Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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NEW PLASMID CONSTRUCTIONS FOR HIGH
LEVEL PRODUCTION OF EURARYOTIC PROTEINS
Technical Field
The present invention is related generally to
the construction of plasmid vectors. Nore partlcularly,
the present invention is related to the construction of
novel plasmids for high level production of eukaryotic
proteins in their natural form in a suitable expression
vector.
Backaround of the Invention
Plasmid system~ for the expression of proteins
of single subunit structure have been known. ~ypical of
such vectors are the ones described by Amann et al, 1983,
Gene, 25:167-178 and Rosenberg et al, 1983, Methods in
EnzYmolo~Y, 101:123-138, respectively. Bacterial
expression of eukaryotic proteins is a tool of ever-
increasing importance in biochemistry and molecular
biology. However, the ma prity of the recombinant
eukaryotic proteins that have been expressed in bacteria
are produced as fusion proteins and not in their native
conformation. Despite advances in plasmid engineering, a
plasmid system for high level expression of proteins with
quarternary structure (that is, proteins formed by more
than one subunit) in their natural form in a bacterial
; 25 expression vector has not heretofore been described.
SUNNARY OF TH~ INVENTION
It is, therefore, an ob~ect of the present
invention to construct novel plasmids for high level
expression of quarternary proteins in their native form,
in a suitable expression vector.
It is another ob~ect of the present invention
to provide plasmids containing artificial operons for the
expression of dimeric or multimeric proteins in their
native (natural) form and to provide expression vectors
of general utility.
It is a further ob~ect of the present invention
to provide high efficiency cDNA expression libraries for
the screening of eukaryotic genes with antibody probes.
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Other ob~ects and advantages of the present
invention will become evident from the following detailed
description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other ob~ects, features and many of
the attendant advantages of the invention will be better
understood upon a reading of the following detailed
de~cription when considered in connection with the accom-
panying drawings wherein:
Figure lA shows a schematic construction of
pLEl01 and pLEl02.
Figure lB ~hows a schematic construction of
pLE103-l from pLE101. The sequence of pLE103-1 between
the BamHI site and the HindIII site is reported above the
scheme of the subcloning ~teps. Arrows indicate the
limits of the synthetic 89-base pair BamHI-Nco I DNA
fragment used for this construction. Only the restric-
tion sites relevant for plasmid construction and the
cloning sites are indicated. Ptrc:trc promoter; 5S: E.
coli rrnB 5S rRNA gene; T: Tl and T2 rrnB terminators;
~10: ~10 phage T7 promoter; UG: mature UG coding
sequence from pUG617; SD: Shine-Dalgarno sequence.
Figure 2A shows the expression of UG in E. coli
BL21(DE3):pLE103-1 as estimated by SDS-polyacrylamide gel
electrophoresis on a 15-25% gradient gel containing 0.1%
SDS. Each lane was loaded with the equivalent of 50 ~
of bacterial culture. Lane 1: purified rabbit UG (1
~g); lane 2: molecular weight standards (BRL, pre-
~stained); lanes 3-4: l hour after induction time, non-
induced culture (3) and induced culture (4); lanes 5-6:
2 hours after induction time, non-induced culture (5) and
induced culture (6); lanes 7-8: 3 hours after induction
time, non-induced culture (7) and induced culture (8).
The arrows indicate the position of the
expressed bands: ~ -lactamase (uppermost arrow) and the
two bands of UG. Aliquots of cultures (l ml) in M9CA
medium were centrifuged at 12000 x g for 2 min. Cells
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were wa~hed with ice-cold PBS and centrifuged as above.
Pellets were lysed in 50 ul of 2X sample buffer contain-
ing 0.2% mercaptoethanol. Samples were boiled for 5 mln.
and ad~usted to a final volume of 100 ul with distilled
water. Aliquots (10 ul) were loaded on a 10-20% gradient
polyacrylamide gel, 1.5 mm thick. Silver staining wa~
performed by means of a Biorad kit, according to the
manufacturer's instructions.
Figure 2B shows the immunoblot of expres~ed
recombinant UG. Each lane of a 15-25% polycrylamide
gradient gel containing 0.1 % SDS was loaded with the
equivalent of 50 ~l of bacterial culture, and protein
bands were transfered overnight to a Nitroscreen West
membrane (DuPont, 0.22 ~ m pore size) at 4C, with a
current of 34 mA. Lane 1: molecular weight standards
` (BRL, prestained); lanes 2-3: 1 hour after induction
time, non-induced culture (2) and induced culture (3);
- lanes 4-5: 2 hours after induction time, non-induced
culture (4) and induced culture (S); lanes 6-7: 3 hours
after induction time, non-induced culture (6) and induced
culture (7); lane 8: purified rabbit UG (250 ng). Note
that both UG monomer and dimer bands appear to be stained
(arrow).
Figures 3A and 3B show the quantitation of
recombinant UG in bacterial lysate supernatants as
detected by RIA. Each point represents the average of
` three determinations, each performed in duplicate.
Figure 4 demonstrates the determination of
recombinant UG molecular weight by means of size-exclu-
sion chromatography on Sephacryl-S200 (Pharmacia). Sam-
ples of pure rabbit UG (140 ~g) and of bacterial lysate
supernatant (400 ~l from bacteria harvested 90 min after
induction) were analyzed. One ml fractions were
collected, diluted 1:100 and assayed for UG by RIA. The
inset shows the calibration of the column with standard
proteins (gel-filtration calibration kit, Pharmacia, plus
horse heart myoglobin, Sigma). Abbreviations: LYS =
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ly~ozyme; MYO = myoglobin; CHT = chymotrypsinogen A; OVA
- ovalbumin; BSA - bovine ~erum albumin. KaV value~ were
calculated a~ de~cribed in Pharmacla technical booklett
Gel Filtratlon in Theory and Practlce. Values on the y
axis repre~ent concentratlons of UG ln the dlluted ~am-
ples, without ad~ustments for the dilutlon factor. The
plot line was obtained by least-square analy~is, followed
by linear regression. R = O.998. The arrow indicate8
the Xav of recombinant and natural UG. Apparent molecu-
lar weight = 17 kd.
Figure 5 shows the results of SDS-polyacryl-
amide gel electrophoresis of E. coli proteins in IPTG
induced and non-induced cultures. Samples were electro-
phoresed using a 15 ~ polyacrylamide gel containing 0.1 %
SDS. Lane a: molecular weight standards (BRL, low
molecular weight standards); lane b: pure rabbit UG (4
g) non-induced culture (control), 3 hours after induc-
tion time; lane c: bacterial lysate supernatant (about
12 g protein) induced culture, 3 hours after induction;
lane d: pooled UG-containing fractions after Sephacryl-
S200 superfine chromatography (about 20 ~g protein) non~
induced culture, 4 hours after induction; lane e: pooled
:.. - . -. :- ~ - --
fractions after CM-Sepharose chromatography (1.8 ~g pro~
tein) induced culture, 4 hour~ after induction; lane f:
pooled fractions after Sephadex-G50 ~uperfine chroma-
tography (1.6 ~g protein) purified rabbit UG, silver
stain.
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Note that the bacterial lysate supernatant
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appears to be more concentrated than the Sephacryl
pool. This is probably due to its hight content in
nucleic acid fragments (A260 ~5)- Note also the presence
of a very abundant band with an apparent molecular weight
of about 25,000 in lane d. This band appears only after
induction with IPTG and probably corresponds to over-
produced ~ -lactamase.
Figure 6 shows the N-terminal sequence of
recombinant UG.
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Figure 7 shows a schematic diagram of pLD101,
below which is shown the sequence of pLD101 between the
Bam HI site and the Hlnd III site.
Figure 8 shows the dose respon~e of natur~l and
recombinant UG as inhibitors of porcine pancreatic
PLA2. Each point represents the average of three determ-
inations, each performed in duplicate.
