ++
HybA 411 2 (95.6) +++
HybO N/A N/A
NapA 1 2 ( 100)
Nape 6 7 (95)
NrfC 43 9 (95) +
Sufi 337 3(96.6) +;-r-
TorA 203 34 (100) +++
WcaM 96 6 (99) ++
YacI~ 72 13 ( 100) ++
YaeI 2 2 (100) -
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YagT 436 235 (95) -
YahJ 684 3 (100) +++
YcbK 367 97 (95) +
YcdB 514 356 (95) -
YdcG 59 27 (100) ++
YdhX 18 4 (95) +
YedY 73 35 (95) -
YfhG 36 7 (95) +
YgfA 8 3 (100)
YggJ 1 2 (100)
YnfE 24 8 (100) +
YnfF 203 101 (95) -
Arithmetic fluorescent means from FACS data of pBADl8-Cm::leader peptide-GFP-
SsrA
constructs in MC4100-P and B1LK0-P cells. Data for the B1LK0-P cells were
calculated
from all the cells (% cells shown) except the small population of highly
fluorescent cells.
EXAMPLE 2
Bacterial Strains and Plasmids Construction
All strains and plasmids used in the following examples are listed in Table 5.
E.
coli strain XLl-Blue (YecAl endAl gyfA96 thi-I lasdRl7 supE44 relAl lac [F'
proAB
lacI9ZdMI5Tn10 (Tet~]) was used for all experiments unless otherwise noted. E.
coli
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XL1-Blue tatB and XLl-Blue tatC were made using pFAT24 (Sargent et al. 1999)
and
pFATl66 (Bogsch et al., 1998) respectively according to established procedure
(Bogsch
et al., 1998). Strains were routinely grown aerobically at 37°C on
Luria-Bertani (LB)
media and antibiotic supplements were at the following concentrations:
ampicillin, 100 p.g
ml-1, chloramphenicol, 25 ~g ml-1.
The plasmids constructed in the following examples were based on pBAD33
(Gunman et al., 1995) and were made using standard protocols Sarnbrook et al.,
2000).
Plasmid pGFP was constructed by cloning the GFPmut2 variant (Crameri et al.,
1996)
using the primers GFPXbaI (5'-GCGATGTCTAGAAGTAAAGG
AGAAGAACTTTTCACT-3', SEQ ID NO:112) and GFPHindlTl (5'-
GCGATGAAGCTTCTATTTGTATAGTTCATCCAT-3', SEQ ID N0:113) which
introduced unique restriction sites of XbaI and Hindla at the 5' and 3' ends
respectively of
the 716-by gfpmut2 gene and enabled cloning of this sequence into XbaI-
HineIIII digested
plasmid DNA. Plasmid pGFPSsrA was made similarly using the primers GFPXbaI and
GFPSsrA (5'-GCGATGAAGCTTGCATGCTTAAGCTGCTAAAGCGTAGTTTTCG
TCGTTTGCTGCGTCGACTTTGTATAGTTCATCCATGCC-3', SEQ ID N0:114) to
introduce the unique SsrA recognition sequence. Plasmid pTorAGFP and
pTorAGFPSsrA
were made by PCR~ amplification of E. coli genomic DNA using primers TorASacI
(5'-
GCGATGGAATTCGAGCTCTTAAAGAGGAGAAAGGTCATGAACAATAACGATCT
CTTTCAG-3', SEQ ID NO:115) and TorAXbaI (5'-
GCGATGTCTAGAAGCGTCAGTCGCCGCTTGCGCCGC-3', SEQ ID N0:116) to
generate a138-by torA cDNA with unique SacI and ~'baI restriction sites at the
5' and 3'
ends respectively. This sequence was then inserted into SacI-XbaI digested
pGFP or
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pGFPSsrA plasmid DNA. All plasmids constructed in this study were confirmed by
sequencing.
TABLE 5: Bacterial Strains And Plasmids
Strain or plasmid Relevent genotype/phenotype Source
E. co.li strains
XLl-Blue Stratagene
XLtatB XLl-Blue with tatB deletion This study
XLtatC XL1-Blue with tatC deletion This study
Plasmids
pFAT24 pMAK.705 carrying tatB deletion allele(Sargent
et
al., 1999)
pFATl66 pMAK705 carrying tatC deletion allele(Bogsch
et al.,
1998)
pGFP Signal sequenceless GFP in pBAD33 This study
pGFPssrA Signal sequenceless GFP tagged with This study
C-terminal
ssrA tag in pBAD33
pTorAGFP TorA leader peptide fused to GFP in This study
pBAD33
pTorAGFPssrA TorA leader peptide fused to ssrA-taggedThis study
GFP in
pBAD33
pB6::GFP Clone B6 leader cloned into pGFP This study
pB7::GFP Clone B7 leader cloned into pGFP This study
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pE2::GFP Clone E2 leader cloned into pGFP This study
pTorAR30Q pTorAGFP with R12Q mutation in leader This study
pTorAR30QGFPssrA pTorAGFPSsrA with R12Q mutation in leader This study
EXAMPLE 3
Flow Cytometric Analysis
Overnight cultures of XLl-Blue cells harboring GFP-based plasmids were
subcultured into fresh LB medium with chloramphenicol and induced with 0.2%
arabinose
in mid-exponential phase growth. After 6 h, cells were washed once with PBS
and 5 ul
washed cells were diluted into 1 ml PBS prior to analysis using a Becton-
Dickinson
FACSort.
EXAMPLE 4
Generation of torA Combinatorial Libraries
A library of random mutants was constructed by error prone PCR of the torA
gene
sequence using 3.32 or 4.82 rnM Mg2+ (Fromant et al., 1995), XL1-Blue genomic
DNA
and the following primers: torASacI (5'-
GCGATGGAATTCGAGCTCTTAAAGAGGAGAAAGGTCATGAACAATAACGATCT
CTTTCAG-3') (SEQ ID N0:117) and torAXbaI (5'-
GCGATGTCTAGAAGCGTCAGTCGCCGCTTGCGCCGC-3') (SEQ ID N0:118). To
construct libraries with 0.5% error rate, 0.22 mM dATP, 0.20 mM dCTP, 0.34 mM
dGTP
and 2.36 mM dTTP were used, whereas 0.12 mM dATP, 0.1 mM dCTP, 0.55 mM dGTP
and 3.85 mM dTTP were used to construct libraries with 1.5% error rate.
Libraries were
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digested with SacI XbaI and lzgated into pGFPssrA between SacI ~baI, placing
the library
upstream of the gfpssrA sequence. Reaction mixtures were electroporated into
electrocompetent XLl-Blue cells (Stratagene), and serial dilutions were plated
on selective
plates to determine the number of independent transformants.
EXAMPLE 5
Library Screening
Transformants were grown at 37°C in LB medium with chloramphenicol,
induced
with 0.2% arabinose for 6 h and diluted 200-fold in 1 ml PBS. FAGS gates were
set based
upon FSC/SSC and FLl/FL2. Prior to sorting, the library cell population was
labeled with
propidium iodide for preferential labeling of non-viable cells. A total of ca.
3x106 cells
were analyzed by flow cytometry and 350 viable cells were collected. The
collected
solution was filtered, and the filters were placed on LB plates with
chloramphenicol. After
a 12 hour incubation at 37°C, individual colonies were inoculated into
LB with
chloramphenicol in triplicate 96-well plates. Following 12 hours of growth at
37°C, cells
were similarly subcultured into triplicate 96-well plates containing LB with
chloramphenicol and 0.2% arabinose and grown for 6 hours at 37°C.
Individual clones
were screened via FACS and fluorescent plate reader (Bio-Tek FL600, Bio-Tek
Instrument,
Winooski, VT) for verification of fluorescent phenotype.
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EXAMPLE 6
Cell Fractionations
The fraction of periplasmic proteins was obtained by spheroplasting bacteria
by
lysozyme-EDTA treatment under isotonic conditions according to the procedure
of Kaback
(1971). Briefly, cells were collected by centrifugation and resuspended to an
OD6on of
in a buffer containing 100 mM Tris-Cl (pH 8.0), 0.5 M sucrose, and 1 mM Na-
EDTA.
Lysozyme (Sigma) was added to 50 ~g/ml, and cells were incubated for 1 h at
room
temperature to generate spheroplasts. The spheroplasts were pelleted by 15 min
of
centrifugation at 3,000 x g, and the supernatant containing periplasmic
proteins was
10 collected for electrophoretic analysis. The pellet containing spheroplasts
was resuspended
in 10 ml of TE (10 mM Tris-Cl [pH 7.5~, 2.5 mMNa-EDTA) and homogenized in a
French
press cell (Carver) at 2,OOOlb/ina. To analyze total proteins of untreated
cells, direct
resuspension in 10 ml of TE followed by subjection to the French press
homogenization
was performed.
EXAMPLE 7
Screening of Signal Peptide Libraries for Improved Export Phenotypes
The plasmid pTorAGFP contains a gene encoding the TAT-specific leader peptide
and the first eight amino acids of the E. coli trimethylamine N oxide
reductase (TorA)
fused to the FACS optimized GFPmut2 gene (Crameri et al., 1996). The TorA-GFP
gene
was placed downstream of the arabinose-inducible promoter pBAD. Cells induced
with
arabinose for 6 hours and analyzed by FAGS gave a mean fluorescence intensity
(MFLl)
above S00 arbitrary units (FIG. 1C). In agreement with previous reports
(Santini et al.,
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2001), cell fractionation by osmotic shock revealed that ca. 40-50% of total
fluorescence
was located in the periplasm of wild type cells while cytoplasmic GFP
accounted for the
remaining 50-60% of total fluorescence. In tatB and tatC mutants where the TAT
pathway
were abolished, greater than 95% of total fluorescence was retained in the
cytoplasm,
thereby demonstrating that TorA-GFP is exported via the TAT pathway.
