Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
132~162
METHOD FOR STABILIZING HETEROLOGOVS PROTEIN
EXPRESSION AND VECTORS FOR VSE TH~REIN
Technical Field of the Invention
The present invention relates generally to the
field of biotechnology. More particularly, the invention
relates to the fields of protein expression and recombi-
nant DNA ~echnology to improve the yield of poorly ex-
pressed mammalian polypeptides in bacterial hosts.
Backqround of the Invention
Many eukaryotic proteins axe not capable of be-
ing expressed in Escherichia coli in any measurable yield,or even if detectable, are not capable of being expressed
at such commercially recoverable levels due to proteolysis
of the foreign protein by the host. Small proteins (e.g.,
peptide~hormones of less than 100 amino acids) appear to
be especially sensitive to degradation. The degree of
proteolysis varies from host to host and protein to
protein. Possibly the highest level of expression of a
eukaryotic protein in E. coli has been observed with yamma
interferon, which was expressed at approximately 60% of
total cellular protein. The high lev~l of expression of a
few eukaryotic proteins has been achieved because they
reach a~concentration in the cell where they can aggregate
into insolubIe masses called inclusion or refractile bvd-
es (e.g., bovine growth honnone; Schoner et al (1985),
~ 3:151-154). In this~form, the eukaryotic
protein is less susceptible to proteolysis.
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~ 32~162
Proteins which do not become insoluble on their
o~n do in some cases form inclusion bodies if joined to
another protein such as a procaryotic protein. A small
number of prokaryotic proteins have been used in this man-
ner: E. coli lacZ, ~E, and recA genes and the lambdacII gene, for example.
Chloramphenicol acetyltransferase (CAT) has been
used as a selectable maxker (resistance to
chloramphenicol), as an easily assayed enzyme to monitor
the efficiency of both eukaryotic and prokaryotic expres-
sion from different promoters (Delegeane, A.~., et al
(lg87) Mol Cell Biol 7:3994-4002), regulatory sequences,
and/or ribosome binding sites, and for gene fusions which
join sequences encoding a eukaryotic protein to the
nucleotide sequence encoding mature, native CAT (Buckley
and Hayashi (1986) Mol Gen Genet 204:120-125; European
Patent Publication 161,937, published 21 November 1985) or
to the carboxy terminal fragment of CAT (usually retaining
CAT activity)~
While the literature establishes that fusion
proteins are useful to express heterologous proteins in
bacteria and that the native CAT gene sequence has been
used for such a purpose, efforts to use a truncated form
of CAT to express or to increase the recoverable yield of
heterologous, mammalian proteins such as amyloid protein
A4-7Sl insert sequence, glucagon-like peptide I,
adipsin/D, and lung surfactant SP-B and 5P-C, have not
been reported. In light of the fact that many important
protein~ cannot be successfully expressed in bacteria in
any commercially recoverable yield, there is a need to
~ develop systems for the bacterial expression and recovery
: of such proteins.
: 35
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Disclo~ure of the Invention
One aspect of the invention concerns a method of
stabilizing heterologous protein expression in a
prokaryotic host comprising:
(a) constructing a hybrid gene comprising in
sequential order, a 3' truncated chloramphenicol
acetyltransferase (C~T) gene sequence fused in frame with
a heterologous gene sequence encoding a mammalian
polypeptide selec-ted from the group consisting of amyloid
protein A4-751 insert sequence, glucagon-like peptide I,
adipsin/D, lung surfac~ant protein SP-B and lung
surfactant protein SP-C; wherein said polypeptide is
normally not recoverable in bacterial expression systems,
and wherein said hybrid gene, upon translation, produces a
fusion protein in a recoverable yield;
(b) providing a vector for expression of said
hybrid gene;
(c) culturing the prokaryotic host transformed
with the expression vector; and
(d) recovering the fusion protein.
A second aspect of the invention concerns a
bacterial expression vector capable of enhancing the level
of expression of non-stable, bacterially produced
heterologous polypeptides comprising a hybrid gene having,
in sequential order, a 3' CAT truncated gene sequence
fused in frame to a heterologous gene sequence encoding a
mammalian polypeptide selected from the group consisting
of amyloid protein A4-751 insert sequence, glucagon-like
peptide I, adipsin/D, lung surfactant protein SP-B and
lung surfactant protein SP~C, wherein said polypeptide is
normally not recoverable in bacterial expression systems;
whereby said truncated CAT gene sequence is capable of
rendering the resulting fusion protein resistant to
proteolytic degradation.
A preferred embodiment ~or both the method and
vectox of the present invention employs a CAT coding
. .
~4~ ~3?,~162
sequence of less than or equal to 180 amino acids,
preferably between 73 and 180 amino acids. Although the
resulting CAT protein is substantially reduced as compzred
to the native CAT protein, surprisingly, it has been found
that the truncated CAT protein substantially contributes
to the stability of the expressed protein and therefore,
permits recovery of an increased yield of the desired
heterologous protein.
Yet another aspect of the invention provides an
improved bacterial expression vector capable of Pnhancing
the level of expression of non-stable, bacterially
produced heterologous polypeptides wherein said vector
contains a hybrid gene having in sequential order, a
modified 3' truncated CAT gene sequence linked to a
heterologous gene sequence. The improvement comprises
altering one or more DNA codons of the truncated CAT gene
to eliminate potential chemical cleavage sites within the
CAT protein.
Other aspects of the invention will be readily
apparent to those of skill in the art from the description
and examples which follow.
Brief Description of the Drawinqs
Figure 1 set3 forth the amino acid and cor-
responding nucleotide sequences for a 241 amino acid (aa)CAT-hANP hybrid protein containing an endoproteinase Glu-C
proteolytic cleavage site. The amino terminal portion of
this hybrid protein encodes the first 210 amino acids of
CAT, which sequence is extensively referred to throughout
the present invention.
Figure 2 illustrates a series of vectors and
synthetic fragments used for cloning and expression of the
CAT-hANF hybrid proteins of the invention Figure 2A
depicts an EcoRI-PstI synthetic fragment containing the E.
coli trp promoter-operator sequence, a ribosomal binding
site, and downstxeam cloning sites. Figure 23 is a
restriction site and function map of plasmid pTrp233.
Figure 2C is a restriction site and function map of
plasmid pCAT21. Figure 2D is an EcoRI-HindIII synthetic
fragment encoding the hANP (102-126) gene preceded by an
endoproteinase Glu-C cleavage site. Figures 2E through G
are restriction site and function maps of plasmids phNF75,
pChNF109, and pChNF121, respectively. Figure 2H depicts a
synthetic 1-73 aa CAT gene sequence contained withi~ NdeI-
HindIII fragment. Figure 2I is a restriction site and
function map of plasmid pChNF142 wherein site-specific
mutagenesis was used to substitute Tyr and Ser codons for
residues 16 and 31, respectively, of the CAT gene.
Figure 3 illustrates two different preparative
SDS-polyacrylamide gels. Figure 3A is an SDS-
lS polyacrylamide gel of the CAT-A4-7Sli hybrid protein.
Lane 1 = molecular size standards; Lane 2 = induced W3110
(pCAPil32); Lane 3 - induced W3110 (pTrp83) vector
control; Lane 4 = uninduced W3110 (pCAPil36); and Lane 5 =
induced W3110 (pCAPil36). Figure 3B is an SDS-
polyacrylamide gel of the CAT-GLP-I hybrid protein. Lane
1 = molecular size standard; Lane 2 = uninduced W3110
(pCGLP139); Lane 3 = induced W3110 (pCGLP139); and Lane 4
= induced W3110 (pTrp83~ vector control.
Figure 4 illustrates the amino acid and cor-
responding nucleotide sequences for a CAT-A4-751i hybrid
protein and a CAT-GLP-I hybrid protein of the invention.
Figure 4A depicts the first 73 codons encoding the amino
terminus of the CAT protein joined in-frame to the
synthetic A4-751i gene preceded by a chemical cleavage and
site encoded by Asn-Gly. Figure 4B depicts the first 73
codons encoding the amino terminus of the CAT protein
joined in-frame to the synthetic GLP-l gene preceded by a
Met codon.
Figure 5 illustrates two plasmids, pCAT73 and
pCAT210,-in which the gene for tetracycline resistance is
restored in these CAT expression vectors.
~ ~2~1 62
Figure 6 is the nucleotide sequence and cor-
responding amino acid sequence of the SP-B expression
construct pC210SP-B from the EcoRI site preceding the trP
promoter region through the HindIII site containing the
translation stop codon. The CAT, linker, and SP-B regions
are identified therein, respectively, by the arrows.