DETAILED DESCRIPTION OF THE INVENTION
The above and various other ob~ects and advan-
tages of the present invention are achieved by a plasmidcomprising circular, double stranded DNA of a molecular
length of about 4406 base pairs containing at least a
gene for ampicillin-re~istance, 10 T7 promoter, ribo-
somal binding site and three cloning sites, NcoI, PstI
and HindIII, wherein a cDNA for protein to be expre~sed
is inserted at NcoI, PstI or HindIII site. Preferred
plasmids are selected from the group consisting of
pLE101, pLE102 and pLE103-1. The plasmid of the present
invention is distinguishable from any other plasmid by
the following features. pLE 101 and 102 contain UG cod-
ing region under the control of ~trc~ promoter, whereas
pLE 103-1 in addition contains (i) a T7 promoter; (ii)
the T7 gene 10, 5'nontranslated region, the UG coding
sequence and the cloning sites NcoI, PstI and Hind III.
To our knowledge, correct intracellular formation of
multimeric structures containing more than one interchain
disulfide bridge has not been reported so far.
The three plasmids (pLE101, pLE102 and pLE103-
1) are able to direct expression of recombinant rabbit
uteroglobin (UG), a homodimeric protein with two inter-
chain disulfide bridges, in E. coli. Among these, the
plasmid pLE103-1, in which the expression of recombinant
UG is controlled by a bacteriophage T7 late promoter, is
by far the most efficient. With pLE103-1, recombinant UG
production reached about 10% of total bacterial soluble
proteins. This protein accumulated in bacterial cells in
dimeric form, as it is naturally found in the rabbit
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uterus. Recombinant UG was purified to near-homogeneity
and its N-terminal amino acid ~equence wa~ confirmed to
be identical to that of its natural counterpart, except
for 2 Ala residues the codons of which were added during
the plasmid construction. This protein was found to be
as active a phospholipa~e A2 inhibitor as natural UG on a
molar basis. ~he plasmid pLE103-1 may be useful to
explore the structure-function relationship of rabbit
uteroglobin. In addition, thi~ plasmid may be useful in
obtaining high level bacterial expression of other
eukaryotic proteins with quaternary structure, as well a~
for other general applications requiring efficient bac-
terial expre~sion of cDNAs.
Blastokinin or UG is a low molecular weight
secretory protein which is found in several organs of the
rabbit. The synthesis and secretion of UG are regulated
by different steroid hormones in different organs. This
protein has several biological properties, which include
immunomodulatory effects, antiiflammatory properties and
an inhibitory activity on platelet aggregation. UG is
thought to play an immunomodulatory/antiinflammatory role
in protecting the wet epithelia of organs which communi-
cate with the external environment. In particular, UG
has been proposed to protect the rabbit embryo from
maternal immunological assault during implantation. A
uteroglobin-like protein has been recently detected in
the human uterus, respiratory tract and the prostate. At
least some of the biological effects of UG may stem from
the phospholipase A2 (PLA2, EC 3.1.1.4) inhibitory pro-
perties of this protein. Because of its PLA2 inhibitoryeffect, UG can prevent liberation of arachidonic acid
from membrane phospholipids, which is the first step of
the arachidonate cascade, leading to the synthesis of
various eicosanoids, some of which are well known media-
tors of inflammation. UG is a homodimeric protein,formed by identical subunits of 70 amino acids each,
joined in antiparallel orientation by two disulfide
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bridges.
A nonapeptide in ~ -helix 3 of UG which may be
the active site, or at lea~t part of an active ~ite, for
the PLA2 inhibitory activity of UG. A~ a prelude to
confirming this observation by site-directed mutagenesis,
a high level bacterial expression of this protein was
obtained. However, the structure of the protein posed a
unique problem, since to our knowledge bacterial expres-
sion of multimeric eukaryotic proteins with two inter-
chain disulfide bridges in their natural form has not
been reported so far. Using plasmid pLE103-1, a high
level expression of recombinant UG (about 9-11% of total
bacterial soluble proteins) was obtained. In this
plasmid, the transcription of UG cDNA is controlled by
the ~10 late promoter of bacteriophage T7. Recombinant
UG in its natural dimeric form is synthesized by E. coli
cells harboring pLE103-1, with no apparent intracellular
accumulation of free subunits. The recombinant protein
was purified to near-homogeneity and its N-terminal amino
acid sequence was found to be identical to that of its
natural counterpart, except for 2 Ala residues the codons
of which were added during the plasmid construction.
Recombinant UG was found to have an identical PLA2
inhibitory activity as that of the natural protein.
Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art
to which this invention belongs. Although any methods
and materials similar or equivalent to those described
herein can be used in the practice or testing of the pre-
sent invention, the preferred methods and materials are
now described. All publications mentioned hereunder are
incorporated herein by reference. Unless mentioned
otherwise, the techniques employed herein are standard
methodologies well known to one of ordinary skill in the
art.
The term "high level" or "high efficiency," as
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used herein, means about a 1000 fold or more synthe~is of
the protein by the plasmid system of the present inven-
tion in an _. coli expression vector, compared to the ATG
vector pKK-233-2.
The term "artificial operon," as used herein,
means a group of foreign gene~, controlled by a common
regulatory sequence, inserted into a plaqmid for the
expression of a quarternary protein.
The term "quarternary proteinn as used herein
means a protein formed by more than one subunit, such as
heterodimeric, homodimeric or multimeric proteins.
The principles and methodologies of the present
invention are now described.
The genetic material of most organisms consists
of DNA. In order to synthesize proteins one helix of DNA
is transcribed" into a single stranded RNA (mRNA) by an
enzyme (RNA polymerase). The mRNA is then "translated
into a protein by intracellular organelles (ribosomes)
which synthesize a polypeptide chain (a chain of amino
acids linked to each other by a peptide bond) according
to the sequence specified by the sequence of codons
(groups of three nucleotides) of the mRNA. The basic
mechanisms of these processes are similar in all orga-
nisms. However, in eukaryotic cells (cells with distinct
nuclear envelope, such as cells of animal and plant
origin, the protozoans and unicellular fungi) the process
is much slower and more complex than in prokaryotic cells
(e.g. bacteria and blue-green algae). In prokaryotes
transcription and translation are simultaneous and
3G coupled, and both processes are much faster than in
eukaryotes. Moreover, growing prokaryotes in a con-
trolled laboratory environment is usually much cheaper
than maintaining eukaryotic cells in culture, because of
the simpler nutrients required and a much higher rate of
growth in prokaryotes.
The Gram-negative bacterium E. coli is by far
the best characterized organism from the molecular and
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genetic point of view, and genetic manipulations in this
organism are relatively easy. For these reason~, several
systems have been developed to produce high quantitie~ of
eukaryotic proteins in Escherichia coli (E. coli) both
for scientific and industrial applications (see for exam-
ple Rosenberg et al, suPra; Crow et al, Gene, 38, 31-38,
1985; Amann et al, supra; Mandecki et al, Gene, 43, 131-
138, 1986). These systems utilize plasmid vectors.
A plasmid is a circular double stranded DNA
molecule which exists intracellularly ~mostly in bacteria
but also found in eukaryotes like the yeast) and repli-
cates independently from the host chromosome. Biologic-
ally, a plasmid does not belongU to the host cell, but
it is rather an endosymbiotic entity. Usually plasmids
encode functions which are useful to the host cell.
Typically, the plasmids used in experimental procedures
contain genes which confer to the host resistance to one
or more class of antibiotics. By growing plasmid-harbor-
ing bacteria in antibiotic-containing medium, an inves-
tigator can ensure that virtually every cell in theculture contains the plasmid which confers antibiotic-
resistance to the host.
