Note: Descriptions are shown in the official language in which they were submitted.
CA 02621608 2008-03-10
METHODS FOR OPTIMIZING THE SECRETION
OF PROTEIN IN PROKARYOTES
Field Of The Invention
[0001] The present invention relates to the field of recombinant protein
production in
prokaryotes such as Escherichia coli.
Background Of The Invention
[0002] Prokaryotes have been widely used for the production of recombinant
proteins.
Controlled expression of the desired polypeptide or protein is accomplished by
coupling the
gene encoding the protein through recombinant DNA techniques behind a
promoter, the
activity of which can be regulated by external factors. This expression
construct is carried on
a vector, most often a plasmid. Introduction of the plasmid carrying the
expression construct
into a host bacterium and culturing that organism in the presence of compounds
which
activate the promoter results in high levels of expression of the desired
protein. In this way,
large quantities of the desired protein can be produced.
[0003] E. coli is the most commonly used prokaryote for protein production.
Many different
varieties of plasmid vectors have been developed for use in E. coli to build
expression vectors.
The different variations employ several different types of promoters,
selectable markers, and
origins of replication where each of the different configurations imparts a
unique property to
the expression vector. In the most common arrangement, the expressed protein
accumulates
in the cytoplasm. While this approach is useful for some proteins, not all
proteins can be
accumulated in the cytoplasm in an active state. Often, when the desired
protein is produced
at high levels relative to the host proteins, is toxic to the host cell, or
has particular structural
properties, the protein accumulates as an insoluble particle known as an
inclusion body.
Proteins which accumulate as inclusion bodies are difficult to recover in an
active form.
[0004] One means of solving this problem is to export the desired protein to
the periplasm
between the inner and outer membranes (Choi et al., 2004; Cornelis, 2000). By
placing a
CA 02621608 2008-03-10
signal sequence in front of the coding sequence of the desired protein, the
expressed protein
can be directed to a particular export pathway (U.S. Patent No. 5,047,334 to
Petro et al., U.S.
Patent No. 4,963,495 to Chang et al.). Known export pathways in E. coli
include the SecB-
dependent (SEC) (Fekkes et al., 1999), the twin-arginine translocation (TAT)
(Sargent et al.,
2005; Fisher et al., 2004), and the signal recognition particle (SRP) pathway
(Koch et al.,
2003; Valent, 2001; Luirink et al., 2004). Translocation in the SEC or TAT
pathway is via a
post-translational mechanism, whereas the SRP pathway translocation is co-
translational.
Proteins translocated by the SEC pathway are unfolded prior to export and then
refolded in the
periplasm. In the TAT pathway, the proteins are translocated in a folded
state.
[0005] The selected export pathway is encoded in the signal sequence placed in
front of the
coding sequence of the desired protein within an expression vector. Currently
available
expression vectors incorporate signal sequences derived from proteins whose
export is
directed through the SEC pathway, such that the proteins accumulate in the
periplasm (Table
1 taken from Choi et al., 2004):
Table 1. Signal sequences used to secrete proteins in E. coli.
Signal Sequences Protein
PeIB Pectate lyase B from Erwinia carotovora
OmpA Outer-membrane protein A
St1I Heat-stable enterotoxin 2
Endo Endoxylanase from Bacillus sp.
PhoA Alkaline phosphatase
OmpF Outer-membrane pore protein F
PhoE Outer-membrane pore protein E
MalE Maltose-binding protein
OmpC Outer-membrane protein C
Lpp Murein lipoprotein
LamB k receptor protein
OmpT Protease VII
LTB Heat-labile enterotoxin subunit B
[0006] Although numerous proteins have been successfully produced by this
method, many
proteins are not exported correctly or in a functional state due to
aggregation in the cytoplasm;
lysis of the cells; incorrect folding; limitations to translocation or
proteolytic degradation
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(Jung et al., 1997; Krebber et al., 1996; Brinkmann et al., 1995; Rodi et al.,
2002; Wulfing et
al., 1993). The efficiency of translocation of a given protein depends on the
signal sequence
used and does not guarantee the secretion of a protein. Since the SEC and TAT
pathways
require the use of chaperone proteins (known to be substrate (protein)-
specific; Baneyx et al.,
2004) to effect translocation, many heterologous proteins when expressed in E.
coli with SEC
signal sequences cannot be exported due to lack of recognition by the host
chaperones. For
SEC-based translocation, the chaperones must retain the substrate protein in a
partially
unfolded state which is not likely possible with every protein. For secretion
using the TAT
pathway, some proteins can not be exported in a fully folded state due to
steric interference.
Protein export using the SRP pathway is likely to be hindered by the nature of
the protein
sequence; for example, where an amino acid sequence with a series of charged
or hydrophobic
residues might be blocked from being translocated due to strong protein-
protein interactions.
Not all proteins can be translocated equally well by any one export mechanism.
[0007] There is thus a need for signal sequences and expression vectors
including such
sequences to facilitate the selection of an appropriate export pathway which
is most suitable
for the production of a desired protein.
Summary Of The Invention
[0008] The present invention relates to a method for producing a recombinant
protein,
polypeptide or peptide of interest through secretion of the recombinant
protein, polypeptide or
peptide to the periplasm or extracellular growth medium. The method utilizes
expression
vectors carrying particular secretory signal sequences to direct the secretion
of the
recombinant protein, polypeptide or peptide to the periplasm or extracellular
growth medium
via the SEC, TAT or SRP export pathways.