Figure 7 is a preparative SDS-polyacrylamide gel
of the CAT:SP-B fusion protein. Lane A = molecular size
standards; Lane B = induced W3110 cells containing pTrp233
vector control; and Lane C = induced pC210SP-B/W3110
cells.
Figure 8 illustrates the nucleotide sequence and
corresponding amino acid sequence of the 251 residue
CAT:SP-C fusion protein from plasmid pC210SP-C. The CAT
qene, linker sequence and SP-B gene are sequentially
identified therein by the arrows.
Figure 9 provides the molecular wsight
determinations for Pach of the CAT:SP-C fusion proteins.
Lane A = molecular size standards; Lane B = induced W3110
cells containing pTrp233 vector control; Lane C = induced
pC106SP-C; Lane D = pC149SP-C; Lane E = pC179SP-C; and
Lane F = pC210SP-C.
Figure 10 provides the cDNA and amino acid
sequences for human adipsin/D.
Modes for CarrYing Out the Invention
A. Definitions
As used herein the term "stabilizing protein
expression~ refers to a property of a fusion protein
responsible for inhibiting proteolysis of a foreign
protein by a recombinant host cell.
~ Insoluble~ as referred to proteins intends a
condition wherein a protein may be recovered only by
extraction with dstergents or chaotropic agents. Usually,
.
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1 ~ 2 ~
insoluble proteins are formed as a consequence of
intracellular aggregation of the cloned gene products.
~ High protein expression" or "enhanced protein
expxession~ refers to a level of expression wherein the
fused protein can comprise 10% or more of the total
protein produced by each cell. A preferred range for high
protein expression levels is from 10-20~ of total cell
protein.
As used herein, ~'non-recoverable" refers to a
level of expression wherein the desired protein may be
detected using sensitive techniques, e.g., Western blbt
analysis, yet the protein is not commercially recoverable
using conventional purification techniques such as SDS
polyacrylamide gel el~ctrophoresis, gel filtration, ion
exchange chromatography, hydrophobic chromatography, af-
finity chromatogrAphy, or isoelectric focusing.
IlMammalian'' refers to any mammalian species, and
includes rabbits, mice, dogs, cats, primates and humans,
preferably humans.
As used herein, the term "heterologous~ proteins
refers to proteins which are foreign to the host cell
transformed to produce them. Thus, the host cell does not
generally produce such proteins on its own.
B. CAT Fusions
CAT encodes a 219 amino acid mature protein andthe gene contains a number of convenient restriction
endonuclease sites (5'-PvuII, EcoRI, DdeI, NcoI, and ScaI-
3l) ~hroughout its length to test gene fusions for high
level expression. These restriction sites may be used for
ease of convenience in constructing the hybrid gene
sequences of the invention or other sites within the gene
sequence may ~e generated using t~chniques commonly known
to those of skill in the art. Any of the resulting CAT
sequences are considered useful so long as the resulting
.
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132~1 62
CAT fusion retains the ability to enhance the expression
of the desired heterologous peptide.
The expression constructs of the invention can
employ most of the CAT-encoding gene sequence or a
substantially truncated portion of the sequence encoding
an N-terminal portion of the CAT protein linked to the
gene encoding the desired heterologous polypeptide. In
one embodiment of the invention, the CAT portion of the
fusion codes for about the N-terminal one-third of the CAT
sequence.
The expression constructs exemplified herein,
which demonstrated enhanced levels of expression for a
~ variety of heterologous proteins, utilize a number of
varying lengths of the CAT protein ranging in size from 73
to 210 amino acids. The 73 ~mino acid CAT fusion
component is conveniently form~d by dige~ting the C~T
nucleotide sequence at the EcoRI restriction site.
Similarly, the 210 amino acid CAT fusion component is
formed by digesting the CAT nucleotide sequence with ScaI.
These, as well as other CAT restriction fragmen~s, may
then be ligated to any nucleotide sequence encoding a
desired protein to enhance expression of the desired
protein.
Significantly, although the expression level of
fusion protein ~approximately 15-20~ of total cell
protein) was similar for the CAT (106 amino acid) - SP-C
fusion and the CAT (210 amino acid) - SP-C fusion, it can
be een that the former case actually represents a
significant increase in expression level for the desired
SP-C polypeptide, since the SP-C polypeptide constitutes a
substantially larger proportion of the total fusion
protein in the former case. The ability to increase
expression level for the desired polypeptide by reducing
the size of the fused CAT protein sequence was quite an
unexpected finding in view of the experience of the prior
art. In general, the prior art experience has been that
. .
~32~62
reduction in size of the bacterial leader sequence does
not result in increased production of the fused
heterologous polypeptide due to a concomitant larger
reduction in the expression level of the fusion protein.
With one exception, the various CAT-heterologous
fusion proteins exemplified herein were found to be
expressed in the range of approximately 10-20% of the
total cell protein. Thus, the versatility of the CAT fu-
sions, that is, the ability to use a variety of CAT coding
sequences having the ability to enhance the expression of
a desired protein, allows great flexibility of choice when
construc~ing CAT hybrid genes.
The reading frame for translating ths nucleotide
sequence into a protein begins with a portion of the amino
terminus of CAT, the length of which varies, continuing
in-frame with or without a linker sequence into the
protein to be e~pressed, and terminating at the carboxy
terminus of the protein. An enzyma~ic or chemical cleav-
age site may be introduced downstream of the CAT sequence
to permit recovery of the cleaved product from the hybrid
protein. Such cleavage sequences are known in the art as
are the conditions under which cleavage can be effected.
Following cleavage, the desired heterologous polypeptide
can be recovered using known techniques of protein
purification. Suitable cleavage sequences include,
without limitation, cleavage following methionine residues
(cyanogen bromide), glutamic acid residues (endoproteinase
Glu-C), tryptophan residues ~N-chlorosuccinimide with urea
or with sodium dodecyl sulfate (SDS)) and cleavage between
asparagine and lysine residues (hydroxylamine).
To avoid internal cleavage within the CAT
sequence, amino acid substitutions can be made using
conventional site specific mutagenesis techniques (Zollerr
M.J., and Smith, M. (1982), Nuc Acids Res 10:6487-6500,
and Adelman, J.P., et al (1983), DNA 2:183-193). This is
conducted using a synthetic oligonucleotide primer com-
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1 ~'2~1 62
plementary to a single-stranded phage DNA to be
mutagenized except for limited mismatching, representing
the desired mutation. Of course, these substitutions
would only be performed when expression of CAT is not
significantly affected. Where there is only one internal
cysteine residue, as in the short CAT sequence, this
residue may be replaced to help reduce multimerization
through disulfide bridges.
C. CAT Fusion Vectors
Procaryotic systems may be used to express the
CAT fusion sequence; procaryotic hosts are, of course, the
most convenient for cloning procedures. Procaryotes most
frequently are represented by various strains of E. coli;
however, other microbial strains may also be used.
Plasmid vectors which contain replication sites, select-
able markers and control sequences derived from a species
compatible with the hos~ are used; for example, E. coli is
typically transformed using derivatives of pBR322, a
plasmid derived from an E coli species by Bolivar et al,
Gene 2:95 (1977). pBR322 contains genes for ampicillin
and tetracycline resistancer and thus provides multiple
selectable markers which can be either retained or
destroyed in constructi.ng the desired vector.
In addition to the modifications described above
which would facilitate cleavage and purification of the
product polypeptide, the gene conferring tetracycline
resistance may be restored to the exemplified CAT fusion
vectorq for an alternative method of plasmid selection and
maintenance.
Although the E. coli tryptophan promoter-
operator sequences have been exemplified in the present
CAT vec~ors, different control sequences can be
substituted for the ~ regulatory sequences and are
considered to be within the scope of the invention. Com-
monly used procaryotic control sequences which are defined
132~1~2
herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding
site sequence, include such commonly used promoters as the
beta-lactamase (penicillinase) and lactose (lac) promoter
systems (Chang et al, Nature 198:1056), the lambda-derived
PL promoter (Shimatake et al, Nature 292:128 (1981)~ and
N-gene rihosome binding site, and the trp-lac ~trc)
promoter system (Amann and Brosius, Gene 40:183 (1985)).