Since the massive production of a foreign pro-
tein usually kills the bacterial host, or at least slows
down its growth, most protein-expressing plasmids, con-
structed so far, contain mechanismsi of regulation. These
plasmids are engineered to contain a very active promoter
(i.e. a DNA region with very high affinity for RNA polym-
erase) and a biochemical mechanism is incorporated which
keeps the promoter from being activated until an exogen-
ous Ninducing" stimulus is given. The coding sequence
for the foreign protein of interest (typically a eukary-
otic cDNA) is inserted into the plasmid in a 3'-direction
from the promoter. This allows the bacteria to be grown
up to an appropriate density, and then induce the produc-
tion of the foreign protein by starting the transcription
of the plasmid promoter. An mRNA is produced from the
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plasmid DNA in a 3'-position of the promoter, up to the
next transcriptional terminator site. At the same time,
the mRNA, which includes the coding sequence for the
foreign protein, is translated by bacterial ribosomes
into a protein.
Demonstrated herein is the construction of a
highly efficient novel plasmid for bacterial expression
of an anti-inflammatory protein, uteroglobin (UG). This
protein ~for review see Niele et al, Endocrine Rev. 8,
474-494, 1987) has several important biological proper-
ties which include immunomodulatory effects, anti-inflam-
matory properties and platelet-antiaggregating activity.
UG is a secretory homodimeric protein (i.e.
formed by two identical monomers). It is significant to
note that the dimeric nature of UG posed a peculiar prob-
lem for bacterial expression, since no other proteins
with quarternary structure (i.e. formed by more than one
subunit) had heretofore been expressed in their natural
form in bacteria. The present invention is the first
successful demonstration of the synthesis of a quar-
ternary protein in its natural form by a plasmid in a
bacterial system (E. coli). The novel plasmid directing
the expression of UG is designated herein as pLE103-1.
NETHODS OF CONSTRUCTION OF PLASMID VECTORS (pLE103-1,
pLE101 AND pLE102) AND EXPRESSION OF UG IN E. COLI
The construction of pLE103-1 is described in
Figs. lA and lB. Plasmid pUG617 was a gift from Dr.
David Bullock (Lincoln College, Canterbury, New
~ Zealand). Plasmid pKK233-2 was kindly provided by Dr. J.
Brosius (Columbia University, NY). E. coli strain tJM105
was purchased from Pharmacia and strain JM109 was a gift
from Dr. J. Messing (University of Minnesota). E. coli
Strain BL21(DE3) was generously provided by Dr. W.
Studier (Brookhave National Laboratory, NY). "Library-
efficient" competent E. coli strain HB101 was purchased
from BRL. All recombinant DNA manipulations were per-
formed according to standard techniques. Restriction
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enzymes, T4 DNA polymerase, T4 DNA liga~e and E. coli DNA
polymerase I large fragment were obtained from either
Pharmacia or BRL. All other reagents were ultra pure
grade from BRL.
First, a 430 bp fragment containing the entire
UG coding ~equence was excised from pUG617 (Chandra et
al, DNA 1:19-26, 1981) by means of digestion with the
restriction enzyme PstI. Digestions with restriction
endonucleases were performed according to the instruc-
tions of the manufacturer (New England Biolab~, Pharmacia
or Bethesda Re~earch Laboratories). pUG617 was a
generous gift from Dr. D. Bullock. This fragment does
not contain the coding sequence for the UG "signal" pep-
tide, i.e. the N-terminal fragment, 21 amino acids long,
which is eliminated from mature UG in eucaryotic cells.
The 430 bp fragment was purified by agarose gel
electrophoresis (Maniatis et al, In Molecular Clonina, a
LaboratorY Manual, Cold Spring Harbor Laboratory, 1982)
and divided into two aliquots: one was kept intact, and
the other was treated with phage T4 DNA polymerase
(Maniatis et al, suPra). This treatment eliminated the
protruding ends left by PstI digestion, producing a 422
bp blunt-ended fragment. The intact fragment was coval-
ently joined by means of phage T4 DNA ligase to PstI-
digested pKR233-2 (Amann et al, Gene 40:183-190, 1985)
purchased from Pharmacia. T4 DNA ligase and 5X T4 DNA
ligase buffer were purchased from Bethesda Research
Laboratories. The ligation reaction contained 0.1 units
of DNA ligase, 4 ul of 5X ligase buffer, 100 ng of frag-
ment and 50 ng of plasmid in a total volume of 20 ul.
The reaction was carried out at 12C for 6 hrs. The
ligated DNA was diluted 1:5 with TE buffer (Maniatis et
al, supra) and 5 ul were used to transform E. coli strain
JM105 (Yanisch-Perron et al, G 33, 103-119, 1985) and
several recombinant clones were obtained. Transformation
and plating of bacteria were carried out as described by
Maniatis et al, supra. Small-scale preparations of
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plasmids of these clones were performed according to
Birnboim et al (Nucleic Acid Res. 7, 1513-1523, 1979),
and the correct orientation of the in~ert DNA wa~ checked
by dige~tion with appropriate re~triction endonuclea~e~
(e.g. AvaI and HindIII). Clones containing the UG coding
region in the appropriate orientation were designated
pLE101.
For the construction of pLE102, the Pst I-
digested ends of the 430 bp DNA fragment were made blunt
by treatment with bacteriophage T4 DNA polymerase.
Plasmid pXK233-2 was digested with Nco I and the cohesive
ends were made blunt by treatment with E. coli DNA poly-
merase I "large fragment". Direct ligation of blunt-
ended UG cDNA fragment into Nco I-digested, blunt-ended
pKK233-2 generated pLE102. The orientation of the insert
was checked by digestion with Ava I and HindIII, and the
expected reconstitution of two Nco I ~ites at both ends
of the insert was verified.
The difference between pLE101 and pLE102 is
that pLE101 three codons (Met-Ala-Ala) are added to the
5' terminus of the codinq sequence of UG DNA, while in
pLE102 only one codon (Net) is added to the 5' ter-
minus. Therefore, pLE101 directs the production of a
recombinant UG having three additional amino acids at the
N-terminus (Met-Ala-Ala) and pLE102 directs the produc-
; tion of a recombinant UG having only one additional amino
acid (Met) at the N-terminus. The presence of a Met
codon at the 5' end of the coding sequence is indispens-
able for translation of the mRNA in both prokaryotic and
eukaryotic cells. In this case, the Met codon is present
in the NcoI site of the vector.
The plasmid pKK233-2 is an expression vector,
i.e., a plasmid capable of producing foreign proteins in
E. coli, provided that the coding sequence for such pro-
teins is inserted in the appropriate restriction sites inthe vector. The expression of foreign proteins in
pKX233-2 is controlled by the artificial utrc" promoter,
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a regulatory region consisting of part of the E. coli
"trp" promoter and part of the E. coli "l~c" promoter.
The "trc" promoter i9 regulated by the lactose operon
repressor protein ("lac repres~or"). When plasmids
carrying the "trc" promoter are introduced into an E.
coli strain which overproduces the lac represqor (lacI
genotype), the expression of the foreign gene is sup-
pressed. Upon addition of a gratuitous inducer (i.e. a
molecule that binds to the lac repressor and inactivates
it, without being metabolized) such as isopropyl-~-thio-
galactopyranoside (IPTG), the lac" repressor is inactiv-
ated and the foreign gene is transcribed by the E. coli
RNA polymerase. ~he resulting mRNA is then translated to
produce the recombinant protein (e.d. UG).
pLE101 and 102, being derivatives of pKK233-2
express the two forms of recombinant UG upon induction
with IPTG. The two plasmids were tested for expression
using E. coli strainY JM105 and JM109 as hosts. Upon
induction with IPTG both plasmids expressed recombinant
UG detectable by RIA in the bacterial lysate super-
natant. The best results were obtained with JM 109 when
bacteria were grown in M9 medium supplemented with 0.001%
thiamine and expression was induced with IPTG early dur-
ing logarithmic growth, i.e. before the optical density
of the culture at 600 nm (OD600) reached 0.4. Under
these conditions, pLE101 expressed about 300 ng UG/ml
supernatant and pLE102 expressed about 120 ng/ml.