A nucleotide sequence encoding a C-terminal SsrA degradation peptide was fused
to the TorA-GFP gene. The resulting gene, pTorA-GFP-SsrA, was also placed
downstream from a pBAD promoter in the vector pTorAGFPSsrA. As a negative
control,
GFP without the leader peptide was fused in frame to the SsrA tag and
expressed from the
plasmid pGFPSsrA. GFP-SsrA-expressing cells showed virtually no appreciable
fluorescence intensity, indicating that cytoplasmic SsrA-tagged GFP is
degraded almost
completely (FIG. lA). Cells expressing TorA-GFP-SsrA were ca. 8 times more
fluorescent
compared to GFP-SsrA expressing cells (FIG. 1B). Expression of TorA-Gfp-SsrA
in tatB
and tatC mutant cells only led to background fluorescence.
Error prone PCR (Fromant et al., 1995) was used to generate libraries of
random
mutants of the TorA leader peptide. Three libraries with expected mutation
frequencies of
0.5, 1.5 or 3.5% nucleotide substitutions were constructed. The mutated TorA
leader
peptides were ligated upstream of the GFP-SsrA sequence in pGFPSrA.
Transformation of
E. coli resulted in libraries consisting of between 106 and 107 independent
transformants.
Sequence analysis of 20 randomly selected clones confirmed the presence of
randomly
distributed mutations within the TorA leader peptide.
FACS-based screening of the three libraries resulted in isolation of a total
of six
clones, 2 from the higher error rate library and four from the lower error
rate libraries. All
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six clones exhibited higher cell fluorescence relative to the parental TorA-
Gfp-SsrA
construct (FIG. 2). The increase in the fluorescence level was between 3 and 6-
fold
relative to what is obtained with the wild type leader peptide. Back
transformation of these
clones into strains XLl-Blue or DHB4 resulted in maintained fluorescence
levels, thus
indicating that the increased fluorescence was conferred by the respective
plasmids and
was not due to an unrelated mutation in the host cell. When the plasmids were
transformed
into tatB or tatC cells the cell fluorescence was abolished, as would be
expected for a
process that is dependent on the TAT export system.
Representative Western blots indicate that periplasmic GFP accumulation by
cells
expressing the B6 and E2 clones was significantly increased relative to those
expressing
wild type construct (lanes 1-3, FIG. 3). Furthermore, there was virtually no
detectable GFP
protein in the cytoplasmic fractions. This was because the presence of the
SsrA tag
resulted in degradation of the protein. Also shown in FIG. 3 were Western
bands of two
fractionation marker proteins, the cytoplasmic marker GroEL and the
periplasmic marker
DsbA. The absence of GroEL in the periplasmic fraction and the high level of
DsbA in
periplasmic fractions confirm that cell fractionation was successful.
Data on the distribution of fluorescence in the cytoplasmic and periplasmic
fractions for two mutant TorA leader peptides are shown in FIG. 3B. Nearly
identical
results were observed for the remaining four clones (B7, Fl, Fl 1 and H2). The
sequences
of the six clones were determined and indicated that in all cases either one
or two single
residue mutations were sufficient to alter the observed export dynamics. In
general, these
mutations occur within or in close proximity to the conserved S/T-R-R-x-F-L-I~
(SEQ ID
NO:l) consensus motif (Table 6).
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It was confirmed that the six mutant TorA leader peptides confer increased GFP
export not only when the protein is tagged with the SsrA tag but also for the
untagged,
proteolytically stable GFP (FIG. 4). This increase in fluorescence was due to
the increased
periplasmic flux of folded GFP protein. Similar results were observed for the
remaining
clones fused to GFP.
A representative Western blot comparing wild type TorA-GfP and TorAB7-Gfp
indicated that cells expressing both constructs accumulated nearly identical
levels of
cytoplasmic GFP (FIG. 5, lanes 3 and 4). However, the amount of exported GFP
was
significantly higher in cells expressing the ToAB7-GFP clone (FIG. 5, lanes 1
and 2).
Further support of this can be seen in the whole cell lysates. The intense
band denoted as
mature (M) GFP represents TorA-Gfp chimeric protein that has been processed
most likely
by signal peptidase I (Berks et al., 2000). Therefore, the intense band
corresponding to
mature GFP accumulated by the TorAB7-GfP construct signifies substantially
more
periplasmic processing of GFP relative to wild type TorAGFP cells (FIG. 5,
lanes 5 and 6).
Similar results were observed for all five remaining clones. As described
above, the
GroEL and DsbA marker proteins confirm successful cell fractionations.
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TABLE 6: Sequences Of Six Clones Exhibiting Increasing TAT-Dependent Secretion
Clone Amino Acid Sequence
m
Wild typeMNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAAQA.ATD
(SEQ m NO:120)
B6 MNNNDLFQTSRRItLLAQLGGLTVAGMLGPSLLTPRR.ATAAQAATDA
(SEQ m No.121)
B7 MNNNDLFQTSRQRFLAQLGGLTVAGMLGPSLLTPRRATAAQAATDA
(SEQ m NO:122)
E2 MfNNNDIFQASRRRFLAQPGGLTVAGMLGPSLLTPRRATAAQAATDA
(SEQ m N0:123)
F 1 l~fI~NNELFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAA QAA TDA
(SEQ m N0:124)
F 11 I~~INNNDLFQTTRRRFLAQLGGLT VAGMLGPSLLTPRRATAA QAA TDA
(SEQ m N0:125)
H2 MNNNDSFQTSRRRFLAQLGGLTVAGMLGPSLLTPRRATAAQAATDA
(SEQ m N0:126)
Twin arginine consensus motif is indicated by underlined amino acids; first 8
residues of
mature TorA protein are indicated by italics; mutations in TorA leader peptide
are
indicated by emboldened letters.
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EXAMPLE ~
Secretion of Folded Recombinant Proteins Containing Multiple Disulfide Bonds
Through The Twin Arginine Translocation Export Pathway
One embodiment of the methods disclosed herein comprises use of a fusion
between a TAT-specific leader peptide and a heterologous polypeptide of
interest. For
example, TAT-specific leader peptide TorA may be fused to alkaline phosphatase
(TorA-
PhoA fusion). Alkaline phosphatase (PhoA) contains two disulfide linkages
that. are
consecutive in the primary sequence so that they are normally incapable of
forming in the
cytoplasm of E. coli strains having reducing environment (e.g. strain DHB4).
Since the
TAT pathway requires folded or at least partially folded substrates, TAT-
dependent
secretion of PhoA in DHB4 cells would be blocked due to the accumulation of
unfolded
PhoA in the cytoplasm.
Hence, there is a need to change the cytoplasm into an oxidizing state for
secretion
through the TAT pathway. Normally, the bacterial cytoplasm is maintained in a
reduced
state due to the presence of reducing components such as glutathione and
thioredoxins that
strongly disfavors the formation of disulfide bonds within proteins. Earlier
work by
Bessette et al. resulted in the engineering of bacterial strains having a
highly oxidizing
cytoplasm that allows efficient formation of disulfide bonds (Bessette et al.,
1999). As
shown in Bessette et al., E. eoli depends on aerobic growth in the presence of
either of the
two major thiol reduction systems: the thioredoxin and the glutathione-
glutaredoxin
pathways. Both the thioredoxins and the glutaredoxins are maintained in a
reduced state by
the action of thioredoxin reductase (TrxB) and glutathione, respectively.
Glutathione is
synthesized by the gshA and gshB gene products. The enzyme glutathione
oxidoreductase,
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the product of the gor gene, is required to reduce oxidized glutathione and
complete the
catalytic cycle of the glutathione-glutaredoxin system.
In a trxB null mutant, stable disulfide bonds can form in normally secreted
proteins,
such as alkaline phosphatase, when they were expressed in the cytoplasm
without a signal
sequence. The two thioredoxins were oxidized in the trxB mutant and served as
catalysts
for the formation of disulfide bonds. Disulfide bond formation was found to be
even more
efficient in double mutants defective in both the thioredoxin (trxB) and
glutathione (gor or
gshA) pathways. Double mutants, trxB gor or trxB gshA, grow very poorly
(doubling time
300 min) in the absence of exogenous reluctant such as DTT and accumulate
suppressor
mutations in the alkyl hydroperoxidase (ahpC) gene. The resulting ahpC* allele
allows
efficient growth in normal (non-reducing media) without compromising the
formation of
disulfide bonds in the cytoplasm. Thus trxB, gor ahpC* mutant strains (such as
E. coli
DR473 or FA113) exhibit the ability to support disulfide bond formation in the
cytoplasm
and also can grow equally well as the corresponding wild-type strain DHB4 in
both rich
and minimal media.
In the present example, DHB4 cells expressing TorA-PhoA were found to exhibit
almost undetectable alkaline phosphatase activity levels while DR473 cells
expressing
TorA-PhoA showed extremely high PhoA activity levels. Fractionation
experiments
confirmed that as much as 50% of the measured PhoA activity in cell lysates
were
attributed to periplasmic accumulation.
Since the major catalyst of disulfide bond formation is a periplasmic protein,
DsbA,
which oxidizes thiols in newly synthesized and translocated proteins, it was
next
determined whether the disulfide bonds were formed in the cytoplasm and
secreted intact
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to the periplasm. The TorA-PhoA construct was expressed in an E. coli dsbA
mutant,
strain DR473 dsbA::kan. A comparison of PhoA activity in the dsbA mutant
versus the
isogenic DR473 parental strain revealed nearly identical activity levels in
whole cell
lysates. This result demonstrates that oxidation of the PhoA protein is
completed in the
cytoplasm, and stable disulfide bonds are able to transverse the inner
membrane as the
protein is directed from the cytoplasm into the periplasmic space (Table 9).
In order to measure the extent of folding necessary for substrate
compatibility with
the TAT secretion pathway, eukaryotic model proteins with increasingly complex
patterns
of disulfide bond formation were tested. The TorA leader peptide was fused to
a truncated
version of tissue plasminogen activator (vtPA) consisting of the kringle 2 and
protease
domains with a total of nine disulfides (TorA-vtPA), or to a heterodimeric
2610 anti-
digoxin antibody fragment with 5 disulfide bonds including an interchain
disulfide linkage
(TorA-Fab). DR473 cells expressing TorA-vtPA and TorA-Fab showed remarkably
high
levels of activities in cell lysates for each of the expressed proteins
relative to DHB4 cells
expressing identical constructs. Activities in DHB4 lysates were virtually
undetectable in
all cases except for vtPA. Fractionation experiments further confirmed that
significant
portion (30-50%) of the overall activities for each of the proteins was found
in the
periplasmic fraction.