Since the general utility of these CAT vectors
have been established with very different mammalian
peptides (ranging in protein size, the presence or absence
of disulfide bonds, and being hydrophobic or hydrophilic
in nature) vec~ors with unique restriction site~ may be
created or substituted for the pBR322-derived vector il-
lustrated in the examples.
D. Heteroloqous Protein Expression
Amino terminal DNA sequences of CAT have beenfused to DNA sequences encoding human polypeptides for
high lev~l expression in the bacterial host E. coli. The
polypeptides described herein are relatively small mam-
malian polypeptides ranging in size from about 30 to 76
amino acid residues. Atkempts to directly express, e.g.,
in a non-fused form, each of these polypeptides in
bacteria have been unsuccessful, most likely due to the
proteolytic degradation which occurs upon translation of
the m~NA product. In the case of extremely hydrophobic
polypeptides, even attempts to express such polypeptides
using beta-galactosidase fusions produced detectable but
very low level amounts of protein.
Examples of polypeptides that have been success-
fully expressed to high level in bacteria using the
truncated CAT fusions include a variety of mammalian
polypeptides including amyloid protein A4-751 insert
sequence, glucagon-like peptide I, adipsin/D, lung
surfactant protein SP5 (SP-C), and lung surfactant SP18
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~.3~162
(SP-B). Preferably, the mammalian protein is of human
origin, although other sources are also contemplated to be
within the scope of this invention. A4-751 is a 57 amino
acid sequence identified within the precursor for the A4
amyloid protein associated with Alzheimex's disease and
shares homology with the Kunitz family of serine
proteinase inhibitors (Ponte, P., et al (1988) Nature
331:525-527; Tanzi, R.E., et al (1988) Nature 331:528-
530). Glucagon-like paptide I (GLP-I, 7-31) is a 31 amino
acid hormone co-encoded in the glucagon gene which is a
potent stimulator of insulin release (Mojsov, S., et al
(1987) J Clin Inves 79:616-619j. Adipsin/D is a serine
protease synthesized in and secreted from adipocytes
(Zusalak, K.M., et al (1985) J Mol Cell Biol 5:419). Lung
surfactant SP-B is a 76 amino acid hydrophobic protein.
Lung surfactant SP-C is a 35 amino acid hydrophobic
protein. Both SP-B and SP-C greatly enhance spreading of
surfactant phospholipids at an air:water interface.
E. Hosts Exemplified
Host strains used in cloning and procaryotic
expression herein are as follows:
For cloning an~ sequencing, and for expression
of construction under control of mos~ bacterial pxomoters,
E. coli strains such as MC1061, DHl, RRl, W3110, MM294, B,
C600hfl, K803, HB101, JA221, and JM101 may be used.
F. General Methods
~ecombinant DNA methods are described in
Maniatis et al (1982), Molecular Cloning, Cold Spring
Harbor Laboratory, Cold Spring Harbor, New York, when not
specifically cited in the following examples. Methods are
also described in the literature for visualizing inclusion
bodies, isolating them from cells, then solubilizing,
purifying, and cleaving the hybrid protein (e.g., Itakura,
X., et al ~1977) Science 19~.1056-1063; Shine, J., et al
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(1980) Nature_285:455-461). Methods are also available,
if necessary, for refolding ~he protein product
(Creighton, T.E., Proceedinqs of Genex-UCLA Symposium,
1985, Kingstones (in press).
Examples
I. Expression of Chloramphenicol AcetYltransferase-Human
Atrial Natriuretic Peptide Hybrid Proteins in Escherichia
coli.
A. Expression vector pChNF109.
Expression vector pChNF109 encodes a 241 amino
acid CAT-hANP hybrid protein containing an endoproteinase
Glu-C proteolytic cleavage site (Fig. 1). Most of the CAT
gene (amino acids 1-210) has been joined in-frame to the
hANP(102-126) gene and cleavage site (26 amino acids)
through a linker sequence (5 amino acids). The hANP
polypeptide comprises abou~ 10~ of the hybrid protein.
This vector was constructed from plasmids pTrp233, pCAT21,
and phNF75 which supplied the plasmid backbone and trp
promoter-operator, the CAT gene, and the h~NP(102-126)
gene and cleavage site, respectively.
1. Construction of ~ChNF109.
Plasmid pTrp233 was constructed by insertion of
a synthetic EcoRI-PstI fragment containing the E. coli trp
promoter-operator sequence, a ribosomal binding site, and
downstream cloning sites into plasmid pKK~33-2-NdeI which
contains strong transcription termination signals, TlT2,
`and the beta-lactamase gene. The synthetic fragment (see
Fig. 2A) was assembled using the method of Vlasuk et al
(1986), J. Biol Chem 261: 4789-4796 and its sequence
confirmed by the method of Sanger et al (1977), Proc Natl
Acad Sci USA 74:5463-5467 in M13mp8 and M13mp9. Plasmid
;~ :
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~23162
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pKK233-2-NdeI (disclosed in U.S. patent 4,764,504 dated
16 August 1988) was digested with EcoRI and PstI, its termini
dephosphorylated using calf intestinal phosphatase, and
ligated with the synthetic EcoRI-PstI fragment. Plasmid
pTrp233 was isolated (Fig. 2B) from E. Coli JA221 transformed
to ampicillin resistance.
Plasmid pCAT21 was constructed by insertion of the
CAT gene (from transposon Tn9, Alton and Vapnek, (1979)
Nature 282:864-869) into plasmid pTrp233 under the control of
the ~ promoter-operator. Plasmid pAL13ATCAT (a plasmid
disclosed in Canadian application Serial No. 576,975 filed
8 September 1988), was digested with NdeI and HindIII and the
approximately 750 bp NdeI-HindIII fragment containing the CAT
gene (with the initiating Met residue encoded at the NdeI
site) was purified using agarose gel electrophoresis. The
CAT gene was ligated with NdeI and HindIII digested pTrp233
using T4 DNA ligase. From E. Coli MC1061 (Casadaban et al
(1980), I Mo] Biol 138: 179-209) ampicillin-resistant
.
transformants, plasmid pCAT21 was isolated (Fig. 2C).
Plasmid phNF75 was constructed by insertion of a
synthetic hANP gene preceded by a proteolytic cleavage site
into plasmid pBgal (Shine et al (1980), Nature 285:456).
Eight oligodeoxyribonucleotides (Fig. 2D) were assembled into
a synthetic hANP(102-126) gene preceded by an endoproteinase
Glu-C cleavage site (method of Vlasuk et al (1986), supra).
The synthetic DNA fragment (with a 5' EcoRI tail and a 3'
blunt end) was ligated with EcoRI and SmaI restriction
endonuclease digested M13mpl9 using T4 DNA ligase for the
purpose of DNA sequencing (method of Sanger e~ al (1977),
~ ) A clone with the correct sequence, M13-hNF7, was
digested with BamHI and BglII, the fragment containing the
hANP gene purified by agarose gel electrophoresis, and the
fragment ligated with BamHI-
~'
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digested and bacterial alkaline phosphatase
dephosphorylated pTrp233 using ~4 DNA ligase. A plasmid
with the insert in the orientation which gives adjacent
HindIII, BamHI and EcoRI sites at the 3' end of the hANP
gene, phNF73, was identified by the siæe of the fragments
generated ~y digestion with HindIII and PvuII. Plasmid
phNF73 was digested with EcoRI, the hANP gene purified
using polyacrylamide gel electxophoresis, and the gene
ligated with EcoRI-digested and bacterial alkaline
phosphatase dephosphorylated plasmid pBgal. From E coli
MC1061 ampicillin-resistant transformants, plasmid phNF75
~Fig. 2E) was identified by the size of the DNA fragments
generated by digestion with PstI and HindIII.
Expression vector pChNF109 was constructed by
insertion of DNA fragments containing CAT, hANP and the
proteolytic cleavage site, and a linker sequence into
plasmid pTrp233. Plasmid phNF75 was digested with EcoRI
and HindIII, the approximately 80 bp EcoRI-HindIII frag-
ment containing hANP was purified by polyacrylamide gel
electrophoresis, and ligated with EcoRI- and HindIII-
digested pTrp233 using T4 DMA ligase. From E. coli MC1061
ampicillin-resistant transformants, plasmid phNF87 was
isolated and digested with BamHI and the fragments were
dephosphorylated using bacterial alkaline phosphatase. A
BamHI cassette containing the trp promoter-operator,
ribosomal binding site, and large amino terminal fragment
of the CAT gene was generated by digesting pCAT21 with
ScaI, attaching BamHI synthetic linkers (5'-CGGATCCG-3')
to the blunt termini using T4 DNA ligase, digesting the
ligation with BamHI and purification of the approximately
740 bp BamHI fragment by agarose gel electrophoresis. The
BamHI cassette and plasmid phNF87 were ligated using T4
ligase and ampicillin-resistant transformants of E. coli
MC161 obtained. Plasmid pChNF109 (Fig. 2F), with the
BamHI cassette in the orientation such that ~he C~T gene
is fused in-frame ~o the endoproteinase Glu-C cleavage
~3~0~&~,
site followed by the hANP gene, was selected on the basis
of DNA fragment size in an EcoRI digest of the plasmid.