Expression of recombinant UG was tested essen-
tially as described by Amann and Brosius for pLE101 and
102 and by Rosenberg et al. for pLE103-1. Expression
experiments were always performed with two identical
cultures, one of which was induced with isopropyl-~
-thiogalactopyranoside (IPTG, Calbiochem) early during
logarithmic growth. Samples from the cultures were with-
drawn before induction and at various times after induc-
tion. Aliquots of the samples were centrifuged in an
Eppendorf microcentrifuge and the bacterial pellets were
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- 14 -
lysed directly in SDS-polyacrylamide gel sample buffer
for electrophoretic analysi~. The remainder of the
samples were centrifuged st 10,000 x g for 10 min. The
bacterial pellets were washed with pho~phate-buffered
saline (PBS, Quality Biologicals) and recentrifuged at
10,000 x g for 10 min. The pellets were resuspended in
buffer L, which consists of 50 mM Tris-HC1 pH 8, 5 mM
EDTA containing 4% glycerol, 250 ~M phenylmethylsulfonyl-
fluoride (Sigma), 0.7 ~g/ml pepstatin A (Calbiochem) and
0.5 ~g/ml leupeptin (Calbiochem). The samples were
flash-frozen in liquid N2, thawed on ice and sonicated
for 20 sec (Heat Systems-Ultrasonics sonicator, setting
4, continuous). Bacterial lysates were centrifuged at
30,000 x g and supernatants were transfered to clean
polypropylene tubes while pellets were resuspended in
buffer L. Aliquots from supernatants and from
resuspended pellets were assayed for UG by RIA.
Purification of recombinant UG was accomplished
as follows. Eight hundred ml of M9 medium (Quality
Biologicals, Gaithersburg, MD) containing 200 g/ml
ampicillin and supplemented with 0.001% thiamine were
inoculated with 2 ml of a saturated culture of
BL21(DE3):pLE103-1 grown in ~he same medium. UG expres-
sion was induced early during logarithmic growth with
IPTG at a final concentration of 0.45 mM. One hundred
minutes after induction, the bacteria were harvested by
-~ centrifugation at 10,000 x g for 10 min. Bacterial
pellets were flash-frozen in liquid N2 in the centrifuge
, bottles, and thawed on ice. The pellets were resuspended
in a total of 10 ml of ice-cold buffer L, and lysed by
three cycles of sonication on ice (1 min each, setting
4.5, continuous).
Recombinant UG was purified from E. coli-lysate
by a modification of the original method published by
Nieto et al. for rabbit UG. Briefly, the bacterial
lysate was centrifuged at 30,000 x g for 30 min. The
suy rnatant ~~ 9 ml) was loaùed on a column of Sephacryl~
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S200 superfine (Pharmacia), 2.5 x 100 cm, equilibrated in
buffer L. The fractlons were assayed for UG by RIA, and
UG-containing fractions were pooled, dialyzed ag~inst
distilled H2O and lyophilized. The lyophilized material
was resuspended in 12 ml of 25 mN ammonium acetate
buffer, pH 4.2 and centrifuged at 27,000 x g. The super-
natant was loaded on a CN-Sepharose Fast Flow column
(Pharmacia), bed volume 10 ml, equilibrated with 25 mM
ammonium acetate, pH 4.2. The column was washed with 3
bed volumes of the same buffer, and eluted with a linear
gradient made of 100 ml of 25 mM ammonium acetate, pH 4.2
and 100 ml of 120 mM ammonium acetate, pH 6Ø UG con-
taining fractions (as determined by RIA) were pooled and
lyophilized. The lyophilized material was resuspended in
2 ml of 10 mM ammonium bicarbonate buffer, pH 8.0, and
loaded onto a column of Sephadex G50 superfine (1.5 x 70
cm) equilibrated in the same buffer. UG containing frac-
tions were pooled and lyophilized. Recombinant UG was
stored in lyophilized form at -70C with dessicant. The
concentration of purified recombinant and natural UG was
estimated spectrophotometrically, using an ~N value of
1800, as published by Nieto et al. Protein concentration
of complex mixtures was determined according to Bradford
by means of a kit from Biorad. SDS-polyacrylamide gel 25 electrophoresis was performed according to Laemmli and
silver ~taining was performed by means of a kit from
Biorad according to the manufacturer's instructions.
The N-terminal sequence of recombinant UG was
determined by an ABI 477A gas phase sequenator following
standard protocols for Edman degradation and analysis of
phenylthiohydantoin derivatives. The sample (0.6 mg
total) was divided into two aliquots. One aliquot was
reduced with dithiothreitol under denaturing conditions
and the Cys residues were pyridylethylated before
sequence analysis. The other aliquot was processed with-
out pretreatment.
Phospholipase A2 assay was performed as previ-
Z~O(~fi1~
-
- 16 -
ously described, with modifications. Briefly, the reac-
tion mixture contained 100 mM Tri~-HCl, pH 8, 100 mM
NaC1, 1 mM Na-deoxycholate, 10 ~M 2-~ 4C~-arachidonyl
phosphatidylcholine (Amersham, 58 mCi/mmole) and 2 nM
porcine pancreatic PLA2 (Sigma) in a total volume of 50
~1. PLA2 was preincubated with either recombinant or
natural UG at 37C for 5 min and the enzymatic reaction
was started by addition of aliquots of the preincubation
mixture to the radioactive substrate. Controls were kept
in which PLA2 was preincubated with buffer only. PLA2
reaction was run at 37C for 30 sec. and stopped by addi-
tion of 50 ~1 of chloroform/methanol 2:1, followed by
50 ~1 of chloroform and 50 ~ 1 of 4 N RCl. Radioactive
arachidonic acid was separated from unhydrolyzed sub-
strate by thin layer chromatography on silica plates(silica gel G, prechanneled, Analtech). The eluent was
petroleum ether/diethyl ether/acetic acid 70:30:1.
Iodine-stained bands comigrating with the arachidonic
acid standard were scraped and counted in a Beckman LS-
9000 liquid scintillation counter.
The maximum level of expression reached withboth pLE101 and 102 was about 2 ng/ml of bacterial
culture, measured by radioimmunoassay with a monospecific
goat anti-UG antiserum. Radioimmunoassay (RIA) for UG
was performed as previously described. Immunoblots
(NWestern~ blots) were performed according to Burnette
using Nitroscreen West membranes (New England Nuclear).
Blots were stained with a goat-anti UG antibody and a
rabbit anti-goat Immunogold-Silver Staining (IGSS) kit
(Janssen). This maximum level was reached in host strain
JN109 (Yanisch-Perron et al, supra) in Luria-Bertani (LB)
broth (Maniatis et al, supra). These results suggested
that immunoreactive UG can be produced in E. coli, and it
is not toxic to the bacterial host at the concentration
reached. The presence of 1 instead of 3 residues at the
N-terminus of the recombinant protein did not modify the
level of expression. Moreover, the level of expression
:' '"
Z~ fiF;~
was not affected by the use of different culture media,
such as M9 minimal medium, supplemented wlth Ca~amino
acids (Difco) or with a mixture of 20 pure amino acid~.
The recombinant proteinis produced by pLE101 and
102 were soluble, being recovered almost totally in the
supernatant after centriguation of the bacterial lysate
at 30,000 x g. The relatively low level of expre~sion
obtained by these plasmids is most probably explained by
the peculiar problem posed by the expression of a dimeric
protein in E. coli. UG i8 a dimeric protein, being
formed by two identical subunits ~oined by two disulfide
bridges. Without being bound to any specific theory, it
is postulated that the intracellular concentration of UG
reached in E. coli depended on the following variables:
i) rate of transcription of the UG gene; ii) rate of
translation of the transcribed mRNA; iii) rate of degrad-
ation of the intracellular protein; and iv) the equi-
librium of dimerization of the intracellular UG monomers.
The dimerization of UG monomers can be
described as UGm + UGm -> UG, where UGm is UG monomer.