In conclusion, these results show efficient secretion of disulfide linked
proteins can
occur via the Tat pathway but only in host cells that are able to fold these
proteins into their
native conformation. Low background levels of active tPA in the periplasm of
DHB4 cells
suggests that this protein is able to at least partially fold in a reducing
cytoplasm. The
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resulting folded proteins with multiple disulfide bonds are then secreted into
the periplasm
as an active homo- (alkaline phosphatase) or heterodimer (2610 antibody
fragment).
EXAMPLE 9
Demonstration of Export of Multidisuliide Proteins by the Bacterial
Twin-Arginine Translocator
An examination was carried out to determine whether the formation of disulfide
bonds in the cytoplasm of trxB gor aphC mutants was sufficient to render
proteins
competent for export via the Tat pathway. This was shown to be the case for
two model
proteins, namely PhoA and Fab fragment raised against digoxin (Fab). PhoA
consists of
two polypeptide chains with a total of two disulfide bonds that are required
for folding and
enzymatic activity while Fab is comprised of two non-identical chains (each
with two
intermolecular disulfide bonds) linked together by an intermolecular disulfide
bond.
Normally, the formation of disulfide bonds in these proteins occurs following
export into
1 S the oxidizing environment of the periplasmic space. However, in the
analysis, it was
demonstrated that proteins with multiple disulfides can be exported via the
Tat system after
they have first folded in an oxidizing cytoplasm and further, that the
transporter
mechanistically requires that the substrate be folded properly for periplasmic
localization.
A. Procedures
Bacterial strains, growth and induction conditions:
The bacterial strains and plasmids used are described in Table 7. Strains DHBA
and DRA were obtained by Pl transduction of the dsbA::kanl allele from JCB571
(MC1000 phoR zihl2::Tn10 dsbA::kan) into E. coli strains DHB4 and DR473,
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respectively. Strain DASD was obtained by P1 transduction of tatB::kan allele
from
MCMTA (MC4I00 tatB::kan) into E. coli strain DR473. Strains FUDDY was obtained
by
P I transduction of the tatC: apec allele from BUDDY (MC4100 tatC: apec) into
E. coli
strain FA113. E. coli strain XL1-Blue (recAl endAl gyrA96 thi-I hsdRl7 supE44
relAl
lac [F' pf~oAB lacl~ZDMlS TialO (Tetr)]) was used for cloning and plasmid
propagation.
For phosphatase assays, cells were subcultured from overnight cultures into
minimal M9
medium [M9 salts with 0.2% glucose, 1 p.g/ml vitamin Bl, 1 mM MgS04, SO p.g/ml
18 amino acids (excluding methionine and cysteine)] at a 100-fold dilution,
and then
incubated at 37°C. For Fab studies, cells were subcultured from
overnight cultures into
fresh LB medium (5% vlv) and then incubated at 30°C. Growth was to mid-
log phase
(OD6op~0.5) and induction of both alkaline phosphatase and Fab was
accomplished by
addition of IPTG to a final concentration of 0.1 mM. Co-expression of DsbC was
induced
using 0.2% arabinose. Antibiotic selection was maintained for all markers on
plasmids at
the following concentrations: ampicillin, 100 p.g/ml; spectinomycin, 100
p.g/ml; and
chloramphenicol, 25 p,g/ml.
Plasmid construction:
Plasmid p33RR was constructed by PCR amplification of the E. coli torA signal
sequence (ssTorA) from E. coli genomic DNA using primers TorASacI and TorAXbaI
described above. Amplified DNA was digested using Sacl and XbaI arid inserted
into the
same sites of pBAD33. Plasrnid p33KK was generated identically as p33RR except
that
mutagenic primer TorAkk (5'-
gcgatggagctcttaaagaggagaaaggtcatgaacaataacgatctctttcaggcatcaaagaaacgttttctggcac
aactc-3')
(SEQ 1D N0:129) was used to PCR amplify the to3A signal sequence. DNA encoding
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signal sequence-Iess phoA (PhoA d2-22) was generated by PCR amplification from
E. coli
genomic DNA using primers Phofor (5'-gcgatgtctagacggacaccagaaatgcctgt-3') (SEQ
ID
N0:130) and Phorev (5'-gcgatgaagcttttatttcagccccagagcggctt-3') (SEQ ID
N0:131). The
amplified phoA DNA was digested with XbaI and HindIQ and inserted into the
same sites
of p33RR and p33KK resulting in plasmids p33RRP and p33KKP, respectively. A
DNA
fragment encoding torA signal sequence (or torA (R11K;R12K) signal sequence)
fused in-
frame to phoA was amplified from plasmid p33RRP (or p33KKP) using primers
TorASacI
(or TorAKK) and Phorev. The PCR amplified DNA was digested with BSpHI and
HindIlI
and inserted into the NcoI-HindIll sites of pTrc99 resulting in plasmid pRRP
(or pKKP).
Construction of alkaline phosphatase fusions to alternate signal sequences
(e.g. ssFdnG,
ssFdoG) was performed identically as described for pTorA-AP. Plasmid pTorA-Fab
was
constructed by PCR amplification of the anti-digoxin dicistronic Fab gene
encoded in
pTrc99-Fab (Levy et al., 2001) using primers Fabfor (5'-
gctgctagcgaagttcaactgcaacag-3')
(SEQ ID N0:132) and Fabrev (5'-gcgatgcccgggggctttgttagcagccggatctca-3') (SEQ
ID
N0:133) and amplification of torA signal sequence was with primers TorASacI
and
TorAover (5'-gcgctgttgcagttgaacttcgctagcagcgtcagtcgccgcttg-3') (SEQ 117
N0:134). The
two PCR products were fused via overlap extension PCR using primers TorASacI
and
Fabrev. The overlapped product was digested with BspHI and XmaI and inserted
into the
NcoT and XmaI sites of pTrc99A. All plasmids were confirmed by sequencing.
Cell fractionations:
The fraction of periplasmic proteins was obtained by ice-cold osmotic shock
(Sargent et al., 1998). Specifically, cells were collected by centrifugation
and resuspended
in buffer containing 30 mM Tris-HCl (pH 8.0), 0.5 M sucrose, 1 mM Na-EDTA and
20
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CA 02465724 2004-05-04
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mM iodoacetamide was used to prevent spontaneous activation of alkaline
phosphatase.
Cells were incubated for 10 min at 25°C followed by centrifugation for
10 min at SOOOxg
and 4°C. Pellets were then resuspended in ice-cold 5 mM MgS04 and kept
on ice for 10
min. Cells were centrifuged as before and the supernatant containing
periplasmic proteins
was collected for electrophoretic analysis. The pellet was resuspended in 10
ml of TE
(10 mM Tris-Cl [pH 7.5~, 2.5 mM Na-EDTA) and 20 mM iodoacetamide and
homogenized in a French press cell at 2,0001b/in2. To analyze total proteins
of untreated
cells, direct resuspension in 10 ml of TE and 20 mM iodoacetamide followed by
subjection
to French press homogenization was performed.
Enzyme activity assays:
Cells expressing alkaline phosphatase were induced for 6 h. Samples were
harvested, treated with 20 mM iodoacetamide and pelleted by centrifugation.
Collected
cells were fractionated as described above. Soluble protein was quantified by
the Bio-Rad
protein assay, using BSA as standard. Activity of alkaline phosphatase was
assayed as
described previously. Briefly, equal amounts of protein were incubated with
200 p.l p-
nitrophenyl phosphaste (pNPP; Sigma) solution (1 fast tablet in 100 mM
Tris~HCl, pH 7.4)
and DA4os was measured to determine rate of hydrolysis by alkaline phosphatase
in each
sample. Fractionation efficiency was monitored using (3-galactosidase as a
cytoplasmic
marker enzyme and was assayed as described previously. Only data from
fractionations in
which the marker enzyme activities were ~5% correctly localized were analyzed.
ELISA:
Assays were performed as follows. Ninety-six-well high binding assay plates
(Corning-Costar) were coated (100 ul/well) with 4 ug ml-1 BSA-digoxin
conjugate or with
52
CA 02465724 2004-05-04
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4 ug ml-1 BSA (100 ul/well). Coated plates were blocked overnight at
4°C with 5% nonfat
dry milk in PBS. The presence of anti-digoxin scFv and Fab antibodies was
detected using
rabbit-anti-mouse IgG (specific to (Fab')2 light chains) diluted 1:2000
followed by goat
anti-rabbit IgG (H + L) conjugated with horse radish peroxidase diluted
1:1000.
Development was with addition of OPD substrate (Sigma) and the reaction was
quenched
by addition of 4.5 N HZS04. Plates were read at 490 nm on a Bio-Tek
Instruments
microplate reader.
Western blotting analysis:
Western blotting was according to Chen et al. (2001). The following primary
antibodies were used: rabbit anti-alkaline phosphatase (Rockland) diluted
1:5,000, rabbit
anti-tPA diluted 1:5,000, rabbit anti-mouse IgG (specific for (Fab')2 light
chains, Pierce)
diluted 1:5,000, monoclonal rabbit anti-DsbA and anti-DsbC (gift from John
Joly,
Genentech) diluted 1:10,000 and monoclonal rabbit anti-GroEL (Sigma) diluted
1:10,000.
The secondary antibody was 1:10,000 goat anti-mouse-HRP and goat anti-rabbit-
HRP.
Membranes were first probed with primary antibodies and, following
development,
stripped in TBS/2% SDS/0.7 M (3-mercaptoethanol. Stripped membranes were re-
blocked
and probed with anti-DsbA, anti-DsbC and anti-GroEL antibody.