[0009] In one aspect, the invention provides an expression vector capable of
directing the
expression and secretion of a protein, polypeptide or peptide in a suitable
host cell, wherein
the expression vector comprises a nucleic acid encoding a fusion protein
comprising YebF, or
a biologically active variant or portion thereof, and the protein, polypeptide
or peptide,
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operably linked to control sequences compatible with the host cell, and a
secretory signal
sequence for directing the secretion of the fusion protein.
[0010] In one embodiment, the expression vector comprises a signal sequence
comprising one
of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ
ID
NO: 9. In one embodiment, the expression vector is comprised of plasmid
pAES30, pAES31,
pAES32, pAES33, pAES34, or pAES35. In one embodiment, the expression vector is
plasmid pAES40. In one embodiment, the expression vector is for use in a
prokaryotic host
cell, for example, Escherichia coli or a strain thereof.
[0011] In another aspect, the invention provides an isolated host cell
transformed by any of
the above expression vectors, so that the cell expresses and secretes a
protein, polypeptide or
peptide encoded by the nucleic acid. In one embodiment, the host cell is a
prokaryotic host
cell, for example, Escherichia coli or a strain thereof.
[0012] In yet another aspect, the invention provides a method of optimized
production of a
protein, polypeptide or peptide comprising the steps of:
(a) choosing an expression vector as described herein, comprising a signal
sequence
associated with one of SEC, TAT, or SRP export pathway, wherein said choice is
made having regard to known information about the protein, polypeptide or
peptide, or
experimental information from expression studies of the signal sequence and
the
protein, polypeptide or peptide;
(b) transforming a suitable host cell with the chosen expression vector;
(c) culturing the transformed host cell under conditions conducive to the
expression
of the protein, polypeptide or peptide to generate a secreted protein,
polypeptide or
peptide; and
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(d) recovering the secreted protein, polypeptide, or peptide from the host
cell, from
the culture medium comprising the host cell, or from an extract obtained from
the host
cell.
[0013] In one embodiment, the expression vector is selected from the group
consisting of
plasmids pAES30, pAES31, pAES32, pAES33, pAES34, pAES35 or pAES40.
Brief Description Of The Drawings
[0014] The invention will now be described by way of an exemplary embodiment
with
reference to the accompanying simplified, diagrammatic, not-to-scale drawings.
[0015] Figures 1A, 1 B and 1 C depict the plasmid map of the expression vector
pAES25.
[0016] Figure 2 depicts the nucleotide sequence of the promoter, translation
start site and
multiple cloning site of plasmid pAES25.
[0017] Figures 3A and 3B depict the plasmid map and signal sequences of the
expression
vectors pAES30-35.
[0018] Figures 4A and 4B depict the plasmid map of the expression vector
pAES40.
[0019] Figure 5 is a graph showing luciferase activity in E. coli strains
harboring signal
sequence-SA-Luc constructs after induction.
[0020] Figure 6 depicts two immunoblots showing the accumulation of YebF-Amy
(left
panel) and YebF-PhoA (right panel) in the growth medium and intracellularly.
[0021] Figure 7 depicts the expression of YebF using different signal
sequences.
[0022] Figure 8 depicts the plasmid map of the expression vector pYebF-Amy2.
Detailed Description Of The Preferred Embodiments
[0023] As will be apparent to those skilled in the art, various modifications,
adaptations and
variations of the foregoing specific disclosure can be made without departing
from the scope
of the invention claimed herein. The various features and elements of the
described invention
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may be combined in a manner different from the combinations described or
claimed herein,
without departing from the scope of the invention.
100241 The present invention relates to a method for producing a recombinant
protein,
polypeptide or peptide of interest through secretion of the recombinant
protein, polypeptide or
peptide to the periplasm or extracellular growth medium. The method utilizes
expression
vectors carrying particular secretory signal sequences to direct the secretion
of the
recombinant protein, polypeptide or peptide to the periplasm or extracellular
growth medium
via the SEC, TAT or SRP export pathways. The expression vectors facilitate the
selection of
the appropriate signal sequence and export pathway which are most suited for
the protein,
polypeptide or peptide to achieve successful secretion.
[0025] To facilitate understanding of the invention, the following definitions
are provided.
[0026] "Expression" refers to transcription or translation, or both, as
context requires.
[0027] An "expression vector" refers to a recombinant DNA molecule containing
the
appropriate control nucleotide sequences (e.g., promoters, enhancers,
repressors, operator
sequences and ribosome binding sites) necessary for the expression of an
operably linked
nucleotide sequence in a particular host cell. By "operably linked/linking" or
"in operable
combination" is meant that the nucleotide sequence is positioned relative to
the control
nucleotide sequences to initiate, regulate or otherwise direct transcription
and/or the synthesis
of the desired protein molecule. The expression vector may be self-
replicating, such as a
plasmid, and may therefore carry a replication site, or it may be a vector
that integrates into a
host chromosome either randomly or at a targeted site. The expression vector
may contain a
selection gene as a selectable marker for providing phenotypic selection in
transformed cells.
The expression vector may also contain sequences that are useful for the
control of translation.
[0028] A "fusion" protein is a recombinant protein comprising regions derived
from at least
two different proteins. The term "fusion protein" as used herein refers to a
protein molecule
in which a protein, polypeptide or peptide of interest is fused to: YebF, a
biologically active
variant of YebF, or a biologically active portion of YebF (herein a "YebF, or
a biologically
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CA 02621608 2008-03-10
active variant or portion thereof'). "Fused", in one context means that
nucleic acid encoding
YebF, or a biologically active variant or portion thereof, is joined in frame
to the nucleic acid
encoding the protein, polypeptide or peptide of interest, to provide for a
single amino acid
chain when transcription and translation occur. In another context, "fused"
may also be a
reference to the joining of a protein, polypeptide or peptide of interest to
YebF, or a
biologically active variant or portion thereof.
