WO1990004026A1 - Nouvelles structures de plasmide pour la production a haut niveau de proteines d'eucaryotes - Google Patents

Nouvelles structures de plasmide pour la production a haut niveau de proteines d'eucaryotes Download PDF

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WO1990004026A1
WO1990004026A1 PCT/US1989/004427 US8904427W WO9004026A1 WO 1990004026 A1 WO1990004026 A1 WO 1990004026A1 US 8904427 W US8904427 W US 8904427W WO 9004026 A1 WO9004026 A1 WO 9004026A1
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protein
plasmid
ple103
recombinant
coli
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PCT/US1989/004427
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Anil Mukherjee
Lucio Miele
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The United States Of America, Represented By The Secretary, United States Department Of Commerce
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the present invention is related generally to the construction of plasmid vectors. More particularly, 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.
  • Plasmid systems for the expression of proteins of single subunit structure have been known. Typical of such vectors are the ones described by Amann et al, 1983, Gene, 25:167-178 and Rosenberg et al, 1983, Methods in Enzymology, 101:123-138, respectively.
  • Bacterial expression of eukaryotic proteins is a tool of ever- increasing importance in biochemistry and molecular biology. However, the majority of the recombinant eukaryotic proteins that have been expressed in bacteria are produced as fusion proteins and not in their native conformation.
  • 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 expression vector has not heretofore been described.
  • Figure 1A shows a schematic construction of pLElOl and pLE102.
  • Figure IB shows a schematic construction of pLE103-l from pLElOl.
  • the sequence of pLE103-l between the BamHI site and the Hindlll site is reported above the scheme of the subcloning steps.
  • 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.
  • 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-l as estimated by SDS-polyacrylaraide gel electrophoresis on a 15-25% gradient gel containing 0.1% SDS. Each lane was loaded with the equivalent of 50 // l of bacterial culture.
  • Lane 1 purified rabbit UG (1 iq ) ;
  • lane 2 molecular weight standards (BRL, pre- stained); lanes 3-4: 1 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: y-lactamase (uppermost arrow) and the two bands of UG.
  • Aliquots of cultures (1 ml) in M9CA medium were centrifuged at 12000 x g for 2 min. Cells were washed 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 min. and adjusted 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 was performed by means of a Biorad kit, according to the manufacturer's instructions.
  • Figure 2B shows the immunoblot of expressed 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 4°C, 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 (5); 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).
  • LYS lysozyme
  • MYO myoglobin
  • CHT chymotrypsinogen A
  • OVA ovalbumin
  • BSA bovine serum albumin.
  • 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 ⁇ .
  • lane e pooled fractions after CM-Sepharose chromatography (1.8 jug pro ⁇ tein) induced culture, 4 hours after induction; lane f: pooled fractions after Sephadex-G50 superfine chroma ⁇ tography (1.6 _u g protein) purified rabbit UG, silver stain.
  • the bacterial lysate supernatant appears to be more concentrated than the Sephacryl pool. This is probably due to its hight content in nucleic acid fragments (A2 >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 P-lactamase.
  • Figure 6 shows the N-terminal sequence of recombinant UG.
  • Figure 7 shows a schematic diagram of pLDl l, below which is shown the sequence of pLDlOl between the Bam HI site and the Hind III site.
  • Figure 8 shows the dose response of natural and recombinant UG as inhibitors of porcine pancreatic PLA 2 . Each point represents the average of three determ ⁇ inations, each performed in duplicate.
  • a plasmid comprising circular, double stranded DNA of a molecular length of about 4406 base pairs containing at least a gene for ampicillin-resistance, 10 T7 promoter, ribo- somal binding site and three cloning sites, Ncol, PstI and Hindlll, wherein a cDNA for protein to be expressed is inserted at Ncol, PstI or Hindlll site.
  • Preferred plasmids are selected from the group consisting of pLElOl, pLE102 and pLE103-l.
  • 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 Ncol, 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 (pLElOl, 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.
  • UG rabbit uteroglobin
  • the plasmid pLE103-l in which the expression of recombinant UG is controlled by a bacteriophage T7 late promoter, is by far the most efficient.
  • 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 uterus.
  • Recombinant UG was purified to near-homogeneity and its N-terminal amino acid sequence was 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 phospholipase A 2 inhibitor as natural UG on a molar basis.
  • the plasmid pLE103-l may be useful to explore the structure-function relationship of rabbit uteroglobin.