B. A strategy for Tat-dependent export of multidisulfide proteins in E. coli
In bacteria the oxidative folding of secreted proteins is catalyzed by the
periplasmic enzyme DsbA which is recycled by the integral membrane protein
DsbB. In
contrast, the thioredoxin and glutaredoxin. pathways maintain the cytoplasm as
a highly
reducing environment, which disfavors cysteine oxidation in proteins. For this
reason,
host proteins requiring disulfide bonds are exported to the periplasmic
compartment, a
53
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
process facilitated almost exclusively by the Sec pathway in E. coli. The
export of such
proteins by the Tat pathway has been problematic because the Tat pathway
normally
accepts as substrates proteins that are already folded. Since proteins that
contain disulfide
bonds in their native state cannot fold in the cytoplasm, these proteins
presumably cannot
be accepted as substrates for Tat export. Indeed, several earlier studies have
demonstrated that proteins requiring disulfide bonds for folding are not
exported via the
Tat pathway.
It had been established previously and confirmed that PhoA fused to the
trimethylamine N oxide reductase A (TorA) leader peptide, or for that matter
other Tat
specific leader peptides, results in negligible alkaline phosphatase activity,
indicating lack
of export. Therefore, it was reasoned that proper folding, including the
formation of
disulfide bonds, in the cytoplasm prior to export would permit export via the
Tat
pathway. To analyze this, the TorA signal sequence was fused to the N-terminus
of E.
coli alkaline phosphatase (AP) devoid of its natural signal sequence. Wild-
type E. coli
cells (DHB4) harboring plasmid pTorA-AP and induced with IPTG (0.1 mM)
produced
large quantities of cytoplasmic AP as detected by Western blotting. However,
there was
no detectable AP in the periplasmic fraction of the DHB4 cells. Activity
measurements
of the same periplasmic fraction confirmed the lack of extracytoplasrnic AP.
As
expected, AP activity in the cytoplasmic fraction of DHB4 cells was almost
entirely
inactive due to its failure to acquire disulfides bonds in the cytoplasm of
this strain. To
determine whether the oxidation state of AP was critical for Tat-dependent
export, a trxB
gor ahpC triple mutant of E. coli (strain DR473) was used to express the
ssTorA-AP
fusion protein. When ssTorA-AP was expressed from plasmid pTorA-AP (0.1 mM
54
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
IPTG) in strain DR473, about 25% of the total enzymatic activity was found in
the
periplasmic space. Western blotting confirmed that partitioning of AP had
occurred. It
should be noted that the quantity of AP in the cytoplasm of DR473 cells was
significantly
greater than in DHB4 cells, suggesting that misfolded AP is more highly
susceptible to
cytoplasmic proteolysis.
In support of this notion, it has been reported that the intracellular
stability of
alkaline phosphatase was decreased in the absence of either one or both of the
disulfide
bonds. Importantly, (3-galactosidase (LacZ) activity in subcellular fractions
was measured
(see above) and only samples with <5% LacZ activity in the periplasm were
analyzed
herein. As a secondary control, cross-reaction of the cytoplasmic chaperone
GroEL with
specific antisera was used as a control for subcellular fractionation.
Overall, it was clear
that the folding status of AP was the maj or determinant in the ability to
export this
protein by the Tat pathway.
C. Export of PhoA is Tat-specific
~ It was recently observed that, in the context of certain Tat signals, AP can
be
exported in a Tat-independent fashion. Therefore, to confirm that export of AP
in DR473
was specific to the Tat pathway, a defective TorA signal peptide mutant in
which the Rl l
and R12 arginine residues were replaced with lysines (R11K;R12K) was fused in
frame
to signal-sequenceless AP to generate plasmid pKK-AP. It is well documented
that
replacement of the two conserved arginines with a pair of lysines within the
Tat
consensus motif (S/T-R-R-x-F-L-K) effectively abolishes translocation
(Cristobal, et al.,
1999). As expected, DHB4 and DR473 cells expressing ssTorA(R11K;R12K)-AP
fusion
protein were incapable of accumulating periplasmic AP. Importantly, the amount
of
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
cytoplasmic AP in DR473 cells was similar irrespective of whether RR or KK was
present within the leader peptide. It is noteworthy that ssTorA(R11K;R12K)-AP
accumulated in the cytoplasm of DHB4 cells to a much lesser extent than ssTorA-
AP in
the same cells. One possible explanation is that a proper Tat signal (Arg-Arg)
targets
even misfolded AP to the cytoplasmic side of the inner membrane. In turn,
membrance
localization sequesters some of the misfolded enzyme from proteolysis. In
contrast, the
defective Lys-Lys leader peptide does not properly interact with the Tat
machinery and as
a result non-targeted AP is more susceptible to cytoplasmic proteolysis. A
similar
phenomena has been observed in plant thylakoids where the N-terminal
presequence on a
large, bulky avidin-bound precursor is available for membrane binding and
initial
recognition by the transport machinery, but the attached avidin signals the
machinery that
the precursor is an incorrectly configured substrate and thus import is
aborted.
Consequently, Muser and Theg proposed that the ~pH/Tat machinery's
proofreading
mechanism must operate after precursor recognition but before the committed
step in
transport.
As an independent confirmation that export was Tat-dependent, P1 transduction
of DR473 with the tatB::kan allele from strain MCMTA was performed to generate
strain
DQ~D (DR473 tatB::kan). As expected, DQ~D cells expressing ssTorA-AP fusion
protein
from plasmid pTorA-AP were unable to accumulate AP in the periplasm as
evidenced by
Western blotting and activity measurements of subcellular fractions. In
addition, AP was
exported in a Tat-dependent fashion when fused to two different signal
sequences from
formate dehydrogenase-N (FDH-N) subunit G (ssFdnG) and FDH-O subunit G
(ssFdoG).
rr,llPCtivelv, these results confirm that the appearance of AP in the
periplasm was
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CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
completely dependent on export via the Tat pathuray and that translocation
could be
accomplished by several different Tat leader peptides.
D, Folding and oxidation occurs in the cytoplasm prior to export
To determine whether PhoA oxidation occurred in the cytoplasm prior to
translocation,
the ssTorA-AP fusion protein was produced in an E. coli dsbA null mutant
(strains DHBA and
DRA). DsbA is the major periplasmic enzyme involved in catalyzing disulfide
bond formation in
newly synthesized proteins normally secreted by the Sec pathway. As a result,
both the DHBA
and DRA mutant strains were completely unable to oxidize periplasmic proteins
due to a null
mutation of dsbA. Unexpectedly, expression of ssTorA-AP from plasmid pTorA-AP
(0.1 mM
IPTG) in strain DRA resulted in nearly identical periplasmic AP accumulation
and activity
compared to that obtained using the DR473 dsbA+ strain. Therefore, the
accumulation of active
AP in the periplasmic compartment was due almost entirely to the export of AP
that had already
been folded and oxidized in the cytoplasm.
To determine whether this phenomenom was specific to the TorA presequence or a
general feature of the Tat export system, 10 known and putative Tat leader
peptides were
analyzed (Table 8). The 10 signal sequences were fused in frame to signal
sequenceless AP,
expressed in six different but genetically related backgrounds and assayed for
periplasmic AP
activity. To establish a baseline for residual periplasmic AP activity, the
constructs were all
expressed in strain DHA (DHB4 dsbA:: kan). Since AP oxidation is prohibited in
both the
cytoplasm and periplasm of this strain, the total AP activity measured in DHA
was found to be
negligible for all leader peptide-AP fusions (Table 9). The periplasmic AP
activity measured in
the remaining S strains was normalized to this baseline level. For comparison,
the amount of
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CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
signal-sequenceless AP (02-22) exported in the same strains was measured and
found to be
negligible in all six backgrounds.
Next, expression of the constructs in wildtype cells (DHB4) resulted in two
distinct
outcomes: 1) the Tat leaders AmiA, FdnG, FdoG, HyaA, HybA and TorA were unable
to export
AP when the cytoplasm was reducing; however 2) certain other Tat leader
peptides (DmsA, Sufl,
YacK and YcbK) could direct AP to the periplasm even though disulfide bond
formation in the
cytoplasm was not possible. This was likely due to Sec-dependent export of AP.
As expected,
nearly all of the leader peptides were able to direct AP to the periplasm of
strain DR473 due in
part to the more oxidizing cytoplasm. The notable exceptions were ssAmiA and
ssHybA, which
were unable to accumulate AP in the periplasm of all the strains tested.
Comparison of AP
activity found in the periplasm of DR473 versus DRA (DR473 dsbA: : kan)
confirmed that in the
cases of ssFdnG, ssFdoG, ssHyaA and ssTorA, export of AP occurred only after
folding and
oxidation were accomplished in the cytoplasm. Expression of the constructs in
strains having an
oxidizing cytoplasm but a defective Tat apparatus (DQjD and DUDDY)
demonstrated that
ssFdnG, ssFdoG and ssTorA directed AP to the periplasm in a Tat-specific
fashion. In contrast,
the export of AP directed by ssSufl, ssYacK and ssYcbK was still able to occur
in tatB and tatC
mutants confirming the earlier DHB4 results and thus the probable use of the
Sec pathway.
Interestingly, export of AP by ssHyaA was blocked in a tatC mutant but not in
a tatB strain,
suggesting that in the context of this leader peptide-AP fusion, Tat export
could occur without
the TatB protein. It should be noted that export of CoIV was similarly
observed to occur in a
tatGdependent, tatB-independent fashion when fused to ssTorA. The quality of
subcellular
fractionations performed for all samples reported in Table 9 was confirmed by
lacZ activity
measurements as well as by protein dot blotting.
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Finally, Western blot analysis and AP activity measurements for both
periplasmic and
cytoplasmic fractions were. performed for the case of ssFdnG-AP expressed in
all six genetic
backgrounds (FIG. 7). It was noted that the total AP activity (periplasmic and
cytoplasmic)
found in DR473/pFdnG-AP was nearly identical to the amount of AP measured in
the cytoplasm
of DR473 expressing the signal-sequenceless version of AP from plasmid
pAID135. It is clear
from this data that in the context of the ssFdnG leader peptide, AP must be
folded and oxidized
prior to translocation by the Tat machinery. To the inventors knowledge, this
is the first evidence
that de n~vo disulfide bonds formed in the cytoplasm are stably maintained
during Tat-dependent
membrane translocation. Whether PhoA is translocated as a monomer (~48 kDa) or
in its active
homodimeric state (~96 kDa) is still unclear, although PhoA is known to fold
rapidly into its
highly stable, native dimeric state. Moreover, the notion that the large
alkaline phosphatase
dimer is compatible with the Tat machinery is supported previous studies
demonstrating that the
142. kDa FdnGH subcomplex of E. coli formate dehydrogenase-N is transported by
the Tat
system.