[0029] A "secreted fusion protein" is the part of the fusion protein that is
secreted into the
growth medium. As is apparent, a secreted fusion protein will likely lack the
amino acids that
comprise the leader sequence of YebF, specifically MKKRGA FLGLLLVSAC ASVF
(included in SEQ ID NO:1).
[0030] A "nucleotide" refers to a ribonucleotide or a deoxyribonucleotide.
"Nucleic acid"
refers to a polymer of nucleotides and may be single- or double-stranded.
"Polynucleotide"
refers to a nucleic acid that is twelve or more nucleotides in length.
[0031] A "nucleotide sequence of interest" refers to any nucleotide sequence
that encodes a
"protein, polypeptide or peptide sequence of interest," the production of
which may be
deemed desirable for any reason, by one of ordinary skill in the art. Such
nucleotide
sequences include, but are not limited to, coding sequences of structural
genes, regulatory
genes, antibody genes, enzyme genes, etc., or portions thereof. The nucleotide
sequence of
interest may comprise the coding sequence of a gene from one of many different
organisms.
[0032] A nucleotide sequence "encodes" or "codes for" a protein if the
nucleotide sequence
can be translated to the amino acid sequence of the protein. The nucleotide
sequence may or
may not contain an actual translation start codon or termination codon.
[0033] A "protein, polypeptide or peptide sequence of interest" is encoded by
the "nucleotide
sequence of interest." The protein, polypeptide or peptide may be a protein
from any
organism, including but not limited to, mammals, insects, micro-organisms such
as bacteria
and viruses. It may be any type of protein, including but not limited to, a
structural protein, a
regulatory protein, an antibody, an enzyme, an inhibitor, a transporter, a
hormone, a
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hydrophilic or hydrophobic protein, a monomer or dimer, a therapeutically-
relevant protein,
an industrially-relevant protein, or portions thereof.
[0034] A "peptide" is polymer of four to 20 amino acids, a "polypeptide" is a
polymer of 21 to
50 amino acids and a "protein" is a polymer of more than 50 amino acids.
[0035] A "portion" when used in reference to a protein refers to fragments of
that protein.
The fragments may range in size from four amino acid residues to the entire
amino acid
sequence of the protein, minus one amino acid.
[0036] "Purified" or "to purify" refers to the removal of undesired components
from a sample.
For example, to purify the secreted protein from growth medium, may mean to
remove other
components of the medium (i.e., proteins and other organic molecules), thereby
increasing the
percentage of the secreted protein.
[0037] The terms "modified", "mutant" or "variant" are used interchangeably
herein, and refer
to: (a) a nucleotide sequence in which one or more nucleotides have been added
or deleted, or
substituted with different nucleotides or modified bases or to (b) a protein,
peptide or
polypeptide in which one or more amino acids have been added or deleted, or
substituted with
a different amino acid. A variant may be naturally occurring, or may be
created
experimentally by one of skill in the art. A variant may be a protein,
peptide, polypeptide or
polynucleotide that differs (i.e., an addition, deletion or substitution) in
one or more amino
acids or nucleotides from the parent sequence.
[0038] In this regard, it is well understood in the art that certain
alterations inclusive of
mutations, additions, deletions and substitutions can be made to a reference
nucleic acid or
protein, whereby the altered nucleic acid or protein retains a particular
biological function or
activity, or perhaps displays an altered but nevertheless useful activity.
Some deletions,
insertions and substitutions will not produce radical changes in the
characteristics in a protein
or nucleic acid. However, while it may be difficult to predict the exact
effect of the
substitution, deletion or insertion in advance of doing so, one skilled in the
art will appreciate
that the effect can be evaluated by routine screening assays. For example
whether a variant
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has a secretory function can be determined by assaying for whether the
variant, or a fusion
protein comprising the variant, is secreted into the medium, by the methods
disclosed herein.
Modifications of protein properties such as redox or thermal stability,
hydrophobicity,
susceptibility to proteolytic degradation, or the tendency to aggregate with
carriers or into
multimers may be assayed by methods well known to one of skill in the art.
[0039] Variants may be created experimentally using random mutagenesis,
oligonucleotide-
mediated (or site-directed) mutagenesis, PCR mutagenesis and cassette
mutagenesis.
Oligonucleotide-mediated mutagenesis is well known in the art using vectors
that are either
derived from bacteriophage M13, or that contain a single-stranded phage origin
of replication.
Production of single-stranded template is described, for example, in Sambrook
et al. (1989).
Alternatively, the single-stranded template may be generated by denaturing
double-stranded
plasmid (or other DNA) using standard techniques. Alternatively, linker-
scanning
mutagenesis of DNA may be used to introduce clusters of point mutations
throughout a
sequence of interest that has been cloned into a plasmid vector (Ausubel et
al., 1990).
Region-specific mutagenesis and directed mutagenesis using PCR may also be
employed to
construct variants according to the invention. With regard to random
mutagenesis, methods
include incorporation of dNTP analogs and PCR-based random mutagenesis.
[0040] "Periplasm" refers to a gel-like region between the outer surface of
the cytoplasmic
membrane and the inner surface of the lipopolysaccharide layer of gram-
negative bacteria.
[0041] "Secretion" refers to the excretion of the recombinant protein that is
expressed in a
bacterium to the periplasm or extracellular growth medium.
[0042] "YebF" is a reference to the protein having the amino acid sequence of
SEQ ID NO: 1.
"Mature YebF" is a reference to the protein having the amino acid sequence of
SEQ ID NO:2.
"yebF" is a reference to a nucleic acid or nucleotide sequence having the
sequence of SEQ ID
NO:3.