The equilibrium constant of this proceqs is Req
UG]itUGm]2. Therefore tUG], i.e. the intracellular
molar concentration of UG, is = Keqx lUGm]2. In other
words, the amount of UG being produced depends on the
second power of the intracellular concentration of UGm.
If it is assumed that the isolated UG monomer, being much
more unstable in solution than dimeric UG, has a much
shorter half-life in the E. coli, the dimerization of
isolated monomers becomes a rate limiting step in the
expression of recombinant UG.
As has been mentioned supra, dimerization is a
second-order process, depending on the squared concen-
tration of UGm. In practice, this means that in order to
obtain efficient production of UG, the intracellular
level of UGm must be kept high enough to allow efficient
dimerization of UGm, because a small decrease in tUGm]
would cause a dramatic decrease in [UG]. This can be
. .,, ,, , ~ . ~ , .
~)n~
- 18 -
obtained by maximizing the rate of transcription and
translation of UGm.
To accomplish this, the regulatory sequence~ of
pLE101 were replaced with synthetic regulatory sequences
identical to those of bacteriophage T7. Such promoters
have been previously shown to direct high level expres-
sion of recombinant proteins in E. coli. Fig. lb shows
the construction of pLE103-1 from pLE101. In pLE103-1
the regulatory sequences originally present in pKK233-2
(lac operator, trc promoter and ribosome binding site)
have been substituted with the synthetic DNA fragment
whose sequence is shown in Figure lB. The synthetic DNA
fragment contained the 010 late promoter of bacteriophage
T7, the 5' non-translated region of T7 gene 10 and the
ribosome binding site from the same gene. The sequence
of this synthetic regulatory region was derived from the
wild-type sequence which has been used by Studier and
coworkers in their Utranslation vectors, with the excep-
tion that the 2 bases preceding the initiation ATG
triplet were substituted with two cytosines. This sub-
stitution was made in order to create an Nco I site
including the initiation triplet. Additionally, a BamHI
site was added at the 5' end. The plasmid obtained in
this way has the same cloning sites, and should have the
same possible applications, of pKK233-2 and related "ATG
vectors", except that expression of the recombinant pro-
tein is controlled by a more specific and very efficient
viral promoter.
The regulatory sequences in pLE101 are derived
from pKK233-3 and consist of the "trc" promoter followed
by an artificial Nribosome binding site" (RBS~. The
latter sequence is thought to control the association of
a bacterial mRNA to the 30S subunit of ribosomes, thereby
regulating the rate of translation of the mRNA. The
regulatory sequences in pLE101 ~as in pKK233-2) are con-
tained in a 285 bp BamHI-NcoI fragment. This fragment
was excised from pLE101 ~Fig. lB) by means of digestion
Z~n~ fi~
-- 19 --
with BamHI and NcoI restriction endonucleases. The
remaining portion of the plasmid was purified by electro-
phoresis on an agarose gel and covalently ~oined to a
completely synthetic 89 bp BamHI-NcoI DNA fragment. The
latter reproduced the sequence of the 10 promoter of
phage T7 (Studier et al, J. Mol. Biol. 189:113-130, 1986;
Rosenberg et al, Gene 56:125-135, 1987), followed by the
5' nontranslated region, including the RBS, of phage T7
gene 10. The synthetic regulatory region ends with an
initiation codon (ATG) contained in the NcoI site. The
recombinant plasmid obtained in this way was designated
pLEl03-1, and it contains the same cloning sites as
pKR233-2, but now under the control of the synthetic T7
Ngene 10-like regulatory region.
Phage T7 promoters are usually not transcribed
by E. coli RNA polymerase, but only by T7 RNA polymerase,
which is highly specific for T7 promoters and does not
recognize E. coli promoters. Therefore, unless an active
T7 RNA polymerase is delivered into the bacterial host,
the basal level of transcription of a foreign gene under
the control of a T7 promoter is negligible. However, T7
RNA polymerase initiates transcription with very high
efficiency and it elongates RNA 5-times faster than E.
coli polymerase. T7 RNA polymerase produces from
plasmids such as pLE103-1 longer transcripts than E. coli
RNA polymerase, which stops at the rrnB terminators
present in pKK233-2 (Fig. l). The length of the RNAs
transcribed by T7 RNA polymerase has been suggested to
protect them from intracellular exonucleolytic degrada-
tion starting from the 3' end, thereby increasing thehalf-life of these RNAs in E. coli.
This means that when an active T7 RNA poly-
merase and a T7 promoter are concomitantly present in an
E. coli cell, transcription from the viral promoter will
successfully inhibit transcription from the host promo-
ters, by limiting the availability of nucleotide triphos-
phates for transcription of E. coli genes. When the T7
Z~ fi~
.
- 20 -
promoter controls the expression of a foreign gene, this
generally leads to a very high accumulation of expre~ed
protein, and eventually to the death of the bacterial
host. An expression vector carrying a T7 promoter, pro-
vided that it is stable in E. coli, usually leads to ahigh expression of proteins which are not toxic for the
bacterial host.
Possible ways of delivering an active T7 RNA
polymerase into the bacterial host are: ~a) by infection
with T7 phage; (b) by infection with a recombinant lambda
phage containing the gene of T7 RNA polymerase by use of
a bacterial host 8uch as BL21(DE3). This strain of E.
coli i8 lysogenic for a lambda phage containing the T7
gene for RNA polymerase. In other words, the chromosome
of this strain of E. coli contains the entire genome of a
lambda phage which in turn has been engineered to contain
the gene for T7 RNA polymerase, cloned under the control
of a ~lac W" E. coli promoter, also artificially inserted
into the lambda genome. The "lac W" promoter, like the
Nlac" promoter, is regulated by a repressor protein which
is inactivated by IPTG.
When BL21(DE3) is exposed to IPTG, it produces
T7 RNA polymerase. If at the same time the ~train con~
tains a plasmid carrying an active T7 promoter, the T7
RNA polymerase produced transcribes any gene cloned down-
stream, i.e. in 3' direction, with respect to the T7
promoter. This is a very convenient system to induce the
expression of any nontoxic protein from plasmids carrying
IT7 promoters. When applicable, the use of BL21(DE3) is
preferable to the other methods of induction, because
infection with T7 causes competition of viral promoters
with the vector promoter, and infection with a recom-
binant lambda phage causes lysis of the bacteria.
Expression of toxic proteins in BL21(DE3) needs addi-
tional measures, such as the introduction in the host ofa T7 lysozyme gene, whose product inhibits the basal
levels of T7 RNA polymerase produced by the lysogenic
`:
-
znn(~fi~
- 21 -
lambda. This provides a mechani~m to keep the expre~sion
of a toxic protein completely repres~ed, 00 that not even
trace amount~ of it are produced until the moment of
induction. Alternatively, methods (a) and (b) vide
su~ra, can be used to induce the production of a toxic
protein in E. coli.
In accordance with the present invention, plas-
mid pLE103-1 was used in BL21(DE3) to express UG, becau~e
it was found that low levels of UG are not toxic for E.
coli as demonstrated by the data obtained with pLelOl and
102. In this strain, phage T7 RNA polymerase is produced
upon induction with IPTG from a recombinant lambda phage
which is integrated into the bacterial chromosome.
BL21(DE3):pLE103-1 expresses recombinant UG upon induc-
tion with IPTG, and the recombinant protein is readilydetectable by polyacrylamide gel electrophoresis.
Fig. 2A clearly shows the time-dependent
appearance in induced bacteria of a protein band of
apparent molecular weight corresponding to that of mature
rabbit UG monomer. The difference in molecular weight
due to the presence of the expected additional three
residues in the recombinant protein is not apparent under
these conditions. The lower molecular weight band
appearing immediately below the putative recombinant UG
band (Fig. 2A) may be a product of partial degradation of
recombinant UG, or an artifact caused by the formation of
an intramolecular disulfide bridge in UG during SDS-
polyacrylamide gel electrophoresis. The appearance of a
pure UG as a "doublet" band due to such an artifact has
been described by Nieto et al. The identity of the
recombinant protein was confirmed by Western blot. That
the new band appearing upon induction is recognized by
anti-UG antibody is shown in Fig. 2B. Interestingly,
both in the control sample of rabbit UG and in the recom-
binant material an immunoreactive band with apparentmolecular weight of about 13,000 is present (Fig. 2B).