  • this plasmid may be useful in obtaining high level bacterial expression of other eukaryotic proteins with quaternary structure, as well as for other general applications requiring efficient bac ⁇ terial expression 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.
  • UG may stem from the phospholipase A 2 (PLA 2 , EC 3.1.1.4) inhibitory pro- perties of this protein. Because of its PLA 2 inhibitory effect, 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 edia- tors of inflammation.
  • UG is a homodimeric protein, formed by identical subunits of 70 amino acids each, joined in antiparallel orientation by two disulfide - 1 -
  • a nonapeptide in d- -helix 3 of UG which may be the active site, or at least part of an active site, for the PLA 2 inhibitory activity of UG.
  • a high level bacterial expression of this protein was obtained.
  • 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.
  • plasmid pLE103-l a high level expression of recombinant UG (about 9-11% of total bacterial soluble proteins) was obtained.
  • UG cDNA is controlled by the _il0 late promoter of bacteriophage T7.
  • Recombinant UG in its natural dimeric form is synthesized by J3.. coli cells harboring pLE103-l, 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 PLA inhibitory activity as that of the natural protein.
  • high level or “high efficiency,” as used herein, means about a 1000 fold or more synthesis of the protein by the plasmid system of the present inven ⁇ tion in an J5_. coli expression vector, compared to the ATG vector pKK-233-2.
  • artificial operon means a group of foreign genes, controlled by a common regulatory sequence, inserted into a plasmid for the expression of a quarternary protein.
  • quarternary protein as used herein means a protein formed by more than one subunit, such as heterodimeric, homodimeric or multimeric proteins.
  • RNA polymerase messenger RNA polymerase
  • ribosomes intracellular organelles
  • a polypeptide chain a chain of amino acids linked to each other by a peptide bond
  • the basic mechanisms of these processes are similar in all orga ⁇ nisms.
  • prokaryotic 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) .
  • prokaryotic cells e.g. bacteria and blue-green algae
  • transcription and translation are simultaneous and coupled, and both processes are much faster than in eukaryotes.
  • 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.
  • E_. coli The Gram-negative bacterium E_. coli is by far the best characterized organism from the molecular and genetic point of view, and genetic manipulations in this organism are relatively easy. For these reasons, several systems have been developed to produce high quantities of eukaryotic proteins in Escherichia coli ( L 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 "belong" 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 the culture contains the plasmid which confers antibiotic- resistance to the host.
  • plasmids 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 mechanisms 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 "inducing" stimulus is given.
  • the coding sequence for the foreign protein of interest typically a eukary ⁇ otic cDNA
  • mRNA is produced from the plasmid DNA in a 3'-position of the promoter, up to the next transcriptional terminator site.
  • the mRNA which includes the coding sequence for the foreign protein, is translated by bacterial ribosomes into a protein.
  • 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 ( L coli) .
  • the novel plasmid directing the expression of UG is designated herein as pLEl03-l. METHODS OF CONSTRUCTION OF PLASMID VECTORS (pLE103-l, pLElOl AND pLE102) AND EXPRESSION OF UG IN E. COLI
  • Plasmid pUG617 was a gift from Dr. David Bullock (Lincoln College, Oxford, New Zealand). Plasmid pKK233-2 was kindly provided by Dr. J. Brosius (Columbia University, NY). jE ⁇ . coli strain JM105 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 J3. coli strain HBlOl was purchased from BRL.
  • UG coding sequence 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 Biolabs, Pharmacia or Bethesda Research 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 Cloning, 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 Pstl- digested pK 233-2 (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- ent and 50 ng of plasmid in a total volume of 20 ul. The reaction was carried out at 12°C for 6 hrs.
  • the ligated DNA was diluted 1:5 with TE buffer (Maniatis et al, supra) and 5 ul were used to transform j ⁇ . coli strain JM105 (Yanisch-Perron et al. Gene 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 plasmids of these clones were performed according to Birnboim et al (Nucleic Acid Res. 7, 1513-1523, 1979), and the correct orientation of the insert DNA was checked by digestion with appropriate restriction endonucleases (e.g. Aval and Hindlll). Clones containing the UG coding region in the appropriate orientation were designated pLElOl.
  • Plasmid pK 233-2 was digested with Nco I and the cohesive ends were made blunt by treatment with JE_. coli DNA poly ⁇ merase I "large fragment”. Direct ligation of blunt- ended UG cDNA fragment into Nco I-digested, blunt-ended pK 233-2 generated pLE102. The orientation of the insert was checked by digestion with Ava I and Hindlll, and the expected reconstitution of two Nco I sites at both ends of the insert was verified.