[0043] Standard recombinant DNA and molecular cloning techniques used herein
are well
known in the art and are described by Sambrook, J., Fritsch, E. F. and
Maniatis, T., Molecular
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Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, Cold
Spring Harbor, N.Y. (1989); Silhavy, T. J., Bennan, M. L. and Enquist, L. W.,
Experiments
with Gene Fusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor,
N.Y. (1984);
and Ausubel, F. M. et al., Current Protocols in Molecular Biology, published
by Greene
Publishing Assoc. and Wiley-Interscience (1990).
[0044] The present invention utilizes expression vectors carrying particular
secretory signal
sequences and a nucleotide sequence encoding a target protein, polypeptide or
peptide to
direct the secretion of the recombinant protein, polypeptide or peptide to the
periplasm or to
extracellular growth medium via the SEC, TAT or SRP export pathways. The
expression
vectors facilitate the selection of the appropriate secretory signal sequence
and export pathway
which are most suited for the protein, polypeptide or peptide to achieve
successful secretion.
[0045] The expression vectors of the present invention can be constructed
using techniques
well known in the art (Sambrook et al., 1989; Ausubel et al., 1990). Briefly,
the nucleotide
sequence encoding the target protein, polypeptide or peptide is placed in
operable combination
with control nucleotide sequences (e.g., promoters, enhancers, repressors,
operator sequences
and ribosome binding sites) to initiate, regulate or otherwise direct
transcription and/or the
synthesis of the desired protein, polypeptide or peptide. The control
nucleotide sequences
include, for example, initiation signals such as start (i.e., ATG);
transcription termination or
stop codons; promoters which may be constitutive (i.e., continuously active)
or inducible; and
enhancers which increase the efficiency of expression. Secretory signal
sequences are
included to achieve secretion of the encoded protein, polypeptide or peptide
from the
cytoplasm into the periplasm or extracellularly.
[0046] It will be appreciated by those skilled in the art that the promoter
which regulates the
transcription of the nucleotide sequence encoding the target protein,
polypeptide or peptide
may be modified to increase or decrease the transcription rates. Likewise, the
plasmid copy
number may be increased or decreased by modifying the origin of replication.
Both of these
modifications would be expected to yield higher or lower levels of expression
and thus higher
or lower levels of accumulated protein, polypeptide or peptide. It will be
appreciated by those
CA 02621608 2008-03-10
skilled in the art that the promoter and copy number variants can be matched
with the
appropriate secretory signal sequence to effect optimum protein production.
[0047] Marker genes are included to allow selection of host cells bearing the
desired
expression vector including, but not limited to, antibiotic (e.g., ampicillin
and kanamycin)
resistance genes, or reporter genes (e.g., luciferase) which catalyze the
synthesis of a visible
reaction product. Ancillary sequences enhancing protein purification may also
be included in
the expression vector.
100481 In one embodiment, the invention provides expression vectors comprising
the basic
plasmid map shown in Figures 1A, 1B and 1C. The base expression vector, pAES25
(SEQ ID
NO: 18) was constructed using standard techniques known in the art by coupling
together the
origin of replication (ori) from pBR322, a plasmid which is one of the most
commonly used
E. coli cloning vectors; the nptll gene (kanamycin-resistance); the lacI gene
(lactose
repressor); a promoter under the control of the lac operator/repressor system;
a translation
start site and a multiple cloning site (MCS). Figure 2 depicts the nucleotide
sequence from
upstream of the promoter through the MCS. The promoter is located upstream of
the
translation start site which lies upstream of the MCS. The signal sequence is
inserted into the
MCS downstream of the promoter and translational start site. The coding
sequence for the
desired protein, polypeptide or peptide is inserted downstream of the signal
sequence. A gene
encoding an easily assayed reporter protein may be included to measure
expression and
protein secretion.
[00491 In one embodiment, the invention provides expression vectors comprising
the plasmid
map shown in Figures 3A and 3B. Each expression vector (pAES30-35) was
constructed to
carry a particular secretory signal sequence (Table 2), but is otherwise
identical to pAES25.
The signal sequences were selected to represent two signals for each of the
SEC, TAT and
SRP export pathways of E. coli (Table 3). Figure 1C shows the plasmid map of
pAES25 with
the control region expanded out. DNA sequences encoding each of the signal
sequences were
synthesized chemically. The expression vectors were made by inserting the
signal sequences
into the MCS at the BamHl (5' end) and SacI (3' end) restriction sites of
pAES25. The
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resulting plasmids retained the reading frame defined by the ATG start codon
of pAES25
(Figure 2).