2. Expression of C~T(1-210)-hANP(102-126)
Hybrid Protein From Plasmid pC NF103.
Plasmid pChNF109 expresses a CAT-hANP(102-126)
hybrid protein under the control of the E. coli ~
promoter-operator. The plasmid was used to transform E.
coli W3110 (ATCC Accession No. 27325) to ampicillin
resistance and one colony was grown in culture overnight
at 37C in complete M9 medium containing M9 salts, 2 mM
MgS04, 0.1 mM CaC12, 0.4% glucose, 0.5~ casamino acids, 40
ug/ml tryptophan, 2 ug/ml thiamine hydrochloride, and 100
ug/ml ampicillin sulfate. The overnight culture was
diluted 100-fold into the same M9 medium described above
(uninduced culture) and into M9 medium in which the
tryptophan had been replaced by 25 ug/ml of 3 beta-
indoleacrylic acid (induced culture).
Expression was assessed after shaking the
cultures for 6 hr at 37C. The uninduced culture had
reached a high cell density (stationary phase) and the
induced culture was still at a low cell density
(exponential phase). Phase-contrast microscopy revealed
cells of normal morphology in the uninduced culture and
elongated cells containing several refractile inclusion
bodies in the induced culture. Total cell protein samples
were prepared by boiling cell pellets in Laemmli buffer
for 5 min and were analyzed by electrophoresis through a
12% SDS-polyacrylamide gel followed by staining of the
protein with Coomassie Blue.
B. ExPression Vector pChNF121.
Expression vector pChNF121 encodes a 99 amino
acid CAT-hANP hybrid protein containing an endoproteinase
Glu-C proteolytic cleavage site (Fig. 4A). Approximately
one-third of the CAT gene (amino acids 1-73) has been
.
-17- ~ ~ r~ ~ ~ g ~
fused to the hANP(102~126) gene and proteolytic cleavage
site (26 amino acids) without an intervening linker. The
hANP polypeptide comprises 25% of the hybrid protein.
This vector was constructed from plasmids pChNF109 and
phNF87 which supplied the amino terminal fragment of the
CAT gene and the hANP gene and proteolytic cleavage site,
respectively.
1. Construction of pChNF121.
Plasmid phNF87 was digèsted with EcoRI, its
termini dephosphorylated with bacterial alkaline
phosphatase, and ligated with an approximately 320 bp
EcoRI fragment containing the trp promoter-operator,
ribosome binding site, and amino-~erminus of the CAT gene. -
This EcoRI cassette was purified from an EcoRI digest of
PChNF109 using agarose gel electrophoresis. Plasmid
pChNFl21 (Fig. 2G) was isolated from the ampicillin-
resistant transformants of E. coli MC1061. On the basis
of the si~e of the DNA fragments from a PvuII digest of
the plasmid, the CAT and hANP genes were inferred to be
fused in-frame to produce a hybrid protein.
2. Expression of CAT(1-73)-hANP(102-126) Hybrid
Protein From Plasmid pChNF121.
Plasmid pChNF121 expresses a CAT-hANP(102-126)
hybrid protein under the control of the E. coli trp
promoter-operator. The plasmid was used to transform E.
coli W3110 (prototroph, 'rrpR~) to ampicillin resistance
and one colony was grown in culture overnight at 37C in
complete M9 medium (see Section A.2.). The overnight
culture was diluted 100-fold into complete M9 medium
(uninduced culture) and into M9 medium with 25 ug/ml 3-
be~a-indole-acrylic acid xeplacing the 40 ug/ml tryp-
tophan (induced culture).
Expression was assessed after shaking ~he
cultures for 6 hr at 37C. The uninduced culture had
,~ ~ ' '
, .
-18- ~ 3 2 ~1 g2
reached a high cell density whereas the induced culture
reached about one-third this density. Phase contrast
microscopy revealed cells of normal morphology in the
uninduced culture and elongated cells with several
refractile inclusion bodies in the induced culture. Total
cell protein samples were prepared by boiling cell pellets
in Laemmli buffer for 5 min. and were analyzed by
electrophoresis through a 12% SDS-polyacrylamide gel fol-
lowed by staining of the protein with Coomassie Blue.
C. Ex~ression Vector pChNF142.
~ xpression ~ector pChNF142 encodes a 99 amino
acid CAT-hANP hybrid protein containing a unique Trp
residue following amino acid xesidue 73 of the CAT
protein, as a si~e for chemical cleavage. Approximately
one-third of the CAT gene ~amino ac,ids 1-73) has been
fused to the hANP(102-126) gene and chemical cleavage site
t26 amino acids). This amino terminal fragment of CAT has
been modified to substitute a Tyr residue for Trp~16] and
a Ser residue for Cys[31] to remove the additional
chemical cleavage site and reduce the multimerization of
the hybrid protein through disulfide bridges. A synthetic
hANP gene preceded by sequence encoding a Trp residue has
been a~sembled for this vector.
1. Construction of pChNF142.
Plasmid pTrp233 was digested with EcoRI, its
termini filled in with E. coli DNA polymerase I, Klenow
fragment, and ligated with T4 DNA ligase (to remove the
EcoRI restriction endonuclease cleavage site). From
ampicillin-resistant transformants of ~. coli MC1061,
plasmid pTrp81 was isolated and shown to resist cleavage
by EcoRI. Plasmid pTrp81 was digested with NdeI and
HindIII, purified by agarose gel electrophoresis, and
ligated with a synthstic CAT gene ~ragment using T4 DNA
ligase. The synthetic NdeI-HindIII CAT gene fragment
.~
, ~ --19--
15~2~162
(Fig. 2H) was assembled from three pairs of oligo-
deoxyribonucleotides as previously described. From
ampicillin-resistant transformants of E. coli MC1061,
plasmid pCAT127 was isolated and shown to contain the
synthetic CAT fragment by digestion with EcoRI and AvaI.
The plasmid was digested with BamHI and HindIII, the
BamHI-HindIII fragment containing CAT was purified by
agarose gel electrophoresis, sequenced by the method of
Sanger et al ~1977), supra, and the correct DNA sequence
confirmed.
Plasmid pC~T127 was digested with EcoRI and
HindIII and ligated using T4 DNA ligase with a pair of
annealed synthetic oligodeoxyribonucleotides encoding
hANP(102-126) preceded by a Trp residue on an EcoRI-
HindIII DNA fragment. Plasmid pChNF142 (Fig. 2I) wasisolated from ampicillin-resistant transformants of
E. coli MC1061. Insertion of the hANP gene was confirmed
by the size of the DNA fragments in a BamHI and HindIII
digest of the plasmid. The sequence of -the hANP gene was
confirmed from an EcoRI-ScaI agarose gel purified fragment
from pChNF142.
2. Expression of CAT(1~73), T~r~l6l Ser[311-
hANP(102-126) pChNF142.
~he expression of a modified CAT-hANP(102-126)
hybrid protein is conducted in substantial accordance with
the teaching of the previous examples A.2 and B.2.
II. E~ression of Chloramphenicol Acetyltransferase--
Am~loid A4 Protein Insert (A4-751i~ Hybrid Proteins
in Escherichla coli.
In the following examples high level expression
of the 57 amino acid insert within the amyloid A4-751
protein was achieved by fusing a synthetic A~-751i gene to
DN~ sequences encoding amino terminal fra~ments of CAT
under the control of the E. coli tryptophan promoter-
-20- ~32a~ 62
operator on a pBR322-derived plasmid. The synthetic A4-
751i gene encodes amino acids 289-345 from amyloid A4-751
protein (Ponte et al (1988), Nature 331:525-527) preceded
by a chemical cleavage site, Asn-Gly. Hydroxylamine
cleavage of the hybrid protein between these two residues
will yield the insert protein with a Gly residue at its
amino terminus.