E~~AMPLE 10
'Hitchhiker' strategy for Tat-mediated export of a folded anti-digoxin
antibody
fragment from the cytoplasm of E. coli.
A considerable portion of the proteins exported by the Tat pathway are enzymes
that acquire cofactors in the cytoplasm prior to export and generally function
in respiratory
or electron transport processes (e.g., E. coli trimethlamine N oxide
reductase). The
acquisition of cofactors in the cytoplasm requires tertiary structure contacts
that occur only
after folding has been largely completed. Along these lines, it has been found
that
membrane targeting and the acquisition of nickel by HybC, the large subunit of
the E. coli
hydrogenase 2, is critically dependent on the export of the small subunit,
HybO which
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CA 02465724 2004-05-04
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contains a Tat-specific leader peptide. The model favored is that the small
and large
subunits of hydrogenase 2 first form a complex in the cytoplasm and the
complex is then
targeted to the membrane by virtue of the leader peptide of the small subunit.
Analogous
to this naturally-occurring complex, it was tested whether a non-physiological
heterodimeric antibody fragment could be exported via the Tat translocator
when folded
properly in the cytoplasm. Surprisingly, it was found that the Tat pathway
could also
export a disulfide linked heterodimer in which only one polypeptide chain was
fused to the
TorA leader peptide (see schematic, FIG. 6).
A Fab antibody fragment specific for the cardiac glycoside digoxin was used
which consisted of two polypeptide chains, the heavy and light chains, linked
together via
a disulfide bond. In addition, the heavy and light chains each contained two
intra-
molecular disulfide bonds. The TorA leader peptide was fused only to the heavy
chain
(VH-CHl) which was co-expressed with the light chain (Vl-C~) from a
dicistronic operon.
In this fashion, the TorA-heavy chain carries the light chain into the
periplasm in a
'piggyback' fashion only if the interchain disulfide bridge is formed first in
the cytoplasm
prior to translocation.
In a mutant strain with an oxidizing cytoplasm (strain DR.A) and lacking
elsbA,
complete Fab protein was exported by the Tat pathway, but only a small
fraction of Fab
was localized (~15=20%) in the osmotic shock fraction as confirmed by Western
blotting.
Earlier, it was reported that the folding yield of the anti-digoxin Fab in the
cytoplasm is
greatly increased by co-expressing a signal-sequenceless version of the
periplasmic
disulfide isomerase DsbC (~ssDsbC) or GroEL. In the present analysis, co-
expression of
~ssDsbC resulted in a significant increase in the amount of Fab in the
periplasm (~50% in
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
the osmotic shock fraction). This may be due to co-expression of chaperones in
the
cytoplasm increasing the amount of protein competent for export presumably
because it
improved the yield of folded protein.
Fab was immunologically probed using a primary antibody that recognizes mouse
light chain sequences. Therefore, the bands seen confirmed that the light
chain was
properly recruited by the heavy chain via intermolecular disulfide bond
formation and
subsequently delivered to the periplasmic space. The localization of the
cytoplasmic
marker protein GroEL and the periplasmic marker protein DsbC demonstrates that
the
subcellular fractionation was successful. The Fab protein in the periplasmic
fraction of
DRA cells was correctly folded and functional as evidenced by its ability to
bind the
antigen, digoxin in ELISA assays.
As with ssTorA-AP fusions, the appearance of Fab in the osmotic shock fraction
was completely abolished in a tatB mutant, when the RR dipeptide in the TorA
leader was
mutated to KID or in DHB4 cells having a reducing cytoplasm. Moreover, when
incubated
under conditions that increase the outer membrane permeability (Chen et al.,
2001), intact
cells expressing Fab antibodies exported into the periplasm via the Tat
pathway could be
specifically labeled with the fluorescent antigen digoxin-bodipy. The
fluorescence of these
cells was 5-fold higher than the background fluorescence observed in DIIA or
DOD
control cells. Overall, these results indicate that: (i) the Tat pathway is
capable of
exporting a fully oxidized Fab across the membrane and (ii) the process is
dependent on
the assembly of the light and heavy chains and the formation of the
intermolecular disulfide
within the cytoplasm prior to export. The transport of oxidized, presumably
fully folded,
Fab molecules into the periplasm provides conclusive evidence for the
hitchhiker mode of
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export suggested previously whereby a polypeptide containing a Tat leader
peptide
mediates the translocation of a second leaderless polypeptide with which it
associates in
the cytoplasm.
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Table 7. Bacterial strains and plasmids used in this study.
E. coli strainRelevant phenotype Source
DHB4 MC1000 phoR ~(phoA) PvuII ~(malF~3 F'[ZaclqZYABoyd et al.
pro] 1987
DHBA DHB4 dsbA::kan This work'
DR473 DHB4 ~trxB gor552..TnlOTet ahpC*..TnlOCm(araCP~aGift
trxB)
DRA DR473 dsbA::kan This work
FA113 DHB4 trxB gor552...TnlOtet' ahpC* Gift
F-, araDl39 d(argF lac) U169 fIbB5301 Casabadan
deoCl ptsF25 relAl and Col
MC4100 rbsR22 rpsL150 thiA 1979
MCMTA MC4100 tatB::kan Gift
DAD DR473 tatB::kan This work
BUDDY MC4100 tatC: apec Gift
FUDDY FA113 tatC: apec This work
Plasmid name Relevant features Source
pTrc99A trc promoter, ColEl ori, Ampr Amersham
Pharma
pTorA-AP E. coli TorA signal fused to PhoA(~2-22)This work
cloned in pTrc99A
pKK-AP as pTorA-AP with R11K;R12K mutation in This work
TorA signal peptide
pFdnG-AP E. coli FdnG signal fused to PhoA(02-22)This work
cloned in pTrc99A
pFdoG-AP E. coli FdoG signal fused to PhoA(~2-22)This work
cloned in pTrc99A
pAID135 Signal sequenceless PhoA (42-22) controlled
by tae promoter
pTrc99-Fab Gene encoding anti-digoxin Fab in pTrc99A
E. coli torA signal fused to gene encoding~s work
anti-digoxin Fab in
pTorA-Fab Trc99A
pKK-Fab as pTorA-Fab with R11K;R12K mutation This work
in TorA signal peptide
pBADdsbC Gene encoding DsbC with optimized RBS
in pBAD33
pBAD~ssdsbC Gene encoding DsbC(02-20) with optimized
RBS in pBAD33
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TABLE 8: Amino acid sequence of leader peptides capable of Tat-dependent
export of alkaline phosphatase
AmiA* MSTFKPLKTLTSRRQVLKAGLAALTLSGMSQAIAK (SEQ ID
N0:33)
DmsA MKTKIPDAVLAAEVSRRGLVKTTAIGGLAMASSALTLPFSRIAHAV
(SEQ
ID N0:43)
FdnG MDVSRRQFFKICAGGMAGTTVAALGFAPKQALAQ (SEQ
ID N0:127)
FdoG MQVSRRQFFKICAGGMAGTTAAALGFAPSVALAE (SEQ
ID N0:45) -
HyaA* MNNEETFYQAMRRQGVTRRSFLKYCSLAATSLGLGAGMAPKIAWAL
(SEQ
ID N0:28)
HybA MNRRNFIKAASCGALLTGALPSVSHAAA (SEQ ID
N0:36)
Sufi MSLSRRQFIQASGIALCAGAVPLKASAA (SEQ ID
NO:38)
TorA MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAA (SEQ
ID N0:128)
YacK MQRRDFLKYSVALGVASALPLWSRAVFAA (SEQ ID
N0:29)
YCbK MDKFDANRRKLLALGGVALGAAILPTPAFAT (SEQ ID
N0:30)
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Table 9. Periplasmic alkaline phosphatase (AP) activity obtained from fusions
between
putative Tat signal peptides of E. coli and leaderless E. coli alkaline
phosphatase*
Leader
__ ___~_ a Periplasmic AP activity
dsbA
wildtype
D2-20a trxB gor altpC
trxB gor ahpC dsbA
AmiAb
trxB gor ahpC tatB
DmsA° trxB gor ahpC tatC
FdnG 1.0 (63)
1.3
FdoG
1.6
HyaA 1.3
1.3
HybAb
1.2
Sufl°
nd
TorA° nd
0.2
YacK
0.2
YcbK nd
0.1
1.0 (35)
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
3.2
8.0
5.0
3.2
2.1
1.0 (32)
1.5
13.1
11.6
0.2
0.2
1.0 (55)
1.7
8.1
7.0
0.3
0.2
1.0 (7)
1.3
11.0
10.9
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3.8
1.2
nd
nd
0.2
0.1
nd
0.1
1.0 (75)
4.3
5.4
6.4
3.5
4.1
1.0 (42)
1.4
10.4
9.6
0.9
0.4
1.0 (25)
3.9
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6.1
5.4
7.4
3.2
1.0 (21)
2.5
6.6
5.8
6.3
3.0
Relative alkaline phosphatase activity calculated by normalizing activity in
sample to activity measured
in DHA control strain. Reported values for alkaline phosphatase activity are
the average of 3 separate
measurements from 2 independent experiments (n=6). Standard error is less than
10% for all reported
data. Values in parenthesis indicate the actual activity measured in the DHA
control strain.
aSignal-sequenceless AP construct
bValues normalized to activity measured in DHA/ssHyaA-AP
°Signal sequence carries a c-region positive charge
nd = not detectable
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*AmiA and HyaA are control Tat leader peptides. Both are incapable of
exporting alkaline
phosphatase under the conditions studies here.
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Georgiou and Valax, Curr. ~pin. Biotechnol., 7(2):190-197, 1996.
Guzman et al., .I. Bacteriol., 177:4121-4130, 1995.
Hockney, Trends BioteclZnol., 12(11):456-463, 1994.
I~aback, Methods Enzynaol., 22:99-120, 1971.
Karzai et al., Nat. Struct. Biol., 7:449-455, 2000.
Meyer et al., Natuf°e, 297:647-650, 1982.