The presence of this band is due to incomplete reduction
r--
znn~fi~
- 22 -
of disulf~de bonds between UG subunlts under the condl-
tions used for sample preparatlon and it con~i~tently
appears when concentrated samples of pure rabbit UG aro
sub~ected to SDS-polyacrylamide gel electrophoresis
followed by immunoblot. This observation gave UB a pre-
liminary indication that the recombinant protein may be
present in the bacteria in dimeric form.
The optimal expression of recombinant UG with
pLE103-1 was obtained with a high concentration of ampi-
cillin in the culture medium (200 g/ml) and a very smallinitial inoculum ~aliquots from a saturated culture were
diluted 400-fold in fresh medium). These conditions were
optimal also for pLE101 and pLE102. It is well known
that saturated cultures of bacteria which harbor plasmids
derived from pBR322 (such as the vectors described in
this paper) contain large amounts of -lactamase. There-
fore, unless the plasmid is extremely stable in the host,
it is essential to maintain a high selective pressure in
order to avoid any growth of plasmid-free bacteria.
Moreover, pLE103-1 contains the bla gene in trascrip-
tional orientation with respect to the T7 promoter.
Thus, induction with IPTG will result in transcription of
a polycistronic mRNA containing the bla coding sequence,
and in overexpression of ~ _lactamase. In fact, T7 RNA
polymerase does not recognize E. coli trancriptional
terminators, such as the Tl and T2 rrnB terminators
present in pLE103-1 (sée Fig. lB). Under appropriate
electrophoretic conditions, we have indeed observed an
overexpression of a protein band of molecular weight
corresponding to that of -lactamase (data not shown).
Table 1 shows the results of an expression
experiment using pLE103-1. The production of UG, as
measured by radioimmunoassay in supernatants of bacterial
lysates, reached 1.9 ug/ml of bacterial culture, or 7.7
ug/mg protein, i.e. about 1000-fold more than the levels
obtained with pLE101 and 102, 3 hours after induction.
This amount of protein synthesis is within the range of
r
-
Z(~ fiF~
- 23 -
preparative scale production, corresponding to approxi-
mately 670 ug/g bacteria. Thus, using p~E103-1 a much
higher level of expression of UG in BL21(DE3) wa8
obtained compared to pLE101 or 102 in JM109.
Fig. 3 shows the quantitation of recombinant
UG, as determined by RIA in supernatants from bacterial
ly~ates. The three plasmids pLE101, pLE102 and pLE103-1
were compared under identical experimental conditions,
except for the host strains, i.e. JM109 for pLE101 and
pLE102, and BL21(DE3) for pLE103-1. It is clear that
with pLE103-1 production of recombinant immunoreactive UG
is much higher (about S0-fold more than with pLE101 and
100-fold higher than with pLE102). With pLE103-1, the
highest absolute concentration of recombinant immunoreac-
tive material was reached 120 min after induction (Fig.
3A). However, when the amount of recombinant UG was
expressed as ~g/mg protein, the maximum level of UG was
reached 90 min after induction (Fig. 3B). More than 99~ r
of immunoreactive UG expressed was recovered in the
supernatant after centrifugation at 30,000 x g. This
indicates that the recombinant protein is soluble.
The molecular weight of the recombinant protein
obtained in accordance with the present invention was
then determined both by polyacrylamide gel electro-
phoresis under denaturing conditions and by size exclu-
sion chromatography under non-denaturing conditions.
Fig. 4 shows the determination of molecular
weight of recombinant UG by size exclusion chromatography
j under nondenaturing conditions. It is evident that
recombinant and natural UG have an identical chromato-
graphic behaviour. Under these conditions, both proteins
have an apparent molecular weight of 17,000, slightly
higher than the theoretical value of 15,800. This is in
agreement with the results of Nieto et al. on purified
rabbit UG. No immunoreactive peak indicating the
presence of isolated UG subunits was observed, although
UG subunits are readily recognized by our antibody in
. ,~
~ fip~
- 24 -
Western blots. These results seem to indicate that in
lysates of induced BL21(DE3):pLE103-1 recombinant UG
exists almost solely in itR natural dimeric ~orm.
Fig. 4 shows that the recombinant protein pro-
duced in E. coli has the same apparent mw (17 kd) as thatof natural UG purified from the rabbit uterus. This mw
is slightly higher than the calculated value of 15.8
kd. Again, this is in agreement with former data on the
chromatographic behavior of native dimeric UG (Nieto et
al, suPra). The three additional amino acids present in
recombinant UG do not affect the chromatographhic proper-
ties of the protein enough to alter its apparent mw. No
peak in a position corresponding to UG monomer was
evident in the chromatogram of bacterial extract.
These results indicate that: (i) sbundant
quantities of dimeric UG can be produced in E. doli by
pLE103-1; (ii) the yield of UG expression obtained with
pLE103-1 is about three orders of magnitude higher than
those obtained with pLE101 and 102; (iii) virtually all
the recombinant protein produced by pLE103-1 is in its
natural dimeric form. This indicates that dimeric pro-
teins can be efficiently expressed in E. coli under the
control of a T7 promoter, to a much higher efficiency
than with vectors based on prior art modified E. coli
promoters. This is then the first time that a eukaryotic
protein with quarternary structure has been efficiently
expressed in its natural form in prokaryotes. ~-
Gel electrophoresis (Fig. 5) showed that upon
induction with IPTG (final concentration 0.4 mM) two new
protein bands appear with apparent molecular weight (mw)
between 5000 and 6000. This corresponds to the electro-
phoretic behavior of the natural UG monomer under these
conditions (Nieto et al, Arch. Biochem. BioPhys 180, 82-
92, 1977). It is postulated that the reducing conditions
of electrophoresis destroy the interchain disulfide
bridges of dimeric UG, showing only the monomer. It is -
known that monomeric UG gives electrophoretic artifacts,
~' '-
., ~. .
Z~n'~P~fi~
- 25 -
appearing as a double band (Nieto et al, suPra). Thi~ i~
presumably due to formation of intrachain di~ulfide bond~
during electrophoresi~. One additional band of higher
apparent mw i9 also induced (Fig. 5). Thi~ correspond~
to the apparent mw of ~ -lactamase (bla gene product),
whose gene lies downstream of the UG gene in pLE103-1.
This indicates that transcription from the T7 promoter
proceeds beyond the UG gene through the bla gene. This
is expected, since T7 RNA polymera~e does not stop tran-
~cription a E. coli transcriptional terminator~, and even
natural T7 terminators have a relatively low efficiency.
Despite the higher complexity of the protein
mixture present in E. coli lysates compared to rabbit
uterine flushings, the very large headstart allowed us to
obtain near-homogeneous recombinant UG by a modification
of the original procedure (Figure 5). The chromato-
graphic properties of recombinant UG in the columns used
for purification were indi~tinguishable from tho~e of the
natural protein. The final yield of the purification wa~
3.2 mg recombinant UG from 800 ml of induced bacteria, as
estimated by W absorption using the published value of
1800 for the ~M. The starting material (bacterial lysate
supernatant) contained a total of about 50 mg protein,
and approximately 5 mg of recombinant UG (data not
shown). Therefore, the final recovery of recombinant UG
can be estimated as about 64~.
y The N-terminal amino acid sequence of reduced
and non-reduced recombinant UG is shown in Figure 6.
Sequence a) represents non-reduced recombinant UG. In
this sequence, Cys 3 was detectable only after in situ
reduction and pyridylethylation in the sequenator
cartridge. Sequence b) represents a sample which was
reduced with a 100-fold molar excess of dithiotreitol in
8 M urea at 56C for 1 hour and Cys residues were
pyridylethylated prior to Edman degradation. Unnumbered
residues were added to the N-terminus as a consequence of
plasmid construction. Numbers indicate positions in the
zn~ fi~
- 26 -
sequence of rabbit UG.