A. Expression Vector pCAPi132.
Expression vector pCAPil32 encodes a 132 amino
acid CAT-A4751i hybrid protein containing a hydroxylamine
cleavage si~e (Fig. 4A). Approximately the amino terminal
third of the CAT gene (amino acids 1-73~ ha~ been joined
in-frame to the A4-751i gene and clea~age ~ite (59 amino
acids). The A4-751i protein comprises about 43~ of the
hybrid protein. This vector was constructed from plasmids
pTrp233 and pChNF121 and the synthetic A4-751i gene and
cleavage site.
1. Construction of PcApil32.
Plasmid pTrp233 was digested with EcoRI and
HindIII, purified by agarose gel electrophoresis, and
ligated with the synthetic gene ~ncoding the A4-751i
protein and cleavage site using T4 DNA ligase. The gene
had been assembled from six oligodeoxyribonucleotides
using previously described techniques and its sequence
(Fig. 4A) co~firmed. Plasmid pAPil31 was isolated from
ampicillin-resistant transformants of E. coli MCl061.
Insertion of the synthetic gene was confirmed by the size
of the DNA fragments from a PvuI and BamHI digest of
plasmid mini-prep DNA.
Plasmid pAPil31 was digested with EcoRI to
lineari~e the vector and its termini dephosphorylated
using bacterial alkaline phosphatase. Plasmid pChNF121
was digested with EcoRI and the approximately 320 bp ~coRI
fragmen~ containing the trp promoter-operator, ribosome
-21-
~ 3~0~
binding site, and amino terminus of the CAT gene ~amino
acids 1-73) was purified by agarose gel electrophoresis.
This EcoRI cassette was ligated with the pAPil31 plasmid
using T4 DNA ligase and ampicillin-resistant transformants
s of MC1061 were obtained. On the basis of ~NA fxagment
size in a PvuII digest of mini-prep plasmid DNA, plasmid
pCAPil32 was isolated with an in-frame fusion of CAT and
A4-751i sequences.
2. Expression of CAT( 1-73)-A4-751i Hybrid
Protein From Plasmid pCAPil32.
Plasmid pCAPil32 expresses a CAT--A4-751i hybrid
protein under the control of the E. coli ~ promoter-
operator. The plasmid was used to transform E. coli W3110
to ampicillin resistance and one colony was grown in
culture overnight at 37C in complete M9 medium. The
overnight culture was diluted 100~fold into complete M9
medium which contains 40 ug/ml tryptophan (uninduced
culture) and into complete M9 medium containing 25 ug/ml
3-beta-indoleacrylic acid instead of tryptophan (induced
culture).
Expression was assessed after shaking the
cultures for 6 hr at 37C. The uninduced culture had
reached a high cell density whereas the induced culture
was at a lower cell density. Phase contrast microscopy
revealed cells of normal morphology in the uninduced
culture and cells with ~pre-inclusion bodies~ in the
induced culture. As used herein, "pre-inclusion bodies"
are defined as less refractile bodies which appear to
convert in time to the more refractile "inclusion hodies~
as the hybrid protein accumulates in the cells. Total
cell protein samples were prepared by boiling cell pellets
in Laemmli buffer for 5 min and then analyæed by
electrophoresis through a 12% SDS-polyacrylamide gel fol-
lowed by staining with Coomassie Blue (Fig. 3~). ThisCAT(1-73)-A4-751i hybrid protein migrates between the
: :
- --22~ ~ 32~6~
lysozyme (14,300 MW) and beta-lactoglobulin (18,400 M~7)
protein standards on this gel. Vsing a Kontes fiber optic
scanner and Hewlett-Packard Integrator to scan the gel,
the hybrid protein was estimated to comprise about 7% of
the total cell protein. This is a moderate expression
le~el of E. coli but A4-751i comprises almost half of the
hybrid protein.
To confirm the presence of A4-751i in the hybrid
protein, Western blot analysis was carried out on an
unstained 12% SDS-polyacrylamide gel o~ these protein
samples. Protein was blotted to nitrocellulose and
incubated with anti-A4-751i serum (prepared against a 16
amino acid synthetic pep~ide containing amino acids 11-26
of the 57 amino acid insert protein). After incubation
with 125I-protein A (Amersham) the blot was placed on X-
ray film at -70C for several days. The synthetic peptide
anti-serum detected the hybrid protein as well as several
other E. coli proteins.
B. Expression vector pCAPil36.
Expression vector pCAPil36 encodes a 274 amino
acid CAT-A4-751i hybrid protein containing a hydroxylamine
cleavage site. Most of the CAT gene (amino acids 1-210)
has been joined in-frame to the A4-751i gene and cleavage
site (59 amino acids) through a linker sequence (5 amino
acids). The A4-751i polypeptide comprises about 21% of
the hybrid protein. This vector was constructed from
plasmids pAPil31 and pChNF109.
1. Construction of pCAPil36.
Plasmid pAPil31 was digested with EcoRI to
linearize the vector and its termini dephosphorylated
using bacterial alkaline phosphatase. From a partial
EcoRI digest of pChNF109 an approximately 740 bp EcoRI
fragment containing the trp promoter-operator, the CAT
gene (amino acids 1 210), and linker sequence was purified
.
.
'
-23~
by agarose gel electrophoresis. This EcoRI cassette and
vector pAPil31 were ligated using T4 DNA ligase and
ampicillin-resistant transformants of E. coli MC1061 were
isolated. From the size of DNA fragments in plasmid mini-
preps digested with BamHI, plasmid pCAPil36 was isolatedwith the CAT gene and the synthetic A4-751i gene in-frame.
2. Expression of CAT(1-210)-A4-751i Hybrid
Protein From Plasmid pCAPil36.
Plasmid pCAPil36 expresses a CAT-A4-751i hybrid
protein under the control of the E. coli trp promoter-
operator. The plasmid was used to transform E. coli W3110
to ampicillin resistance and one colony was grown in
culture overnight at 37C in complete Mg medium. The
overnight culture was diluted 100-fold into the same M9
medium (uninduced culture) and into M9 complete medium
containing 25 ug/ml 3-beta-indoleacrylic acid instead of
tryptophan (induced culture).
Expression was assessed after shaking the
cultures for 5 hr at 37C. Both the uninduced and inducqd
cultures reached high cell densities. Phase contrast
microscopy revealed cells of noxmal morphology in the
uninduced cultures and cells containing inclusion bodies
or pre-inclusion bodies (50:50) in the induced cultures.
Total cell protein samples were prepared by boiling cell
pellets in Laemmli buffer for 5 min and were analyzed by
electrophoresis through a 12% SDS~polyacrylamide gel fol-
lowed by staining with Coomassie Blue (Fig. 3A). This
CAT-A4-751i hybrid protein migrates between the alpha-
ch~motrypsinogen (25,700 MW) and ovalbumin (43,000 MW)protein standards on this gel. Using a Kontes fiber optic
scanner and Hewlett-Packar~ In~egrator to scan the gel,
the hybrid protein was estimated to comprises about 15% of
total cell protein. This is moderately high level expres-
sion for E. coli.
(*) Trademark
.
,~:''"
.~
- -24- ~ 2
To confirm the presence of A4-751i ln the hybrid
protein, Western blot analysis was carried out on an
unstained 12% SDS-polyacrylamide gel of these protein
samples. Using the method described above ~section II.
A.2.), the synthetic peptide anti-serum detected the
hybrid protein as well as se~eral other E. coli proteins.
ssion of Chloramphenicol Acetyltransferase--
Glucaqon-L e PePtide I ~-37) Hybrid Protein in
Escherichia coli.
In the following example, high level expression
of the 31 amino acid GLP-I(7-37) was achie~ed by fusing a
synthetic GLP-I gene to DNA sequences encoding an amino
terminal fragment of CAT under the control of the E. coli
tryptophan promoter-operator on a pBR322-derived plasmid.
The synthetic gene encodes amino acids 7-37 of GLP-l
(Mojsov et al (1987), J. Clin Invest 79:616-619) preceded
by a Met residue. Treatment with cyanogen bromide
releases the insulinotropic peptide.
A. Expression Vector PcGLpl39.
Expression vector pCGLP139 encodes a 105 amino
acid CAT-GLP-I hybrid protein containing a cyanogen
bromide clea~age site (Fig. 4B). Approximately the amino
terminal third of the CAT gene (amino acids 1-73) has been
joined in-frame to the GLP-l gene and cleavage site (32
amino acids). The GLP-I peptide comprises about 30~ of
the hybrid protein. This vector was constructed from
plasmids pTrp233 and pChNF109 and the synthetic GLP-I gene
and cleavage site.