Nielsen et al., Magn. Reson. Med., 37(2):285-291, 1997.
Pugsley, Microbiol. Rev., 57:50-108, 1993.
Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 ed. Cold Spring
Harbor
Laboratory Press, Cold Spring Harbor, NY, 2000.
Samuelson et al., Nature, 406:637-641, 2000.
'70
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WO 03/040335 PCT/US02/35618
Santini et al., J. Biol. Chern., 276:8159-8164, 2001.
Sargent et al., EMBO J., 17:3640-3650, 1998.
Sargent et al., J. Biol. Chem., 274:36073-36082, 1999.
Schatz and Dobberstein, Science, 271:1519-1526, 1996.
Settles et al., Sciefzce, 278:1467-1470, 1997.
Stuart and Neupert, Nature, 406:575-577, 2000.
Thomas et al., Mol. Microbiol., 39:47-53, 2001.
Weiner et al., Cell, 93:93-101, 1998.
Yahr and Wickner, EMBO J., 20:2472-2479, 2001.
~1
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
SEQUENCE LISTING
<110> GEORGIOU, GEORGE
DELISA, MATTHEW
$
<120> ENGINEERING OF LEADER PEPTIDES FOR THE SECRETION
OF
RECOMBINANT PROTEINS IN BACTERIA
<130> CLFR:019W0
<140> UNKNOWN
<141> 2002-11-05
<150> 60/337,452
1$ <151> 2001-11-05
<160> 134
<170> PatentIn Ver. 2.1
<210> 1
<211> 6
<212> PRT
<213> Artificial Sequence
2$
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 1
Arg
Arg
Xaa
Phe
Leu
Lys
1 5
3$ <210> 2
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 2
4$ Ser Arg Arg Arg Phe Leu Lys
1 5
<210> 3
$0 <211> 7
<212> PRT
<213> Artificial Sequence
<220>
$$ <223> Description of Artificial Sequence: Synthetic
Peptide
<400> 3
Ser Arg Arg Xaa Phe Leu Xaa
25227985.1
1/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
1 5
<210> 4
$ <211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 4
Thr Arg Arg Xaa Phe Leu Xaa
IS 1 5
<210> 5
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 5
Ser Arg Arg Xaa Xaa Leu Lys
1 5
<210> s
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 6
Ser Arg Arg Xaa Xaa Leu Ala
1 5
<210> 7
<211> 7
<212> PRT
<213> Artificial Sequence
so
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 7
Thr Arg Arg Xaa Xaa Leu Lys
1 5
2/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<210> 8
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> s
Thr Arg Arg Xaa Xaa Leu Ala
1 5
<210> 9
<211> 7
<2l2> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 9
2$ Ser Arg Arg Xaa Xaa Leu Thr
1 5
<210> 10
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 10
Ser Arg Arg Xaa Xaa Ile Lys
1 5
<210> 11
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 11
Ser Arg Arg Xaa Xaa Ile Ala
1 5
<210> 12
<211> 7
<212> PRT
3/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
$ Peptide
<400> 12
Ser Arg Arg Xaa Phe Ile Xaa
1 5
<210> 13
<211> 7
<212> PRT
1$ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 13
Ser Arg Arg Xaa Phe Met Lys
1 5
<210> 14
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
3$ <400> 14
Ser Arg Arg Xaa Phe Val Lys
1 5
<210> 15
<211> 7
<212> PRT
<213> Artificial Sequence
4$ <220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 15
$0 Ser Arg Arg Xaa Phe Va1 Ala
1 5
<210> 16
$$ <211> 7
<212> PRT
<213> Artificial Sequence
<220>
4/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 16
Ser Arg Arg Gln Phe Leu Lys
1 5
<210> 17
to <211> 6
<212> PRT
<2l3> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 17
Arg Arg Xaa Phe Leu Ala
1 5
<210> 18
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 18
Arg Arg Xaa Phe Leu Lys
1 5
<210> 19
<211> 30
<212> DNA
4~ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 19
gcgatggagc tcttaaagag gagaaaggtc 30
5~ <210> 20
<211> 12
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 20
5/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
gcgatgtcta ga 12
<210> 2l
$ <211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 21
Ser Arg Arg Xaa Phe Met Lys
1$ 1 5
<210> 22
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
~$ Peptide
<400> 22
Ser Arg Arg Xaa Phe Val Lys
1 5
<210> 23
<211> 7
<212> PRT
3$ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 23
Ser Arg Arg Xaa Phe Val Ala
1 5
4$
<210> 24
<211> 7
<212> PRT
<213> Artificial Sequence
$0
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
$$ <400> 24
Ser Arg Arg Gln Phe Leu Lys
1 5
6/34
CA 02465724 2004-05-04
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<210> 25
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 25
Met Pro Phe Lys Lys Leu Ser Arg Arg Thr Phe Leu Thr Ala Ser Ser
1 5 10 15
Ala Leu Ala Phe Leu His Thr Pro Phe Ala Arg Ala Leu
20 25
<210> 26
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 26
Met Thr Trp Ser Arg Arg Gln Phe Leu Thr Gly Val Gly Val Leu Ala
1 5 10 l5
Ala Val Ser Gly Thr Ala Gly Arg Val Val Ala Lys
20 25
<210> 27
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 27
45. Met Lys Glu Ser Asn Ser Arg Arg Glu Phe Leu Ser Gln Ser Gly Lys
1 5 10 15
Met Val Thr Ala A1a Ala Leu Phe Gly Thr Ser Val Pro Leu Ala His
20 25 30
Ala Ala
<210> 28
<211> 46
<212> PRT
<213> Artificial Sequence
7/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 28
Met Asn Asn Glu Glu Thr Phe Tyr Gln Ala Met Arg Arg Gln Gly Val
1 5 10 15
Thr Arg Arg Ser Phe Leu Lys Tyr Cys Ser Leu Ala Ala Thr Ser Leu
20 25 30
Gly Leu Gly Ala Gly Met Ala Pro Lys Ile Ala Trp Ala Leu
35 40 45
20
<210> 29
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 29
Met Gln Arg Arg Asp Phe Leu Lys Tyr Ser Val Ala Leu Gly Va1 Ala
1 5 10 15
Ser Ala Leu Pro Leu Trp Ser Arg Ala Val Phe Ala Ala
20 25
<210> 30
<211> 31
3$ <212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 30
Met Asp Lys Phe Asp Ala Asn Arg Arg Lys Leu Leu Ala Leu Gly Gly
1 5 10 15
Val Ala Leu Gly Ala Ala Ile Leu Pro Thr Pro Ala Phe Ala Thr
20 25 30
$~ <210> 31
<211> 32
<212> PRT
<213> Artificial Sequence
$5 <220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 31
8/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
Met Arg His Ile Phe Gln Arg Leu Leu Pro Arg Arg Leu Trp Leu Ala
1 5 10 15
Gly Leu Pro Cys Leu Ala Leu Leu Gly Cys Val Gln Asn His Asn Lys
20 25 30
<210> 32
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence:
Synthetic
Peptide
0 <400> 32
Met Gln Tyr Lys Asp Glu Val Asn Glu Pro.Ser Arg
Asn Gly Arg Arg
1 5 10 15
Leu Leu Lys Val Ile Gly Ala Leu Ala Gly Ser Pro
Ala Leu Cys Val
~5 20 25 30
Ala His Ala Gln
35
30
<210> 33
<211> 35
<212> PRT
<213> Artificial Sequence
35
<220>
<223> Description of ArtificialSequence:
Synthetic
Peptide
40 <400> 33
Met Ser Thr Phe Lys Pro Thr Leu Thr Ser Arg Gln
Leu Lys Arg Val
1 5 10 15
Leu Lys Ala Gly Leu Ala Thr Leu Ser Gly Met Gln
Ala Leu Ser Ala
t~.520 25 3 0
Ile Ala Lys
50
<210> 34
<211> 45
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
9/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<400> 34
Met Lys Lys Asn Gln Phe Leu Lys Glu Ser Asp Val Thr Ala Glu Ser
1 5 10 15
$ Val Phe Phe Met Lys Arg Arg Gln Val Leu Lys Ala Leu Gly Ile Ser
20 25 30
Ala Thr Ala Leu Ser Leu Pro His Ala Ala His Ala Asp
35 40 45
<210> 35
<211> 31
<212> PRT
1$ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 35
Met Ser Gly Leu Pro Leu Ile Ser Arg Arg Arg Leu Leu Thr Ala Met
1 5 10 15
2$ Ala Leu Ser Pro Leu Leu Trp Gln Met Asn Thr Ala His Ala Ala
20 25 30
<210> 36
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
3$ <223> Description of Artificial Sequence: Synthetic
Peptide
<400> 36
Met Asn Arg Arg Asn Phe Ile Lys Ala Ala Ser Cys Gly Ala Leu Leu
1 5 10 15
Thr Gly Ala Leu Pro Ser Val Ser His Ala Ala Ala
20 25
$0
<210> 37
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
$$ <400> 37
Met Asp Arg Arg Arg Phe Ile Lys Gly Ser Met Ala Met Ala Ala Val
1 5 10 15
Cys Gly Thr Ser Gly Ile Ala Ser Leu Phe Ser Gln Ala Ala Phe Ala
10/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
20 25 30
Ala
<210> 38
<211> 28
<212> PRT
1~ <213> Artificial Sequence
<220>
<223> Description of Sequence:
Artificial Synthetic
Peptide
<400> 38
Met Ser Leu Ser Arg Arg PheIle G1nAla Ser Ile Ala
Gln Gly Leu
1 5 10 15
~ Cys Ala Gly Ala Val Pro LysAla SerAla Ala
Leu
25
<210> 39
2$ <211> 49
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Sequence:
Artificial Synthetic
Peptide
<400> 39
Met Ser Asn Gln Gly Glu ProGlu AspAsn Arg Gly Lys
Tyr Va1 His
1 5 10 15
Glu Pro.His Asp Leu Ser ThrArg ArgAsp Leu Lys Val
Leu Ile Ser
20 25 30
4~ Ala Ala Thr Ala Ala Thr ValVal TyrPro His Thr Leu
Ala Ser Ala
35 40 45
Ala
<210> 40
<211> 45
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 40
Met Ser Trp Ile Gly Trp Thr Val Ala Ala Thr Ala Leu Gly Asp Asn
1 5 10 15
11/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
Gln Met Ser Phe Thr Arg Arg Lys Phe Val Leu Gly Met G1y Thr Val
20 25 30
Ile Phe Phe Thr Gly Ser Ala Ser Ser Leu Leu Ala Asn
$ 35 40 45
<210> 41
<211> 38
<212> PRT
<213> Artificial Sequence
<220>
<223> Description ArtificialSequence: Synthetic
of
1$ Peptide
<400> 41
Met Thr Gly Asp Thr Leu His Ser His Gly Ile Asn
Asn Ile Arg Arg
1 5 10 15
Asp Phe Met Lys Cys Ala Leu Ala Ala Thr Met Gly
Leu Ala Leu Ser
20 25 30
Ser Lys Ala Ala Glu
Ala
2$ 35
<210> 42
<211> 47
<212> PRT
<213> Artificial Sequence
<220>
<223> Description ArtificialSequence:
of Synthetic
3$ Peptide
<400> 42
Met Met Lys Ile Thr Thr Ala LeuMet Lys A1a Ile
His Glu Glu Ser
1 5 10 15
Arg Arg Ser Leu Lys Thr Ala LeuGly Ser Leu Leu
Met Ser Ala Ala
20 25 30
Ser Ser Ala Phe Leu Pro Ser GlnMet Val Arg Ala
Thr Phe Ala
4$ 35 40 . 45
<210> 43
<211> 46
$0 <212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