Table 2. Signal sequences
SEQ ID NO:4 "PhoA" AAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACA
AAAGCG
SEQ ID NO:5 AAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGC
"OmpA" GCAGGCG
SEQ ID NO:6 AAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCG
"DsbA"
SEQ ID NO:7 CGCGTACTGCTATTTTTACTTCTTTCCCTTTTCATGTTGCCGGCATTTTCG
"TorT"
SEQ ID NO:8 TCACTCAGTCGGCGTCAGTTCATTCAGGCATCGGGGATTGCACTTTGTGCAGGC
"Suff" GCTGTTCCACTGAAGGCCAGCGCAGCAGATCTACTAGT
SEQ ID NO:9 AACAATAACGATCTCTTTCAGGCATCACGTCGGCGTTTTCTGGCACAACTCGGC
"TorA" GGCTTAACCGTCGCCGGTATGCTGGGTCCGTCATTGTTAACGCCGCGACGTGCG
ACGGCAGCAGATCTACTAGT
Table 3. Signal sequences used to demonstrate differential secretion
Signal Pathway Amino Acid Sequence for Each Signal Sequence Resulting Plasmid
Sequence (Alternate Plasmid
Name)
PhoA Sec MKQSTIALALLPLLFTPVKTA (SEQ ID NO: 10) pAES25PhoASGL
(pAES32)
OmpA Sec MKKTAIAIAVALAGFATVAQA (SEQ ID NO: 11) pAES25OmpASGL
(pAES3 1)
DsbA SRP MKKIWLALAGLVLAFSASA (SEQ ID NO:12) pAES25DsbASGL
(pAES30)
TorT SRP MRVLLFLLSLFMLPAFS (SEQ ID NO:13) pAES25TorTSGL
(pAES35)
Sufl Tat MSLSRRQFIQASGIALCAGAVPLKASA (SEQ ID NO:14) pAES25SuflSGL
(pAES33)
TorA Tat MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA pAES25TorASGL
(SEQ ID NO: 15) (pAES34)
[0050] Knowledge about the primary or secondary structure of the target
protein can be used
to select the appropriate signal sequence and expression vector likely to
achieve successful
export of the protein. For example, if the target protein is known to have
fast folding
properties or once folded, is difficult to unfold, then the co-translational
mechanism (SRP
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CA 02621608 2008-03-10
pathway) may be most suitable. It will be appreciated by those skilled in the
art that, due to
the degeneracy of the genetic code, modifications can be made to the
nucleotide sequences
encoding the signal sequences. Such modifications would alter the amino acid
sequence such
that the leader is more effective in delivering the target protein to the
export machinery of the
cell. In one embodiment, the modifications comprise replacement of specific
amino acid
residues of the sequences listed in Table 2 with other amino acid residues, or
an increase or
decrease in the length of the leader. It is expected that these sequence
modifications could be
carried on an expression vector similar to, but not necessarily identical, to
pAES25. Further,
modifications to the nucleotide sequence of genes encoding proteins involved
in the export
functions could increase their capacity to translocate proteins. Further, co-
expression of these
same proteins on expression vectors could increase the relative number of
export machinery
proteins and consequently could lead to higher export capacity.
[0051] In one embodiment, the invention provides the above expression vectors
carrying
signal sequences and a target gene to direct the secretion of the recombinant
protein,
polypeptide or peptide into the periplasm (as described in Example 1) or
extracellular growth
medium.
[0052] In one embodiment, the invention provides expression vectors carrying
signal
sequences, a target gene, and a carrier gene to direct the secretion of the
recombinant protein,
polypeptide or peptide into growth medium. In one embodiment, the invention
comprises
fusion of the target protein, polypeptide or peptide to the E. coli protein
YebF, which is
transported to the growth medium. YebF is a small (10.8 kDa) soluble
endogenous protein
which is naturally secreted into growth medium by E. coli cells. It
effectively transports both
small and large prokaryotic and eukaryotic proteins to the extracellular
medium in an active
form (U.S. Patent Application Publication No. US/2006/0246539 Al to Weiner et
al.; Zhang
et al., 2006). In Zhang et al., the E. coli expression vector pMS 119 was used
to construct the
plasmid, pYebFH6/MS, which expresses wild-type YebF protein under the control
of an
IPTG-inducible promoter and with a C-terminal hexa-His affinity tag. Analysis
of the
subcellular localization of the YebFH6 protein after induction showed that the
protein
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accumulated in the medium. To demonstrate that YebF could facilitate the
export of other
proteins, C-terminal fusions were made by inserting the coding sequences for
mature alkaline
phosphatase (E. coli phoA), a-amylase (Bacillus subtilis X-23, amy) and the
human IL-2,
between the C-terminal residue of YebF and the His tag. After induction, all
three proteins
were found to accumulate in the medium, indicating that the YebF protein could
effect
extracellular transport of the fused protein. Importantly, cytoplasmic
proteins did not leak into
the medium. YebF thus represents a potentially useful tool for facilitating
the extracellular
export of recombinant proteins.
[0053] In one embodiment, the invention provides an expression vector
comprising the
plasmid map shown in Figures 4A and 4B. The expression vector, pAES40,
facilitates the use
of YebF as a carrier protein for the extracellular production of a recombinant
protein,
polypeptide or peptide. pAES40 carries the yebF nucleotide sequence, variant
or portion
thereof, which encodes the YebF protein or a biologically active variant or
portion thereof.
pAES40 was constructed by replacing the sequences extending from the Xhol to
the HindlII
sites of pYebFH6/MS (Zhang et al., 2006) with the sequences shown in Figure
4B. The yebF
nucleotide sequence; origin of replication (oriV) from ColEl, a plasmid which
is one of the
most commonly used E. coli cloning vectors; the ApR gene (ampicillin-
resistance); the lacI
gene (lactose repressor); a promoter under the control of the lac
operator/repressor system; a
translation start site and a MCS were coupled together using standard
techniques known in the
art. The C-terminal amino acids of YebF are encoded by the sequence "CTC GAG"
(SEQ ID
NO: 16), with the remainder of the sequence depicting the reading frame of
YebF (Figure 4B).
An enterokinase proteolytic cleavage site "GAC GAT GAC GAT AAG" (SEQ ID NO:
17)
was placed between the MCS and the end of YebF to permit removal of the YebF
sequences
after export. A hexa-His sequence was placed at the end of the MCS to provide
a His6 tag if
needed; for example, for purification from the medium by affinity
chromatography, or for
identification with an antibody. The fusion of a target protein, polypeptide
or peptide linked
to the carboxyl end of YebF which is transported to growth medium thus
facilitates the
extracellular export of the desired protein, polypeptide or peptide, as
described in Example 2.
14
CA 02621608 2008-03-10
The use of YebF as a carrier for recombinant proteins provides a tool to
circumvent toxicity
and other contamination issues associated with protein production in E. coli.