1. Construction_of pCGLP139.
Plasmid pTrp233 was digested with EcoRI and
HindIII, purified by agarose gel electrophoresis, and
ligated with the synthetic gene using T4 DNA ligase. The
gene had been assembled from four oligodeoxyribo-
25- 1~2~2
nucleotides and its sequence (Fig. 4B) confirmed. From
ampicillin-resistant transformants of E. coli MC1061,
plasmid pGLP138 was isolated. Insertion of the synthetic
gene was confirmed by the failure of plasmid mini-prep DNA
to be cut by PstI.
Plasmid pGLP138 was digested with EcoRI to
linearize the vector, its termini dephosphorylated using
bacterial alkaline phosphatase, and ligated with the EcoRI
cassette from plasmid pChNF109 using T4 DNA ligase
Plasmid pChNF109 had been digested with EcoRI and the ap-
proximately 320 bp EcoRI fragment containing the trp
promoter-oper~tor, ribosome binding site, and an amino
terminal fragment of the CAT gene purified by agarose gel
electrophoresis. Plasmid pCGLP139 was isolated from
ampicillin-resistant transformants of MC1061. On the
basis of DNA ragment size in an AvaI and PvuII digest of
plasmid mini-prep DNA, the fusion of CAT and GLP-I
sequences was confirmed to be in-frame.
2. Expression of_CAT(1-73)-GLP-I(7-37) Hybrid
Protei.n From Plasmid pCGLP139.
Plasmid pCGLP139 expresses a CAT-GLP-I hybrid
protein under the control of the E. coli trp promoter-
operator. The plasmid was used to transform E. coli W3110
to ampicillin resistance and one colony was grown in
culture overnight at 37C in complete M9 medium. The
overnight culture was diluted 100-fold into complete M9
medium which contains 40 ugtml tryptophan (uninduced
culture) and into complete M9 medium in which 25 ug/ml 3-
beta-indoleacrylic acid has been substituted for the
tryptophan tinduced culture).
Expression was assessed after shaking the
cultures for 6 hr at 37C. The uninduced culture had
reached a high cell density whereas the induced cul~ure
was at a lower cell density. Phase contrast microscopy
revealed cells of normal morphology in the uninduced
-26- 132~162
culture and elongated cells with three or more refractile
inclusion bodies in the induced culture. Total cell
protein samples were prepared by boiling cell pellets in
Laemmli buffer for 5 min and were analyzed by
electrophoresis through a 12% SDS-polyacrylamide gel fol-
lowed by staining with Coomassie Blue (Fig. 3B). This
CAT(1-73~-GLP-I(7-37) hybrid protein migrates between the
bovine trypsin inhibitor (6,200 MW) and lysozyme (14,300
MW) protein standards. Using a Kontes fiber optic scanner
and Hewlett-Packard Integrator to scan the gel, the hybrid
protein was estimated to comprise about 20~ of the total
cell protein. (Considering the number of inclusion bodies
observed per cell, all of the hybrid protein may not have
been solubilized in the Laemmli buffer, and this estimate
may be low.) This is high level expression for E. coli.
The molecular weight of the hybrid protein is as
predicted for this gene fusion. Amino acid composition
analysis of the purified hybrid protein or protein
sequencing of the peptide after cyanogen bromide cleavage
can be performed to confirm its expression.
IV. CAT Fusion With Human SP-B and SP-C.
The mature forms of both human SP-C and SP-B are
expressed as fusions with portions of bacterial CAT. The
surfactant peptides are ~oined to the carboxy terminus of
the CAT sequences through a hydroxylamine-sensitive
asparagine-glycine linkage. The CAT-surfactant fusions
are expressed from the tryptophan promoter of the bacte-
rial vector pTrp233.
A. Expression Vector pC210SP-B.
SP-B expression vector pC210SP-B encodes a fu-
sion protein of 293 residues in which 210 amino acids of
CAT are joined to the 76 amino acids of SP-B through a
linker of 7 amino acids containing ~he hydroxylamlne-
sensitive cleavage site. Cleavage of the fusion with
-27- ~ 3~162
hydroxylamine releases a 77 amino acid SP-B product
containing the 76 residue mature form of SP-B, plus an
amino-terminal glycine residue.
To construct pC210SP-B, the short EcoRI-HindIII
segment containing ANF sequences was removed from
pChNF109, and replaced by a portion of human SP-B cDNA #3
extending from the PstI site at nucleotide (nt) 643 (Fig.
6) to the SphI site at nt 804. The EcoRI site wa~ joined
at the PstI site through two complementary
oligonucleotides encoding the hydroxylamine sensitive
cleavage site as well as the amino-terminal residues of
mature SP-B (oligo ~2307: 5 ' -AAT TCA ACG GTT TCC CCA TTC
CTC TCC CCT ATT GCT GGC TCT GCA-3' and oligo #2308: 5'-GAC
CCA GCA ATA GGG GAG AGG AAT GGG GAA ACC GTT G-3'). The
lS ~I site was joined to the HindIII site of PTrp233
through a second se~ of complementary nucleotides encoding
the carboxy~terminal residues of ma~ure SP-B (oligo #3313:
5'-ACC TTA CCG GAG GAC GAG GCG GCA GAC CAG CTG GGG CAG CAT
G-3' and oligo #3314: 5'-CTG CCC CAG CTG GTC TGC CGC CTC
GTC CTC CGG TA-3').
The expression plasmid was used to trans~orm E.
c _ stain W3110 to ampicillin resistance. Rapidly grow-
ing cultures of pC210SP-B/W3110 in M9 medium were made 25
ug/ml IAA (3-beta indoleacrylate, Sigma I-1625) to induce
the Trp promoter. By 1 hr after induction, refractile
cytoplasmic inclusion bodies were seen by phase contrast
microscopy inside the still-growing cells. 5 hr after
induction, the equivalent of 1 O.D.550 of cells were
pelleted by centrifugation, then boiled for 5 min in SDS
sample buffer for electrophoresis in a 12~ SDS-
polyacrylamide gel followed by staining with Coomassie
Blue (Fig. 7). Lane A = molecular size standards; Lane B
= induced W3110 cells containing pTrp233 vector control;
and Lane C = induced pC210SP-BJW3110. The predicted
molecular weight of the CAT:SP-B ~usion protein is 45,000
daltons. The hybrid CAT:SP-B protein was es~imated to
- -28- ~3~62
comprise 15-20~ of the total cell protein in the induced
cultures.
B. CAT Fusions with SP-C.
A series of vectors wer0 constructed encoding
fusion proteins in which mature human SP-C was fused to
the carboxy termini of different portions of CAT through a
hydroxylamine-sensitive asparagine-glycine linkage.
~Iydroxylamine cleavage of the fusion protein produced by
each cons~ruct releases a mature SP-C of 35 amino acids
which lacks the amino-terminal phenylalanine residue seen
in a portion of natural human SP-C.
1. pC210SP-C.
The amino acid sequence of the 251 residue fu-
sion protein encoded plasmid pC210SP-C. The 210 amino
acids of CAT are joined to 35 amino acids of mature SP-C
through a linker of 6 amino acids. The mature SP-C
portion of the total fusion protein comprises 14%.
In Fig. 8 is shown the nucleotide sequence of
pC210$P-C, in which th~ EcoRI-HindIII fragment of pC210SP-
B containing SP-B sequences has been replaced by a segment
of human SP-C cDNA #18 extending from the A~LI site at
nucleotide 123 to the AvaII site at nucleotide 161. The
EcoRI site of the CAT vector was joined to the SP5 ApaLI
site through two complementary oligonucleotides encoding
the hydroxylamine sensitive cleavage site as well as the
amino-terminal residues of mature SP-C (oligo #2462: 5'-
AAT TCA ACG GCA TTC CCT GCT GCC CAG-3' and oligo #2463:
5'-TGC ACT GGG CAG CAG GGA ATG CCG TTG-3'). The A~aII
site of SP-C was joined to the HindIII site of pC210SP-B
through a second set of complementary nucleo~ides encoding
the carboxy-terminal residues of mature SP-C and a stop
codon (oligo #2871: S'-AGC TTA GTG GAG ACC CAT GA& CAG GGC
3S TCG CAC AAT CAC C~C GAC ~AT GAG-3' and oliqo #2372: 5'-GTC
.