$$ Peptide
<400> 43
Met Lys Thr Lys Ile Pro Asp Ala Val Leu Ala Ala Glu Val Ser Arg
1 5 10 15
12/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
Arg Gly Leu Val Lys Thr Thr Ala Ile Gly Gly Leu Ala Met Ala Ser
20 25 30
Ser Ala Leu Thr Leu Pro Phe Ser Arg Ile Ala His Ala Val
35 40 45
<210> 44
1O <211> 44
<212> PRT
<213> Artificial Sequence
<220>
<223> Description ArtificialSequence:
of Synthetic
Peptide
<400> 44
Met Ser Lys Asn Arg Met Gly Ile Ser Arg Arg Thr
Glu Val Leu Val
ZO 1 5 10 15
Lys Ser Thr Ala Gly Ser Ala Leu Ala Ala Gly Gly
Ile Leu Phe Ser
25 30
ZS Leu Pro Phe Thr Arg Asn Ala Ala Ala Val
Leu Ala
35 40
<210> 45
~ <211> 34
<212> PRT
<213> Artificial Sequence
<220>
3$ <223> Description of Artificial Sequence: Synthetic
Peptide
<400> 45
Met Gln Val Ser Arg Arg Gln Phe Phe Lys Ile Cys Ala Gly Gly Met
1 5 10 15
Ala Gly Thr Thr A1a Ala Ala Leu Gly Phe A1a Pro Ser Val Ala Leu
20 25 30
45 Ala Glu
<210> 46
So <211> 62
<212> PRT
<213> Artificial Sequence
<220>
55 <223> Description of Artificial Sequence: Synthetic
Peptide
<400> 46
Met Thr Asp Tyr Ala Ser Phe Ala Lys Val Ser Gly Gln Ile Ser Arg
13/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
1 5 10 15
Leu Leu Val Thr Gln Leu Arg Phe Leu Leu Leu Gly Arg Gly Met Ser
20 25 30
Gly Ser Asn Thr Ala Ile Ser Arg Arg Arg Leu Leu Gln Gly Ala Gly
35 40 45
Ala Met Trp Leu Leu Ser Val Ser Gln Val Ser. Leu Ala Ala
50 55 60
<210> 47
<211> 7
IS <212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 47
Arg Arg Arg Gly Phe Leu Thr
1 5
<210> 48
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 48
Gln Arg Arg Arg Ala Leu Thr
1 5
<210> 49
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 49
Thr Arg Arg Glu Phe Ile Lys
1 5
5$ <210> 50
<211> 7
<2l2> PRT
<213> Artificial Sequence
14/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
$ <400> 50
Ser Arg Arg Ser Phe Met Lys
1 5
<210> 51
<211> 7
<212> PRT
<213> Artificial Sequence
1$ <220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 51
Gly Arg Arg Arg Phe Leu Arg
1 5
<210> 52
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 52
Ser Arg Arg Gln Phe Phe Lys
3$ 1 5
<210> 53
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
4$ Peptide
<400> 53
Ser Arg Arg Arg Phe I,eu Gln
1 5
$0
<210> 54
<211> 54
<212 > DNA
$$ <2l3> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
15/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<400> 54
gcgatggagc tcttaaagag gagaaaggtc atgccattta aaaaactctc ccga 54
<210> 55
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
1$ <400> 55
gcgatggagc tcttaaagag gagaaaggtc atgacctggt ctcgtcgc 48
<210> 56
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
2$ <223> Description of Artificial Sequence: Synthetic
Primer
<400> 56
gcgatggagc tcttaaagag gagaaaggtc atgaaagaaa gcaatagc 48
<210> 57
<211> 57
<212> DNA
3$ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 57
gcgatggagc tcttaaagag gagaaaggtc atgaataacg aggaaacatt ttaccag 57
4$ <210> 58
<211> 48
<212> DNA
<213> Artificial Sequence
$0 <220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 58
$$ gcgatggagc tcttaaagag gagaaaggtc gtggggagac gacgcgga 48
<210> 59
<211> 48
16/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 59
gcgatggagc tcttaaagag gagaaaggtc atgcaacgtc gtgatttc 48
<210> 60
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 60
gcgatggagc tcttaaagag gagaaaggtc atgtcccggt cagcgaaa 48
<210> 61
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 61
3$ gcgatggagc tcttaaagag gagaaaggtc atggacaaat tcgacgct 48
<210> 62
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 62
gcgatggagc tcttaaagag gagaaaggtc atgcgacaca tttttcaa 48
<210> 63
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
17/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<400> 63
gcgatggagc tcttaaagag gagaaaggtc atgcagtata aagatgaaaa cgg 53
<210> 64
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 64
gcgatggagc tcttaaagag gagaaaggtc atgagcactt ttaaacca 48
<210> 65
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 65
gcgatggagc tcttaaagag gagaaaggtc atgaaaaaga atcaattttt aaaagaatc 59
<210> 66
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 66
gcgatggagc tcttaaagag gagaaaggtc atgagcggct tacctctt 48
<210> 67
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 67
gcgatggagc tcttaaagag gagaaaggtc atgattcggc aacgtcgt 48
$5
<210> 68
<211> 48
<212> DNA
18/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 68
gcgatggagc tcttaaagag gagaaaggtc atgatcaggg aggaagtt 48
1~
<210> 69
<211> 62
<212> DNA
<213> Artificial Sequence
<2zo>
<223> Description of Artificial Sequence: Synthetic
Primer
ZO <400> 69
gcgatggagc tcttaaagag gagaaaggtc gtgaacagac gtaattttat taaagcagcc 60
tc 62
2$ <210> 70
<211> 48
<212> DNA
<213> Artificial Sequence
3O <220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 70
35 gcgatggagc tcttaaagag gagaaaggtc atggatcgta gacgattt 48
<210> 71
<211> 48
4~ <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:. Synthetic
45 Primer
<400> 71
gcgatggagc tcttaaagag gagaaaggtc atgtcactca gtcggcgt 48
<210> 72
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
19/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<400> 72
gcgatggagc tcttaaagag gagaaaggtc atgagcaacc aaggcgaa 48
$ <210> 73
<2l1> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 73
gcgatggagc tcttaaagag gagaaaggtc atgtcatgga tagggtgg 48
<210> 74
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 74
gcgatggagc tcttaaagag gagaaaggtc atgactggag ataacacc 48
<210> 75
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 75
gcgatggagc tcttaaagag gagaaaggtc atgaaactca gtcgtcgt 48
<210> 76
4$ <211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 76
gcgatggagc tcttaaagag gagaaaggtc atgatgaaaa tccataccac agaggcg 57
<210> 77
<211> 52
<212> DNA
20/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 77
gcgatggagc tcttaaagag gagaaaggtc atgaaaacga aaatccctga tg 52
<210> 7a
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 7a
gcgatggagc tcttaaagag gagaaaggtc atgtccaaaa atgaacgaat ggtg 54
<210> 79
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 79
gcgatggagc tcttaaagag gagaaaggtc atggacgtca gtcgcaga 48
<210> eo
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> ao
gcgatggagc tcttaaagag gagaaaggtc atgcaggtca gcagaagg 48
$0 <210> 81
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 81
21/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
gcgatggagc tcttaaagag gagaaaggtc atgacagatt atgcgtcttt cgctaaagtt 60
<210> 82
$ <211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 82
gcgatggagc tcttaaagag gagaaaggtc atgatttcac gccgccga 48
<210> 83
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 83
gcgatgtcta gagctttgtc gggcgggaag 30
3~ <210> 84
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 84
~ gcgatgtcta gaattgatat tcaacgtttt cgccac 36
<210> 85
<211> 30
4$ <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
$~ Primer
<400> 85
gcgatgtcta gatagggtgc cagctaccgc 30
<210> 86
<211> 30
<212> DNA
<213> Artificial Sequence
22/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
S
<400> 86
gcgatgtcta gagcgcggtt tgttctccag 30
<210> 87
<211> 30
<212> DNA
<213> Artificial Sequence
IS <220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 87
gcgatgtcta gatacgcgcc cgatatggtt 30
<210> 88
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence. Synthetic
Primer
<400> 88
gcgatgtcta gataacgttg ggcgttctgc 30
<210> 89
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
4S <400> 89
gcgatgtcta gagcgcaacc gcacgccaga 30
<210> 90
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 90
gcgatgtcta gagcgtgggg tagagagtgt 30
23/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<210> 91
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<~223> Description of Artificial Sequence: Synthetic
Primer
<400> 91
gcgatgtcta gacgtatcaa tggctggctt 30
<210> 92
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 92
gcgatgtcta gacgcacttt gcgttttttg 30
<210> 93
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 93
gcgatgtcta gattttaaaa gttcgtcttt gg 32
<210> 94
<211> 30
<212> DNA
4$ <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
SO
<400> 94
gcgatgtcta gaaaaccagc taagcagatc 30
5$ <210> 95
<211> 30
<212> DNA
<213> Artificial Sequence
24/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
$ <400> 95
gcgatgtcta gaattgggat caatagccgc 30
<2l0> 96
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 96
gcgatgtcta gagaatacag cgaccgtatg 30
<210> 97
<211> 30
<212> DNA
~5 <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 97
gcgatgtcta gatttaccgc ccttctcttc 30
<210> 98
<211> 30
<212> DNA
<213> Artificial Sequence
0 <220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 98
gcgatgtcta gatggcgggc ggttttcagc 30
<210> 99
<211> 30
$0 <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 99
gcgatgtcta gaggcaatat cagaatctgc 30
25/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<210> 100
<211> 30
<212> DNA
S <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> loo
gcgatgtcta gacggttgct gttgcccggc 30
<210> 10l
<211> 30
<2l2> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 101
2$ gcgatgtcta gaagctgccg gaacgcttgc 30
<210> 102
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 102
gcgatgtcta gacttttctt gcctcgtgtt 30
<210> 103
<211> 30
<212> DNA
<213> Artificial Sequence
.