  • pLElOl three codons (Met-Ala-Ala) are added to the 5' terminus of the coding sequence of UG DNA, while in pLE102 only one codon (Met) is added to the 5' ter ⁇ minus. Therefore, pLElOl 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.
  • the Met codon is present in the Ncol site of the vector.
  • the plasmid p 233-2 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 in the vector.
  • the expression of foreign proteins in p K233-2 is controlled by the artificial "trc” promoter. a regulatory region consisting of part of the E_. coli "trp” promoter and part of the _E_. coli “lac” promoter.
  • the "trc” promoter is regulated by the lactose operon repressor protein ("lac repressor").
  • coli strain which overproduces the lac repressor (lad genotype), the expression of the foreign gene is sup ⁇ pressed.
  • a gratuitous inducer i.e. a molecule that binds to the lac repressor and inactivates it, without being metabolized
  • IPTG isopropyl-B-thio- galactopyranoside
  • the "lac" repressor is inactiv ⁇ ated and the foreign gene is transcribed by the E_. coli RNA polymerase.
  • the resulting mRNA is then translated to produce the recombinant protein (e.d. UG) .
  • pLElOl 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 strains JM105 and JM109 as hosts.
  • 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 (ODg Q g) reached 0.4.
  • pLElOl expressed about 300 ng UG/ml supernatant and pLE102 expressed about 120 ng/ml.
  • the remainder of the samples were centrifuged at 10,000 x g for 10 min.
  • the bacterial pellets were washed with phosphate-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-HCl pH 8, 5 mM EDTA containing 4% glycerol, 250 iHL phenylmethylsulfonyl- fluoride (Sigma), 0.7 / _g/ml pepstatin A (Calbiochem) and 0.5 jUg/ml leupeptin (Calbiochem).
  • the samples were flash-frozen in liquid N 2 , 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.
  • UG 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-l grown in the 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 N 2 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) .
  • M9 medium Quality Biologicals, Gaithersburg,
  • 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 supernatant ( ⁇ 9 ml) was loaded on a column of Sephacryl- S200 superfine (Pharmacia), 2.5 x 100 cm, equilibrated in buffer L. The fractions were assayed for UG by RIA, and UG-containing fractions were pooled, dialyzed against distilled H 2 0 and lyophilized.
  • the lyophilized material was resuspended in 12 ml of 25 mM ammonium acetate buffer, pH 4.2 and centrifuged at 27,000 x g.
  • the super ⁇ natant was loaded on a CM-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 6.0.
  • 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 -70°C with dessicant.
  • the concentration of purified recombinant and natural UG was estimated spectrophotometrically, using an M 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 electrophoresis was performed according to Laemmli and silver staining 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 A 2 assay was performed as previ- ously described, with modifications. Briefly, the reac ⁇ tion mixture contained 100 mM Tris-HCl, pH 8, 100 mM NaCl, 1 mM Na-deoxycholate, 10 / _M 2-[l- 1 C]-arachidonyl phosphatidylcholine (Amersham, 58 mCi/mmole) and 2 nM porcine pancreatic PLA 2 (Sigma) in a total volume of 50 ⁇ l . PLA was preincubated with either recombinant or natural UG at 37°C for 5 min and the enzymatic reaction was started by addition of aliquots of the preincubation mixture to the radioactive substrate.
  • PLA was preincubated with either recombinant or natural UG at 37°C for 5 min and the enzymatic reaction was started by addition of aliquots of the preincubation mixture to the radioactive substrate.
  • the maximum level of expression reached with both pLElOl 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 ("Western" 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).
  • IGSS Immunogold-Silver Staining
  • UG is a dimeric protein, being formed by two identical subunits joined 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 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.
  • dimerization is a second-order process, depending on the squared concen ⁇ tration of UGm.
  • pLElOl the regulatory sequences of pLElOl 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-l from pLElOl.
  • the regulatory sequences originally present in p K233-2 (lac operator, trc promoter and ribosome binding site) have been substituted with the synthetic DNA fragment whose sequence is shown in Figure IB.
  • the synthetic DNA fragment contained the rilO 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 "translation" 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 pLElOl are derived from p K233-3 and consist of the "trc” promoter followed by an artificial "ribosome binding site” (RBS). The latter sequence is thought to control the association of a bacterial mRNA to the 3OS subunit of ribosomes, thereby regulating the rate of translation of the mRNA.