,
'' ' '
. -29-
~3~al62
CTC ATC GTC GTG GTG ATT GTG GGA GCC CTG CTC ATG GGT CTC
CAC TA-3').
2~ ~C179SP-C.
The amino acid sequence of the 217 residue fu-
sion protein encoded by pC179SP-C is a slight modification
of the sequence shown in Fig. 8. In pC179SP-C, the 179
amino acids of CAT are joined to 35 amino acids of mature
SP-C through a linker of 3 amino acids (Glu, Phe, Asn).
SP-C portion of the total fusion protein comprises 16%.
To construct pC179SP-C, a portion of the CA~
sequence was removed from pC210SP-C. S~arting with
pC210SP-C, a DNA fragment extending from the Ncol site at
nt 603 (Fig. 8) to the EcoRI site at nt 728 was removed,
and the NcoI and EcoRI cohesi~e ends were rejoined with
two complementary oligonucleotides (oligo #3083s 5'~CAT
GGG CAA ATA T~A TAC GCA AG-3' and oligo #3084: 5'-AAT TCT
TGC GTA TAA TAT TTG CC-3'). In effect, 31 residues of
CAT, and 3 residues of the linker polypeptide are missing
in the new fusion protein encoded by vector pC179SP-C.
3. pC14gSP-C.
The amino acid sequence of the 187 residue fu-
sion protein encoded by pC149S~-C is a slight modification
of the sequence shown in Fig. 8. In plasmid pC149SP-C,
the 149 amino acids of CAT are ~oined to 35 amino acids of
mature SP~C through a linker of 3 amino acids (Glu, Phe,
Asn). The SP-C portion of the total fusion protein
comprises 18.7%.
To construct pC1495P-C, a portion of the CAT
segment of pC210SP-C extending from the DdeI site at nt
523 (Fig. 8) to the EcoRI site at nt 728 was removed and
replaced by a~set of two complementary oligonucleotides
(oligo #3082: 5' TCA GCC:AAT CCC:G-3' oliqo #3081: 5'-AAT
35~ TCG GGA TTG GC-3').
:: :
,. .
,
.
_30_ ~3 233 1 ~2
4. pC106SP-C.
The amino acid sequence of the 144 residue fu-
sion protein encoded by pC106SP-C is a slight modification
of the sequence shown in Fig. 8. In plasmid pC106SP-C,
the 106 amino acids of CAT are joined to 35 amino acids of
mature SP-C through a linker of 3 amino acids (Glu, Phe,
Asn). The SP-C portion of the total fusion protein
comprises 24%.
pCl06SP-C was constructed by replacing the EcoRI
fragment of pC210SP-C (nt 302 to nt 728, Fig. 8) with two
sets of complementary oligos which were annealed, then
ligated together through a region of homology (oligo
#3079: 5 '-AAT TCC GTA TGG CAA TGA AAG ACG GTG AGC TGG TGA
TAT GGG ATA GTG TTC ACC CTT GT-3' was annealed with oligo
15 #3085: 5'-ACA CTA TCC CAT ATC ACC P,GC TCA CCG TCT TTC ATT
GCC ATA CGG-3'; oligo #3080: 5 ' -TAC ACC GTT TTC CAT GAG
CAA ACT GAA ACG TTT TCA TCG CTC TGG G-3' was annealed with
oligo #3078: 5~-AAT TCC CAG AGC GAT GAA AAC GTT TCA GTT
TGC TCA TGG AAA ACG GTG TAA CAA GG& TGA-3').
5. Expression From SP-C Vectors.
Each SP-C expression vectox was used to
transform E. coli strain W3110 to ampicillin resistance.
Rapidly growing cultures of expression strains were
induced as described above. By 1 hr after induction,
refractile cytoplasmic inclusion bodies were seen by phase
contrast microscopy inside the still-growing cells. 5 hr
after induction, the equivalent of 1 O.DL550 of cell~ were
pelleted by centrifugation, then boiled for 5 min in SDS
sample buffer for electrophoresis in a 12% SDS-
polyacrylamide gel followed by staining with Coomassie
Blue. The results are provided in Fig. g wherein Lane ~ =
molecular size standards, Lane B = induced W3110 cells
containing pTrp233 vector control; Lane C = induced
pC106SP-C; Lane D = pC149SP-C; Lane E = pC179SP-C; Lane F
= pC210SPWC. The hybrid CAT:SP-C protein produced by each
. , . , ~ .
i ~J~16~
vector is estimated to comprise 15-20~ of the total cell
protein in the induced cultures.
v. Improved CAT Vectors for Expression of ~ybrid Proteins
in Escherichia Coli.
In the following examples, the basic CAT gene
fusion vector has been improved in several ways: (1)
unique cloning sites are created for insertion of the gene
to be expressed, (2) the CAT gene is modified to optimize
cleavage andtor purification of the peptides, and (3) the
gene conferring resistance to tetracycline is restored to
provide an alternatlve method for plasmid selection and
maintenance.
A. Expression Vectors ~CAT73 and PCAT210.
Expression vector pCAT73 contains genes confer-
ring resistance to both ampicillin and tetracycline,
unique EcoRI and HindIII cloning sites for insertion of
genes to be expressed, and the amino terminal fragment (1-
73) of the CAT gene. The cleavage site, included with the
inserted ~ene, may not be unique. This plasmid is
constructed from plasmids pBR322, pTrp233, pCAT21, and
oli~odeoxyribonucleotides. Expression vector pCAT210 dif-
fers from pCAT73 in that it contains the larger amino
terminal fragment (1-210) of the CAT gene from which the
EcoRI site at the sequence encoding residues 72 and 73
(Glu-Phe) ha-c been removed. ~An alternative codon choice
preserves the Glu and permits the use of unique EcoRI and
HindIII cloning sites.) Other DNA fragments encoding the
amino terminus of the CAT gene, smaller than 73 amino
acids or between 73 and 210 amino acids may also be
constructed by insertion of an EcoRI site at the desired
fusion point.
-32-
132~6~
l. Construction of pCAT73.
Restoration of the gene for tetracycline resist-
ance requires restoring the BamHI-HindIII-EcoRI fragment
of pBR322 to the CAT expression vector. Since the unique
cloning sites desired for this vector are EcoRI and
HindIII, this must be done in a manner which removes these
sites but retains resistance to tetracycline. Since
insertion of DNA at the HindIlI site upstream of the cod-
ing region often prevents gene expression, this site i5
removed by creating a point mutation at the HindIII site.
Plasmid pBR322, was digested with EcoRI and HindIII and
the vector backbone gel purified. The backbone was
ligated with synthetic EcoRI-HindII fragments, which are
formed by annealing pairs of oligonucleotides using T4 DNA
lS ligase. The fragments contain the normal EcoRI-HindIII
sequence with the exception of point mutations (G or C) at
the first adenine of the recognition sequence 5'-AAGCTT-
3'. An intermediate plasmid was isolated from ampicillin-
resistant and tetracycline-resistant E. coli MC1061
transformants whose plasmid mini-prep DNA was not digested
by HindIII.
A BamHI-EcoRI fragment no longer containing a
HindIII site was purified from agarose gel electrophoresis
from a BamHI and EcoRI digest of plasmid pTetHl. The
fragment was ligated using T4 DNA ligase with plasmid
pTrp233 which was also digested with ~amHI and EcoRI and
agarose gel purified. Transformed wi~h the ligation,
colonies of E. coli MC1061 were selected for ampicillin
and/or tetracycline resistance. Plasmid pTrpT233 was
resistant to both antibiotics.
In an alternate embodiment, digestion of
pTrpT233 with EcoRI, blunting of the termini with DNA
polymerase I, Klenow fragment, and ligation with T4 DNA
ligase will eliminate the EcoRI site (which does not
affect resistance to tetracycline). Tetracycline-
resistant plasmid pTrpT234 which has lost undesirable
~3201 6~
HlndIII and EcoRI sites is isolated from colonies of E.
coli MC1061 transformed with this ligation.
The CAT gene is obtained as an NdeI-HindIII
fragment purified by agarose gel electrophoresis of an
NdeI-HindIII digest of pCAT21. Plasmid pTrpdeltaHind was
digested wikh NdeI and HlndIII, purified by agarose gel
electrophoresis, and ligated with the CAT gene using T4
DNA ligase. From ampicillin (or tetracycline) resistant
transformants of E. coli MC10Sl digested with EcoRI and
HindIII to verify incorporation of the CAT gene, plasmid
pCAT73 (Fig. 5A) is isolated.