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 103
gcgatgtcta gaaaccgatt cggccatctc 30
<210> 104
SS <211> 30
<212> DNA
<213> Artificial Sequence
<220>
26/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 104
gcgatgtcta gactgaccaa caacggcgcg 30
<210> 105
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
1$ Primer
<400> 105
gcgatgtcta gattctaccg gagcctctgc 30
<210> 106
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 106
gcgatgtcta gatggaatgg cgctatcgac 30
<210> 107
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 107
gcgatgtcta gatttttcgc gggcctgttg 30
<210> 108
<21l> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
5$
<400> 108
gcgatgtcta gataatttgt agtttcgcgc ctg 33
27/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<210> 109
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
IO <400> l09
gcgatgtcta gacagtttat actgccgggt ttc 33
<210> 110
1$ <211> 30
<212> DNA
<213> Artificial Sequence
<220>
ZO <223> Description of Artificial Sequence: Synthetic
Primer
<400> 110
gcgatgtcta gacgccacga cctggctg.ac 30
2$
<210> 111
<211> 30
<212> DNA
30 <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
3$
<400> 111
gcgatgtcta gagctcgtgg ctatcgtcgc 30
4O <210> 112
<211> 36
<212> DNA
<213> Artificial Sequence
4$ <220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 112
$O gcgatgtcta gaagtaaagg agaagaactt ttcact 3~
<210> 113
<211> 33
$$ <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
28/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
Primer
<400> 113
gcgatgaagc ttctatttgt atagttcatc cat 33
<210> 114
<211> 81
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
1$
<400> 114
gcgatgaagc ttgcatgctt aagctgctaa agcgtagttt tcgtcgtttg ctgcgtcgac 60
tttgtatagt tcatccatgc c 81
<210> 115
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 115
gcgatggaat tcgagctctt aaagaggaga aaggtcatga acaataacga tctctttcag 60
<210> ll6
3$ <211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 116
gcgatgtcta gaagcgtcag tcgccgcttg cgccgc 36
<210> 117
<211> 60
<212> DNA
$0 <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 117
gcgatggaat tcgagctctt aaagaggaga aaggtcgtga aacaaagcac tattgcactg 60
29/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<210> 118
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 118
gcgatgaagc ttttatttca gccccagagc ggctt 35
<210> 119
1$ <211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 119
Ala Ala Asn Asp Glu Asn Tyr Ala Leu Ala Ala
1 5 10
<210> 120
<211> 45
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 120
Met Asn Asn Asn Asp Leu Phe Gln A1a Ser Arg. Arg Arg Phe Leu Ala
1 5 ~ 10 15
Gln Leu Gly Gly Leu Thr Val Ala Gly Met Leu Gly Pro Ser Leu Leu
20 25 30
Thr Pro Arg Arg Ala Thr Ala Ala Gln Ala Ala Thr Asp
35 40 . 45
<210> 121
<211> 46
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 121
Met Asn Asn Asn Asp Leu Phe Gln Thr Ser Arg Arg Arg Leu Leu Ala
1 5 10 15
30/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
Gln Leu Gly Gly Leu Thr Val Ala Gly Met Leu Gly Pro Ser Leu Leu
20 25 30
S Thr Pro Arg Arg Ala Thr Ala Ala Gln Ala Ala Thr Asp Ala
35 40 45
<210> 122
1~ <211> 46
<212> PRT
<213> Artificial Sequence
<220>
1$ <223> Description of Artificial Sequence: Synthetic
Peptide
<400> 122
Met Asn Asn Asn Asp Leu Phe Gln Thr Ser Arg Gln Arg Phe Leu Ala
1 5 10 15
Gln Leu Gly Gly Leu Thr Val Ala Gly Met Leu Gly Pro Ser Leu Leu
20 25 30
Thr Pro Arg Arg Ala Thr Ala Ala Gln Ala Ala Thr Asp Ala
35 40 45
<210> 123
<211> 46
<212> PRT
<213> Artificial Sequence
<220>
35 <223> Description ArtificialSequence:
of Synthetic
Peptide
<400> 123
Met Asn Asn Asn Ile Phe AlaSerArg Arg Arg Phe Leu
Asp Gln Ala
1 5 10 15
Gln Pro Gly Gly Thr Val GlyMetLeu Gly Pro Ser Leu
Leu Ala Leu
20 25 30
4S Thr Pro Arg Arg Thr Ala GlnAlaAla Thr Asp A1a
Ala Ala
35 40 45
<210> 124
$~ <211> 46
<212> PRT
<213> Artificial Sequence
<220>
55 <223> Description of Artificial Sequence: Synthetic
Peptide
<400> 124
Met Asn Asn Asn Glu Leu Phe Gln Ala Ser Arg Arg Arg Phe Leu Ala
31/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
1 5 10 15
Gln Leu Gly Gly Leu Thr Val Ala Gly Met Leu Gly Pro Ser Leu Leu
20 25 30
$
Thr Pro Arg Arg Ala Thr Ala Ala Gln Ala Ala Thr Asp Ala
35 40 45
l~ <210> 125
<211> 46
<212> PRT
<213> Artificial Sequence
1$ <220>
<223> Description of Sequence:
Artificial Synthetic
Peptide
<400> 125
2o Met Asn Asn Asn Asp Leu GlnThr ThrArg Arg Arg Phe Leu
Phe Ala
1 5 10 15
Gln Leu Gly Gly Leu Thr AlaGly MetLeu Gly Pro Ser Leu
Val Leu
20 25 30
2$
Thr Pro Arg Arg Ala Thr AlaGln AlaAla Thr Asp Ala
Ala
35 40 45
3~ <210> 126
<211> 46
<212> PRT
<213> Artificial Sequence
3$<220>
<223> Description of Artificial Sequence:
Synthetic
Peptide
<400> 126
4~Met Asn Asn Asn Asp Ser GlnThrSer Arg Arg Arg Phe
Phe Leu Ala
1 5 10 15
Gln Leu Gly Gly Leu Thr AlaGlyMet Leu Gly Pro Ser
Va1 Leu Leu
20 25 30
4$
Thr Pro Arg Arg Ala Thr AlaGlnAla Ala Thr Asp Ala
Ala
35 40 45
$~ <210> 127
<211> 34
<212> PRT
<2l3> Artificial Sequence
$$ <220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 127
32/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
Gly Cys Gly Ala Thr Gly Gly Ala Gly Cys Thr Cys Thr Thr Ala Ala
1 5 10 15
Ala Gly Ala Gly Gly Ala Gly Ala Ala A1a Gly Gly Thr Cys Ala Thr
$ 20 25 30
Gly Ala Ala Cys Ala Ala Thr Ala Ala Cys Gly Ala Thr Cys Thr Cys
35 40 45
Thr Thr Thr Cys Ala Gly Gly Cys Ala Thr Cys Ala Ala Ala Gly Ala
50 55 60
Ala Ala Cys Gly Thr Thr Thr Thr Cys Thr Gly Gly Cys Ala Cys Ala
65 70 75 80
1$
Ala Cys Thr Cys
<210> 128
<211> 40
<212> PRT
<213> Artificial Sequence
2$ <220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 128
3~ Gly Cys Gly Cys Thr Gly Thr Thr Gly Cys Ala Gly Thr Thr Gly Ala
1 5 10 15
Ala Cys Thr Thr Cys Gly Cys Thr Ala Gly Cys Ala Gly Cys Gly Thr
25 30
3$
Cys Ala Gly Thr Cys Gly Cys Cys Gly Cys Thr Thr Gly
35 40 45
40 <210> 129
<211> 84
<212> DNA
<213> Artificial Sequence
4$ <220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 129
$0 gcgatggagc tcttaaagag gagaaaggtc atgaacaata acgatctctt tcaggcatca 60
aagaaacgtt ttctggcaca acts 84
<210> 130
$$ <211> 32
<212> DNA
<213> Artificial Sequence
<220>
33/34
CA 02465724 2004-05-04
WO 03/040335 PCT/US02/35618
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 130
$ gcgatgtcta gacggacacc agaaatgcct gt 32
<210> 131
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
1$ Primer
<400> 131
gcgatgaagc ttttatttca gccccagagc ggctt 35
<210> 132
<211> 27
<212> DNA
<213> Artificial Sequence
2$
<220>
<223> Description of ArtificialSequence:Synthetic
Primer
<400> 132
gctgctagcg aagttcaact gcaacag 27
<210> 133
3$ <211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence:Synthetic
Primer
<400> 133
gcgatgcccg ggggctttgt tagcagccggatctca 36
4$
<2l0> 134
<211> 45
<212> DNA
$0 <213> Artificial Sequence
<220>
<223> Description of ArtificialSequence:Synthetic
Primer
$$
<400> 134
gcgctgttgc agttgaactt cgctagcagcgtcagtcgccgcttg 45
34/34