The recombinant UG produced by BL2(DE3)spLE103-
1 is totally dimeric and no accumulation of free subunit~
could be detected. This might indicate that i) the rate
of association of the subunits is very high and/or ii)
free subunits are highly unstable and rapidly degraded in
the bacterial host. To our knowledge, this is the first
report of high level bacterial expression of a full-
length dimeric eukaryotic protein with two interchain
disulfide bridges in its natural quaternary structure.
Our results demonstrate that a recombinant protein can
form correct quaternary structures during overexpression
in E. coli even when correct formation of two interchain
disulfide bridges is essential for its structure, pro-
vided that the rate of intracellular accumulation of
subunits and the rate of association of free subunits arehigh enough.
Non-covalent self-association of recombinant
eukaryotic proteins in E. coli ha~ been described for
human tumor necrosis factor and rat liver aldehyde
dehydrogenase. However, in the first case most of the
recombinant protein appeared in the insoluble fraction
due to incorrect folding and in the second case the high
~` efficiency of expression was suggested to be due to
unique features of the 5' non-translated region of the
cDNA (which contained a potential prokaryotic Shine-
Dalgarno sequence) and its relationship with the lac
promoter present in pUC8.
! - In contrast, pLE103-1, the 5'-non translated
region and the Shine-Dalgarno sequence are built in the
vector, so that theoretically any open reading-frame
could be expressed in place of the UG cDNA. Until
recently, it was generally believed that the intracellu-
lar environment of E. coli is not conducive to the forma-
tion of quaternary structures which require formation ofinterchain disulfide bridges. Correct folding and assem-
bly of heterodimeric fragments of immunoglobulins after
,` zn~(P~
- 27 -
proteolytic processing of fusion precursors and eecretion
into the perlplasmic space of E. coll has been recently
described. In one case the sssembly lnvolved the forma-
tion of one interchain di~ulfide bridge. Our findinge
confirm and extend this observation further, demonstrat-
ing that: i) quaternary structures containing more than
one interchain disulfide bridge can also be properly
assembled in E. coli and ii) at least in the case of UG,
correct assembly of quaternary structure can take place
in the bacterial cytoplasm without the need for correct
proteolytic processing of a precursor protein and trans-
membrane transport of the product. It should be noted
that natural UG is a secretory protein which is synthe-
sized as a precursor that naturally undergoes transmem-
brane transport and proteolytic processing.
Our data indicate that assembly of quarternarystructure in E. coli does not necessarily require the
construction of a fusion precursor protein with a bacter-
ial secretion signal sequence. Such constructions can be
advantageous if secretion of correctly processed recombi-
nant protein in the medium is achieved, but require a
precise "in frame" fusion between the bacterial signal
sequence and the eukaryotic coding sequence. This may
require extensive manipulations on vector and/or insert
DNA.
All in all, our observations seem to suggest
that if a sufficiently high level of intracellular
accumulation of recombinant protein(s) i8 obtained, the
possibility of formation of multimeric structures involv~
ing disulfide bridges depends essentially on the phys-
ical-chemical factors controlling the folding of the
protein(s) and the interaction between subunits. Thus,
it may be-possible to obtain efficient bacterial expres-
sion of eukaryotic multimeric proteins other than UG,
provided that: i) the vector/host system used insures a
high efficiency of expression and intracellular accumula-
tion of the product(s); and ii) the tertiary and quarter-
fi~q
- 28 -
nary struc~ure of the recombinant protein(s) are thermo-
dynamically stable and the kinetics of folding and
assembly are not too slow. The latter conditions
obviously depend on the particular protein(s) being
expressed.
The results of Edman degradation experiments on
recombinant UG, together with the chromatographic and
electrophoretic data (see above) strongly support the
hypothesis that the dimeric structure of recombinant UG
is stabilized by two disulfide bridges identical to those
of natural UG. In theory, it is possible that recombi-
nant UG could form inverted" dimers in which the two
disulfide bridges ~oin Cys 3 and 3'; 69 and 69'. How-
ever, this possibility is made unlikely by the fact that
besides the two disulfide bridges, several other stereo-
specific intermolecular contacts (Van der Waals interac-
tions and H-bonds), contribute to the stabilization of
the UG dimer. Moreover, the identical properties of
recombinant and natural UG as PLA2 inhibitors further
support the hypothesis that the two proteins are struc-
turally identical, with the exception of the two addi-
tional Ala residues in recombinant UG.
With the vector/host system used in this study,
considerable overexpression of ~ -lactamase along with UG
was observed. Simultaneous overexpression of a recombi-
nant protein and ~ -lactamse has also been described with
other vectors based on T7 promoters. Therefore, these
systems are able to support overexpression of two dif-
ferent polypeptides and could be used for the construc-
tion of artificial operons to express heterodimericproteins. The vector pLE103-1 could be a useful addition
to the already existing expression plasmids based on T7
promoters. In fact, after excision of the UG coding
sequence from the Pst I site, pLE103-1 can be converted
into a general purpose expression vector, which we have
denominated pLD101. The cloning sites Nco I, Pst I and
HindIII give to pLE103-1 the same potential applications
:
, .
: : ~
;
Z(?~ fi~
- 29 -
of "ATG vectors a with the advantage of the high
ef~iciency and specificity of the T7 promoter. In
particular, the Nco I site (CCATGG) i9 frequently present
in eukaryotic translational ~tarts.
Moreover, after restriction endonuclea~e diges-
tion the Nco I site can be easily "filled inn with E.
coli DNA polymerase I large fragment. This process
reconstitutes the ATG triplet, thereby allowing "blunt"-
ended DNAs to be cloned in-frame directly into the
"filled-in" NcoI site. In addition, the presence of the
Pst I and HindIII sites allows Nforced" or "directional"
cloning. Finally, the presence of the Pst I site, and
the relative positions of the three cloning sites allow
this plasmid to be used for the construction of cDNA
libraries by several different methods.
The data obtained on the biochemical properties
of recombinant UG have already yielded some valuable
information on the structure/function relationship of
this protein. Since the recombinant protein appears to
fold correctly in E. coli, the presence of the "leader
peptide" which is physiologically present in rabbit preUG
is not necessary for the folding of UG during transla-
tion. Furthermore, the addition of two Ala residues at
the N-terminus of recombinant UG does not affect its
activity as a PLA2 inhibitor.
Of course, as demonstrated for UG, similar
procedure can be applied for the expression in E. coli of
any homodimeric, heterodimeric or multimeric protein by
inserting the appropriate coding sequences for such pro-
teins in place of UG coding sequence. It is noted that
many proteins of medical importance fall in this group,
including human hormones such as insulin, thyroid stimu~
lating hormone, the gonadotropic hormones FSH and LH,
human chorionic gonadotropin and the like. All these
proteins are dimeric consisting of two relatively small
subunits, whose genes can be assembled from totally syn-
thetic DNA fragments and then inserted in tandem into an
Z5~n~fiR
- 30 -
appropriate expression vector carrying the T7 promoter,
such as p~E103-1.
As 3hown by the results presented herein, this
vector directs the synthesis of both subunits, which then
associate within the bacterial cell to produce the com-
plete protein. The overexpression of both UG and ~ -
lactamase by pLEI103-1 shows that T7 RNA polymerase can
easily transcribe ad~acent genes into polycistronic mRNAs
(i.e. mRNAs containing more than one gene) and thereby
direct the expression of two different polypeptide
chains. This, of course, allows the construction of
artificial "operons" (i.e. groups of ad~acent genes con-
trolled by a common regulatory sequence) in T7 expression
vectors for the expression of dimeric or multimeric pro-
teins in E. coli.