[0054] Following assembly of any of the above expression vectors of the
present invention,
various techniques are available for introducing the expression vector into an
appropriate host
cell for expression of the recombinant protein, polypeptide or peptide. A
"host cell" refers to
a cell, irrespective of the type, which expresses a nucleotide sequence
encoding the protein,
polypeptide or peptide within any of the expression vectors of the present
invention and
secretes the protein, polypeptide or peptide into the periplasm or
extracellular medium. In one
embodiment, the host cell is a prokaryote. In one embodiment, the host cell is
E. coli. It will
be appreciated by those skilled in the art that specific mutant strains of E.
coli may permit
higher levels of protein export. In one embodiment, the host cell is E. coli
strain HB101,
DH5a, JM109, HMS174, BLR or TOP10.
[0055) Non-limiting examples of techniques for introducing the expression
vector into the
host cell include electroporation, microinjection, liposome fusion,
lipofection, lipopolyamine-
mediated transfection, calcium-phosphate-DNA co-precipitation, biolistics,
particle
bombardment, polybrene-mediated transfection and other suitable techniques.
The expression
vector may become integrated into the genome of the host cell into which it is
introduced, or
may be present as unintegrated vector. Host cells carrying the expression
vector are identified
through the use of the selectable marker, and the presence of the gene of
interest is confirmed
by hybridization, PCR, antibodies, or other techniques.
[0056] The host cells are grown in growth medium until such time as is desired
to harvest the
secreted protein, polypeptide or peptide. The time required depends upon a
number of factors
relating to the bacterial expression system being used and to the target
protein, polypeptide or
peptide being produced. The rate of growth of a particular bacterial strain or
species; the rate
at which the secreted target protein, polypeptide or peptide accumulates in
the periplasm or
extracellular medium; the stability of the secreted target protein,
polypeptide or peptide; and
the time at which bacterial lysis begins to occur (which will contaminate the
medium) are
CA 02621608 2008-03-10
non-limiting examples of the types of considerations that will affect when the
secreted target
protein, polypeptide or peptide is harvested from the periplasm or
extracellular medium.
[0057] In the case of intracellular production, the cells are harvested and
the protein,
polypeptide or peptide is released from the periplasm into the extracellular
medium by
inducing outer membrane leakage or rupturing the cells using mechanical
forces, ultrasound,
enzymes, chemicals and/or high pressure. Following secretion into the medium
(for example,
via YebF), the protein, polypeptide or peptide may be extracted from the
medium. Depending
upon the level of purity required, which will again depend upon the
application for which the
secreted recombinant protein, polypeptide or peptide will be used, the
secreted protein may be
further purified, for example by chromatography (e.g., affinity
chromatography), precipitation,
ultrafiltration, electrophoresis, or other suitable techniques.
[0058] The present invention provides significant advantages over current
techniques of the
prior art. Since the invention, in one embodiment, incorporates use of
exported proteins, there
is a significantly lower level of contamination, endotoxin, host cell proteins
and nucleic acids,
making purification easier and thus lowering production cost and durations.
Importantly, the
invention enables the production of proteins which might otherwise not be
expressed due to
toxicity and folding errors. The invention may be used for rapid production of
proteins at a
commercial scale, adapted to high throughput protein production, or readily
employed in
automated systems.
[0059] In one embodiment, the invention comprises a method of optimizing
protein secretion.
This is accomplished by expressing the protein of interest using a set of
expression vectors
that are designed to direct the export of the target protein to the periplasm
or extracellular
matrix using each of the three main protein secretion pathways of E. coli. The
construct that
secretes the most target protein identifies the optimal export pathway.
[0060] The Examples provided below are not intended to be limited to these
examples alone,
but are intended only to illustrate and describe the invention rather than
limit the claims that
follow.
16
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100611 Example 1- Secretion of Target Proteins into the Periplasm
[0062] To demonstrate that different proteins are exported differentially, the
genes encoding
alkaline phosphatase (PhoA) from E. coli and a streptavidin-luciferase hybrid
protein (SA-
Luc) were inserted downstream of the signal sequences. Each of these proteins
is a marker for
secretion into the periplasm. PhoA is only enzymatically active when exported
to the
periplasm (Manoil et al., 1990), and is non-functional if it accumulates in
the cytoplasm.
When SA-Luc (a heterologous semi-synthetic protein) is not exported, the
protein forms
inclusion bodies and does not exhibit biotin binding (streptavidin portion) or
luminescent
activity (luciferase portion).
100631 Each of the expression vectors (carrying PhoA and SA-Luc fused to each
of the signal
sequences) was introduced into E. coli strain DH5a (phoA mutant). Expression
of each
protein was measured. For PhoA, the cells were cultured on solid medium
containing
isopropyl-thiogalactopyranoside (IPTG) to induce expression, and 5-bromo-4-
chloro-3-indol-
phosphate (a substrate of PhoA) to measure enzyme activity and thus indicate a
Pho+
phenotype. When the PhoA protein was fused to the phoA (Sec), sufl (TAT) and
torA (TAT)
signal sequences, Pho+ cells were observed with the phoA signal sequence
construct, in
contrast to a marginal positive signal for the sufl and torA signals (data not
shown). The
ompA (Sec), dsbA (SRP) and torT (SRP) signal sequences did not yield Pho+
cells, indicating
a lack of export. For PhoA, only the native signal sequence (Sec pathway)
appeared to yield a
high level of protein export.
[0064] For SA-Luc, cultures were grown from single colonies to late
exponential phase, and
expression was induced by the addition of IPTG. After three hours post-
induction, the cells
were harvested and the enzyme activity was determined for pre- and post-
induction samples.