2. Construction of PCAT210.
The BamHI-HindIII fragment containing the trp
promoter-operator, ribosome binding site, and CAT gene is
purified by agarose gel electrophoresis from a BamHI and
HindIII digest of plasmid pCAT21. Site specific
mutagenesis is carried out on the fragment using M13 and
mutagenic oligodeoxyribonucleotides to convert the GAA
codon for Glu to GAG (also to Glu) within the EcoRI site,
5'-GAATTC-3'. One such plasmid, M13-CATdR, is digested
with ScaI to linearize the vector and ligated with an
EcoRI linker (for the same reading frame as in pCAT73)
using T4 DNA ligase. From the transfectants, M13-CATR1,
is isolated and digested with NdeI and HindIII. The new
CAT gene is purified by agarose gel electrophoresis and
ligated using T4 DNA ligase with NdeI~HindIII-digested
plasmid pTrpT234. Plasmid pCAT210 (Fig. SB) is isolated
from ampicillin (or tetracycline) resistant transformants
f E coli MC1061.
B. Expression Vectors pCAT73-T and ~CAT73-M.
Expression ~ectors pCAT73-T and pCAT73-M are
examples in which the amino acid sequence of CAT has been
altered using site specific mutagenesis techniques to
facilitate purification of the product protein. In these
, .
-34-
:~ 3 2 ~
cases, the Trp residue at position 16 may be substituted
with Tyr and the Met residue at position 67 may be
substituted by Ile or Leu to eliminate potential chemical
cleavage sites within CAT. In addition, the Cys at posi-
tion 31 may also be subs~ituted using a conservative aminoacid alteration, that is, substitution with an amino acid
which does not adversely affect biological activity.
Preferred residues include alanine, serine, leucine,
isoleucine and valine, most preferred is serine. These
la~ter alterations are intended to reduce multimerization
through disulfide bridges.
C. Expression of Modified CAT-GLP-1
Plasmid pTrpdeltaHind contains the restored TetR
gene from pTrp233 (although the HindIII site has been
eliminated), the Trpl6 to Tyr, Cys3l to Ser, and Met67 to
Leu substltutions in the CAT gene sequence, and the GLP 1
gene (taught in Example III) fused in-frame to the
modified CAT gene -through a methione residue. The vector
was used to transform several E. coli strains including
~3110, MC1061, DHl, MM294 and RR1.
E. coli RRl transformants were more stable and
appeared to have better induction/repression control of
the Trp promoter than any of the other hosts. An
alternative construction for this vector includes
reversing the TetR gene (to avoid the back-to-b~ck
placement of the Tet~ and Trp promoters in the present
construct) to alleviate the stability problems observed
using bacterial hosts other than RRI transformants.
VI. Construction of pTrpCAT72:Adipsin/D.
The coding sequence for mature human adipsin/D
was fused to pCAT72 to produce a fusion protein suitable,
for example, to generate antisera agains~ human adipsin/D.
,, ,~
-35-
:L~2~162
A. Construction of pTrpCAT7? Q3Sl
Plasmid pCAT72 Q3Sl was cons~ructed to eliminate
Asn residues at which secondary cleavages can occur during
hydroxylamine release of peptides fused to CAT. The A~n
residues at amino acid positions 26, 51 and 78 of CAT were
changed to Gln residues. At the same time, the single Cys
at position 31 was changed to Ser to decrease the amount
of aggregation seen with many CAT fusion proteins.
The vector pCAT72 Q3Sl was constructed as fol-
lows: Oligos CAT72-1 through 6 (below) were annealed and
ligated into pUC-9 which had been cleaved with NdeI ~nd
EcoRI. In this way, the mutated CAT72 was joined to the
polylinker region of the pUC plasmid. CAT72 Q3Sl with the
polylinker was then removed from pUC by cleavage with NdeI
and HindIII, and inserted into pTrp233 b~tween NdeI and
HindIII to yield pTrpCAT72 Q3Sl.
CAT72-1
TATGGAGAAA AAAATCACTG GATATACCAC CGTTGATATA TCCCAATGGC
~60 70
ATCGTAAAGA ACATTTTGAG GCATTTCA
AT72-2
10 20 30 40 50
CAAAATGTTC TTTACGATGC CATTGGGATA TATCAACGGT GGTATATCCA
TGATTTTTT TCTCCA
CAT72-3
TCAGTTGCT CAATCTACCT ATCAGCAGAC CGTTCAGCTG GATATTACGG
60 70 80
CCTTTTTAAA GACCGTAAAG AAACAGAAGC
CAT72-4
S0
CTTTACGGTC TTTAAAAAGG CCGTAATATC CAGCTGAACG GTCTGCTGAT
AGGTAGATTG AGCAACTGAC TGAAATGCCT
-36-
1320~
CAT72-5
ACAAGTTTTA TCCGGCCTTT ATTCACATTC TTGCCCGCCT GATGCAGGCT
CATCCGG
CAT72-6
AATTCCGGAT GAGCCTGCAT CAGGCGGGCA AGAATGTGAA TAAAGGCCGG
7G
ATAAAACTTG TGCTTCTGTT T
B. Construction of pTrpCAT72 Q6S3
Starting with pCAT72 Q3Sl, pCAT153 Q6S3 was
constructed to change the Asn residues at positions 130,
141 and 148 of CAT to Gln residues, and to change the Cys
residues at 91 and 126 to Ser residues.
Pla~mid CAT72 Q3Sl in pUC-9 was clea~ed with
EcoRI. Oligos CAT153-1 through 6 (below) were annealed
and ligated into pCAT72 to ~ive pCAT153 Q6S3. The
modified pCAT153 was then removed from pUC by cleavage
with NdéI and HindIII, and the resulting fragment inserted
into pTrp233 to give pTrpCAT153 Q6S3.
CAT153-1
AATTTCGTAT GGCAATGAAA GACGGTGAGC TGGTGATATG GGATAGTGTT
CACCCTTCTT ACACCGTTTT CCATGAGCAA
CAT153-2
~ 10 20 30 40 50
AAA~CGGTGT AAGAAGGGTG AACACTATCC CATATCACCA GCTCACCGTC
TTTCATTGCC ATACGA
CAT153-3
~ 10 20 30 40 50
ACTGAAACGT TTTCATCGCT CTGGAGTGAA TACCACG~CG ATTTCCGGCA
~ ~0 70 80
GTTTCTACAC ATATATTCGC AAGATGTGGC
~37~ 132~62
CAT153-4
GCGAATATAT GTGTAGAAAC T5CCGGAAAT CGTCGTGGTA TTCACTCCAG
AGCGATGAAA ACGTTTCAGT TTGCTCATG~
CAT153-5
-10 ~0 30 40 50
GTCTTACGGT GAACAGCTGG CCTATTTCCC TAAAGGGTTT ATTGAGCAGA
TGTTTTTCG~ CTCAGCCCAG CCCG
CAT153~6
- ~ 10 20 30 4~ 5)
AATTCGGGCT GGGCTGAGAC ~A~AAACATC TGCTC~AT~ ACCCTTTA~G
7~ 80
GAAATA~GCC AGCTGTTCAC CGTAAGACGC CACATCTT
~, ~
Next, the human adip~in/D.cDNA hg31-40 (Figure
10) wa~ constructed. The BamHI-~yI fragment containing
th~ mature coding region was gel puri~iod and in3erted
into pUC-9 which had been cleaved with BamHI and HlndIII.
The StyI end of the cDNA ~a~ ~oined to the HindIII end of
pUC using two oligos (#3886 S'-CATGGGTGCCGGGGCCTGA-3~ and
#3887 5'-AGCTTCAGGCCCCGGCACC-3'). 8y inserting the BamHI-
~yI fragment of adipsin/D into pUC in this way, the cod-
ing sequence of adipsin/D wa~ placed in fra~e with the
EcoRI site of pVC~9. 1~he EcoRI-HindIII fragment of thi~
construct w~s removed from pUC-9 and in~ertsd into
pTrpCAT72 between the EcoRI ~ite and the HindIII sites to
yield pTrpCRT72:Adipsin/D.
This con~truct gave 10-15~ lavel~ of fusion
protein upon induction in ~3110 c~llY.
~odifications of the above de~cribed mode~ for
carrying out the invention that are obvious to thoso of
skill in the ar~ of molecular biol~gy, protein chemi~try,
cell biology, or rela~ed field~ are in~ended to be within
the scope of the following claim~.
~1