II Immunoglobulins (antibodies) are a class of
multimeric proteins of enormous biological and medical
interest that could be expressed in E. coli using the
system of the present invention. This is aceomplished by
cloning cDNAs for an immunoglobulin light ehain and heavy
chain into the T7 veetor of the present invention with a
;~ synthetie N spaeer" sequenee containing a prokaryotic
RBS. Furthermore, genetic manipulations in plasmids
eould then allow the construetion of mutant antibodies of
altered antigen speeificity, both for praetieal u~es and
for detailed studies of the moleeular basis of antibody
speeifieity.
Excision of the UG gene from pLE103-1 by means
of digestion with PstI, followed by ligation of the plas-
mid, generated pLD101 (Fig. 7). pLD101 is a novel T7
expression vector with three cloning sites: NcoI, PstI
and HindIII. The complete sequence of the synthetic
regulatory element containing the T7 ~10 promoter and the
gene 10 leader region and RBS is also shown in Fig. 7.
This figure shows the main features of pLD101. It is a
cireular double stranded DNA with a moleeular length of
4406 base pairs (bp). It carries the gene for ampicil-
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- 31 -
lin-resistance (Ampr) and an incomplete remnant of the
tetracycline resistance gene (Tet9). The ~10 T7 promoter
is located between these two genes, followed by the T7
gene 10 non-translated region and RBS. This whole regu-
latory region, shown in Fig. 7, is 89 bp long and it isfollowed by the three cloning ~ites NcoI, P~tI and
HindIII. The nucleotide sequence of the regulatory
region and the cloning sites is shown in the lower part
of Fig. 7. The 5S rRNA gene (5S) and the Tl and T2 E.
coli rRNA terminators (T) are from pKK233-2.
This recombinant plasmid pLD101 provides the
advantages of both T7-promoter vectors and of ATG vectoræ
in a single system.
It may be pointed out that the pre-existing
translation vectors using the T7 promoter have one
restriction site (NdeI) into which foreign genes can be
inserted. Another site (BamHI) is placed downstream, but
insertion of a coding sequence in this site results in
expression of a fusion protein containing 14 additional
amino acids. The NdeI site contains the start codon
ATG. However, use of a NdeI site presents some distinct
disadvantages: (i) NdeI is a rare site on eukaryotic
DNA; (ii) the restriction enzymie NdeI is a very unstable
enzyme (half life of 15 min at 37C, according to manu~
facturer specifications) and doe~ not work well unless
the substrate DNA is thoroughly purified (see New England
Biolabs catalog, 1987); (iii) after reætriction cut, NdeI
leaves a 2 bp overhang (AT) which is more difficult to
ligate than 4 bp overhangs (in fact, ligation of NdeI
ends relies on the formation of an AT-TA base pairing,
which is held together by only 4 hydrogen bonds, and is
rather unstable); ~iv) when NdeI ends are made Nblunt" by
treatment with the "Klenow" fragment of DNA polymerase,
the ATG is not reconstituted. This prevents blunt-ended
fragments to be attached directly "in frameN to a
Ublunted'' NdeI end reconstructing the start codon ATG.
Finally, in some cases the presence of a relatively long
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- 32 -
extraneous peptide at the N- terminus ~f an expre~ed
protein i9 not acceptable. Thi~ limit~ the applications
of the downstream ~amHI site to cases where the addi-
tional 14 residue~ do not interfere with studied struc-
tural or functional features ofthe expressed protein.
In contrast, the NcoI site present in the novel
construct of the present invention allows a greater ver-
satility of cloning, for the following reasons: (i) the
NcoI cite (CCATGG) is frequently present in eudaryotic
translational starts. The consensus sequence derived
from 211 mRNAs is in fact CC~ CATG(G) (Kosak, Nucleic
Acid Res. 12, 857-872, 1984). This means that many
eudaryotic cDNAs can be directly cloned in the NCoI site,
producing an unfused recombinant protein; (ii) NcoI is a
lS stable enzyme, and it also works properly on partially
purified DNA< such as plasmid "minipreps;" (iii) after
cut, NcoI leaves a 4 bp overhang, which allows easier
ligations; (iv) when a cut NcoI site is "filled inN with
Rlenow fragment, the ATG is reconstituted. This allows
NBluntN-ended DNAs to be cloned in-frame directly by
attaching them to a Nfilled-inN NcoI site. This situa-
tion also results in production of an unfused recombinant
protein, provided that the inserted Nblunt-endedN frag-
ment is in the correct reading frame (see, for example,
construction of pLE102).
The presence of the PstI and HindIII sites
allows "forced" or NdirectionalN cloning (i.e. using an
insert with an NcoI or blunt end and a PstI orHindIII
end, so that it can be inserted into the plasmid only in
one orientation). Cloning inserts into the PstI or
HIndIII results in the insertion of only two (PstI), or
three (HindIII) alanine residues after N-terminal
methionine (see for example construction of pLE101 and
pLelO3-1).
Finally, the presence of the PstI site, and the
relative positions of the three cloning sotes om ATG
vectors and in pLDlOl allow use of these plasmids for
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- 33 -
cDNA construction by different methods, such as the homo-
polymeric tailing methods of Land et al (Nucleic Acld
Res. 9, 2251-2266, 1981); Okayama et al (Mol. Cell. Biol.
2, 161-170, 1982); Heidecker et al (Nucleic Acid Re~. 11,
4891-4906, 1983) and the double-linker method of Helfman
et al (Proc. Natl. Acad. Sci. USA 80, 31-35, 1983).
Moreover, constructing cDNA libraries in pLD101 provides
the benefit of high efficiency expression libraries,
particularly useful for antibody screening.
Fig. 8 shows does-response curves of recombi-
nant and natural UG as PLA2 inhibitors. ~oth protein~
were tested in the range of concentrations which have
been reported to be optimal for the PLA2 inhibitory
activity. It is evident that the two curves are essen-
tially identical. This indicates that purified recombi-
nant UG is as potent a PLA2 inhibitor as the natural
protein. The slightly lower percent inhibition obtained
in the present study with UG, with respect to previously
published data is most likely due to differences in the
assay procedure, particularly the change in PLA2 source
and batch. With the batch of PLA2 currently used in our
laboratory, inhibition observed with UG and other poly-
peptide inhibitors rarely exceeds 40%. This may be due
to contaminant protease(s) present in some batches of
PLA2 that may cause degradation of these inhibitors.
In summary, with respect to previously avail-
able ATG vectors, the plasmid constructs of the present
invneiton provide much higher efficiency of expression;
and with respect to previously available T7 promoter
vectors, the plasmids of the present invention provide
~i) the NcoI site; (ii) the PstI site; (iii) the HindIII
site; (iv) the feasibility of direct or directional clon~
ing of blunt-ended cDNAs while conserving the ATG; and
(v) the feasibility of direct construction of expression
libraries.
Of course, it should be clear to one of ordi-
nary skill in the art that the plasmids constructed in
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- 34 _
accordance with the present invention csn be utilized for
expression in any strain of E. coli of a ~uitable geno-
type. Screening of cDNA expre~ion libraries with anti-
body can be performed by any standard methodology well
known in the art and described in ~uch texts as Davis et
al, 1986, Basic Nethods in Molecular Biology, Elsevier
publication.
A deposit of pLE101, 102 and 103-1 has been
made at the American Type Culture Collection (ATCC),
12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A.
on October S, 1988 under accession numbers 67815, 67816
and 67817, respectively. These deposits shall be viably
maintained, replacing if they became non-viable, for a
period of 30 years from the date of the deposit, or for 5
years from the last date of request for a sample of the
deposits, whichever is longer, and made available to the
public without restriction in accordance with the provi-
sions of the law. The Commissioner of Patents and Trade-
marks, upon request, shall have access to the deposits.
It is under~tood that the examples and embodi-
ments described herein are for illustrative purposes only
- ~ and that various modifications or changes in light there-
of will be suggessted to persons skilled in the art and
arè to be includes within the spirit and purview of thi~
application and scope of the appended claims.
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