Figure 5 shows the relative increase in luciferase activity after induction.
Luciferase activity
was significantly higher when either the sufl (TAT) and TorA (TAT) were used,
but
significantly lower when the SEC or SRP signal sequences were used. Little or
no luciferase
was produced after induction when the signal sequences for the SEC and SRP
pathways were
fused to SA-Luc. These experiments demonstrated that the type of signal
sequence used and
17
CA 02621608 2008-03-10
the protein export pathway to which the recombinant protein is directed have a
profound
effect on the level of target protein which accumulates.
[0065] Example 2- Recombinant Protein Production Utilizing YebF
[0066] The YebF export function works in several commonly used strains of E.
coli for the
expression of heterologous proteins including HB 101, HMS 174, BLR and TOP 10.
The
plasmids pYebF-AmyH6/MS ("YebF-Amy") and pYebF-PhoAH6/T7 ("YebF-PhoA") were
constructed according to U.S. Patent Application Publication No. US
2006/0246539 Al to
Weiner et al. (the contents of which are incorporated herein by reference in
its entirety) E.
coli strains carrying these plasmids were cultured from single colonies in 2
ml Terrific Broth
medium (Tartof and Hobbs, 1987) supplemented with 100 g/ml ampicillin
overnight at 30 C.
The overnight cultures were subcultured into 50 ml of fresh medium and
incubated at 30 C
until the OD600 reached -0.6. A 6 mi sample was removed and IPTG was added to
the
remainder of the culture to a final concentration of 0.05 mM. The incubation
continued for 22
hours. Samples were removed at 3, 8 and 22 hours post-induction and treated as
follows: a 1
ml sample was microfuged for 2 min., the culture supernatant reserved and the
cells
suspended in water to give 10 OD per ml. The remaining 5 ml sample was
centrifuged to
separate the cells from the medium. The periplasmic and cytoplasmic fractions
were prepared
by cold osmotic shock and lysozyme/freeze-thaw, respectively. The
corresponding fractions
from the parent strains, MS 119 (HB 101 /pMS 119) and YebF (HB 101 /pYebFH6)
were
prepared similarly. Proteins which accumulated in whole cell extracts and the
culture
supematant were analyzed by immunoblot and enzyme assay. For the immunoblot,
equal
volumes of medium or whole cell extracts prepared in SDS-PAGE loading dye were
loaded
onto 4-20% acrylamide gradient gels. The separated proteins were
electroblotted to
nitrocellulose. The His-tagged proteins were detected using monoclonal anti-
His tag
antibody. The enzyme assays were performed using cell-free extracts and
culture supernatants
as described in Zhang et al. (2006).
[0067] Both fusion proteins, YebF-Amy and YebF-PhoA, appeared in the bacterial
growth
medium and intracellularly following induction (Figure 6). The proteins
exhibited a time-
18
CA 02621608 2008-03-10
dependent increase in export level following induction with IPTG. Their
appearance within
the cells preceded their accumulation in the medium, suggesting a rate-
limiting process. The
increase in enzyme activity for both fusion proteins paralleled the immunoblot
(data not
shown). The immunoblot showed that the proteins may have undergone a
processing event
beyond the expected removal of the signaling peptide such that the amino-
terminal portion of
each YebF was removed (the antibody used was an anti-His tag which is a C-
terminal
epitope). The basis processing is unknown, but peptide sequence analysis of
the purified
proteins revealed the resulting N-termini to be identical.
[0068] Accumulation of the fusion proteins in shake flask cultures was 20-50
mg/L,
suggesting that with a fully optimized fermentation process, production levels
could reach
well over 100 mg/L. Further, strains harboring expression vectors which
produce YebF-Amy
and YebF-PhoA exhibited a growth impaired phenotype when cultured on medium
containing
1 mM but not at 50 M IPTG. In contrast, a strain expressing a YebF-GFP fusion
was fully
inhibited by 50 M IPTG. In addition, the coding sequences of human GM-CSF and
y-
interferon were subcloned into pYebFH6/MS and showed that the GM-CSF protein
was
exported but that the interferon protein was not. As with the Amy and PhoA
fusions, the GM-
CSF construct exhibited a growth impaired phenotype when the cultures were
induced with
IPTG. These data taken with the observation of residual target protein in the
cell lysates
suggest a limitation which prevents full translocation of proteins that are
over-expressed.
100691 To determine if the export block of heterologous proteins fused to YebF
can be
alleviated using alternative export pathways, a set of vectors was constructed
where the wild-
type signal sequence of YebF was replaced with alternative signal sequences.
The nucleotide
sequence encoding the full-length YebF protein was subcloned into the MCS at
the BamHI (5'
end) and Sacl (3' end) restriction sites of pAES25 to form "pAES25-YebF." The
nucleotide
sequence encoding the mature YebF protein was subcloned into the MCS at the
Sacl (5' end)
and KpnI (3' end) restriction sites of the pAES30 vector (to form
"pAES25YebFDSbA") and
pAES31 vector (to form "pAES25-YebFopA"). Each plasmid was introduced into E.
coli
strain TOP10. The resulting strains were analyzed for their ability to express
and export YebF
19
CA 02621608 2008-03-10
protein to the bacterial growth medium. The experiments were performed in 25
ml shake
flask cultures at 30 C as described above. After 22 hours post-induction, the
accumulation of
YebF in the bacterial growth medium was determined by SDS-PAGE and immunoblot
analyses.
[0070] Figure 7 shows the relative level of YebF accumulation in the medium. A
portion of
the stained gel corresponding to the location of YebF is shown above the
graph. The x-axis
labels correlate to the respective gel lane. The relative level of YebF
accumulation was
determined from the scanned gel image using TotalLab v2003.02 imaging software
(Nonlinear Dynamics Ltd., UK). By exchanging the wild-type signal sequence of
YebF for
SEC (ompA)- or SRP (dsbA)-directed signal sequences, the level of YebF
accumulation was
increased by 46- and 35-fold, respectively. These data suggest that not only
is YebF suitable
for directing the translocation of recombinant proteins to the bacterial
growth medium, but
that by applying alternative signal sequences (which direct the proteins to
the different export
pathways), a significant increase in protein accumulation can be achieved.
[0071] Example 3 - Identification of Mutations Affecting YebF Export
[0072] In order to identify mutations affecting YebF export, a simple genetic
screen was
devised. The screen uses medium containing Azure-starch to determine the
amylose
degrading phenotype of colonies. Starch degradation is indicated by the
formation of a clear
zone around the Amy-positive colonies. Amylase-negative colonies do not form
clear zones
and the medium remains unifonnly blue in color. Wild-type E. coli is Amy-
negative, whereas
strains expressing the YebF-Amy fusion are Amy-positive. To develop the strain
needed to
screen for cis- and trans-acting mutations of YebF export function, a plasmid
carrying a YebF-
Amy fusion was prepared. This construct, designated pYebF-Amy2 (Figure 8),
differs from
the original YebF-Amy fusion in that the wild-type yebF gene can be removed
and replaced
with DNA fragments containing mutations in the yebF coding sequence while
retaining the
integrity of the YebF-Amy fusion. This strain, when cultured on medium
containing Azure-
starch in the presence of 50 M IPTG, forms clear zones around the colony
whereas the parent
strain harboring pYebFH6 does not. Moreover, the strain harboring pYebF-Amy2
is able to
CA 02621608 2008-03-10
grow on medium with 1% starch as the sole carbon source in contrast to the
parent strain
which can not. This screen is the basis for selecting cis- and trans-acting
mutants.
[0073] Mutations were introduced into the wild-type yebF coding sequence using
error-prone
PCR. The resulting amplification products were subcloned into pYebF-Amy2
(Figure 8) by
exchanging the wild-type yebF for mutant yebF. The library of constructs was
introduced into
E. coli strain DH5a and the transformants scored on Azure-starch containing
solid medium.
Three mutant phenotypes were identified, (1) clear zones with a reduced area
relative to the
parent plasmid, (2) no clear zones, and (3) clear zones with an increased area
(Table 4). DNA
sequence analysis of the yebF portion of the plasmid purified from isolates
representing each
of the three phenotypes revealed the following: Strains showing no clear zone
carried
termination codons in the yebF sequence. Strains exhibiting a reduced or
increase clear zones
were amino acid substitutions at various locations within the yebF coding
sequence.
Table 4. Phenotype and corresponding mutations in YebF mutants.
Clone No. Phenotype YebF Sequence Mutations
2 No Halo K34term; Q78P; V93A; Gl l lterm
3 Halo G67S; V891
5 Halo C108S
7 Halo I80T
8 No Halo M1L(no start codon); S461; ins A
9 Halo G111R
21 No Halo K64term; del T
22 Halo K34E; E 114G
28 Halo L8S
31 Large Halo N24K
32 No Halo S27G, W75term
34 Small Halo Q82K, S89term
37 Small Halo D62V, D84V, I100T
[0074] References
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Choi, J. H. and Lee, S. Y. (2004) Export and extracellular production of
recombinant proteins
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Patent No. 5,047,334, issued September 10, 1991.
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Valent, Q. A. (2001) Signal recognition particle mediated protein targeting in
Escherichia
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[0075] All publications mentioned in this specification are indicative of the
level of skill of
those skilled in the art to which this invention pertains. All publications
are herein
incorporated by reference to the same extent as if each individual publication
was specifically
and individually indicated to be incorporated by reference.
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SEQUENCE LISTING
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gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag 960
tggcagggcg gggcgtaatt tttttaaggc agttattggt gcccttaaac gcctggggta 1020
atgactctct agcttgaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt 1080
tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccctctag 1140
attacgtgca gtcgatgata agctgtcaaa catgagaatt gtgcctaatg agtgagctaa 1200
cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 1260
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt 1320
ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg 1380
agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat 1440
ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga 1500
gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat 1560
ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt 1620
ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg 1680
attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa 1740
tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag 1800
tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc 1860
aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc 1920
cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc 1980
tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg 2040
atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga 2100
ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg 2160
aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg 2220
gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac 2280
atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta 2340
tcatgccata ccgcgaaagg ttttgcacca ttcgatggtg tcggaatttc gggcagcgtt 2400
gggtcctggc cacgggtgcg catgatctag agctgcctcg cgcgtttcgg tgatgacggt 2460
gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 2520
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gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc 2580
atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc 2640
agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa 2700
aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 2760
ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 2820
gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 2880
aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 2940
gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 3000
ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 3060
cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 3120
cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 3180
gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 3240
cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 3300
agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg 3360
ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 3420
ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 3480
gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 3540
cacgttaagg gattttggtc atggagatgc gtgatctgat ccttcaactc agcaaaagtt 3600
cgatttattc aacaaagccg ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca 3660
accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat 3720
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 3780
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 3840
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 3900
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc 3960
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 4020
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 4080
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 4140
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 4200
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 4260
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 4320
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 4380
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 4440
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tgttggaatt taatcgcggc ctcgagcaag acgtttcccg ttgaatatgg ctcataacac 4500
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat 4560
cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttccccc atgacattaa 4620
cctataaaaa taggcgtatc acgaggccct ttcgtcttca c 4661
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