  • the regulatory sequences in pLElOl (as in pK 233-2) are con ⁇ tained in a 285 bp BamHI-Ncol fragment. This fragment was excised from pLElOl (Fig. IB) by means of digestion with BamHI and Ncol restriction endonucleases.
  • the remaining portion of the plasmid was purified by electro ⁇ phoresis on an agarose gel and covalently joined to a completely synthetic 89 bp BamHI-Ncol 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 Ncol site.
  • the recombinant plasmid obtained in this way was designated pLE103-l, and it contains the same cloning sites as pKK233-2, but now under the control of the synthetic T7 "gene 10-like" regulatory region.
  • Phage T7 promoters are usually not transcribed by JE_. 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.
  • T7 RNA polymerase initiates transcription with very high efficiency and it elongates RNA 5-times faster than j ⁇ . coli polymerase.
  • T7 RNA polymerase produces from plasmids such as pLE103-l longer transcripts than _E_. coli RNA polymerase, which stops at the rrnB terminators present in pKK233-2 (Fig. 1).
  • 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 the half-life of these RNAs in E_. coli.
  • 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 such as BL21(DE3).
  • This strain of E_. coli is lysogenic for a lambda phage containing the T7 gene for RNA polymerase.
  • 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 "lacUV" E_. coli promoter, also artificially inserted into the lambda genome.
  • the "lacUV” promoter like the "lac” promoter, is regulated by a repressor protein which is inactivated by IPTG.
  • BL21(DE3) When BL21(DE3) is exposed to IPTG, it produces T7 RNA polymerase. If at the same time the strain 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 T7 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.
  • BL21(DE3) was used in BL21(DE3) to express UG, because it was found that low levels of UG are not toxic for _E_. coli as demonstrated by the data obtained with pLelOl and 102.
  • 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-l expresses recombinant UG upon induc- tion with IPTG, and the recombinant protein is readily detectable 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 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.
  • induction with IPTG will result in transcription of a polycistronic mRNA containing the bla coding sequence, and in overexpression of ⁇ > -lactamase.
  • T7 RNA polymerase does not recognize E_. coli trancriptional terminators, such as the Tl and T2 rrnB terminators present in pLE103-l (see Fig. IB).
  • E_. coli trancriptional terminators such as the Tl and T2 rrnB terminators present in pLE103-l (see Fig. IB).
  • Table 1 shows the results of an expression experiment using pLE103-l.
  • 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 pLElOl and 102, 3 hours after induction.
  • This amount of protein synthesis is within the range of preparative scale production, corresponding to approxi ⁇ mately 670 ug/g bacteria.
  • Fig. 3 shows the quantitation of recombinant
  • UG as determined by RIA in supernatants from bacterial lysates.
  • the three plasmids pLElOl, pLE102 and pLE103-l were compared under identical experimental conditions, except for the host strains, i.e. JM109 for pLElOl and pLE102, and BL21(DE3) for pLE103-l. It is clear that with pLEl03-l production of recombinant immunoreactive UG is much higher (about 50-fold more than with pLElOl and 100-fold higher than with pLE102). With pLE103-l, the highest absolute concentration of recombinant immunoreac- tive material was reached 120 min after induction (Fig. 3A) .
  • 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 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 Western blots. These results seem to indicate that in lysates of induced BL21(DE3) :pLE103-l recombinant UG exists almost solely in its natural dimeric form.
  • Fig. 4 shows that the recombinant protein pro- prised in E_. coli has the same apparent w (17 kd) as that of 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 chrom togram of bacterial extract.
  • 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 56°C 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 sequence of rabbit UG.
  • the recombinant UG produced by BL2(DE3) :pLE103- 1 is totally dimeric and no accumulation of free subunits 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 are high enough.
  • Non-covalent self-association of recombinant eukaryotic proteins in E_. coli has been described for human tumor necrosis factor and rat liver aldehyde dehydrogenase.
  • 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.
  • pLE103-l can be converted into a general purpose expression vector, which we have denominated pLDlOl.
  • the cloning sites Nco I, Pst I and Hindlll give to pLE103-l the same potential applications of "ATG vectors" with the advantage of the high efficiency and specificity of the T7 promoter.
  • the Nco I site CCATGG
  • the Nco I site can be easily "filled in” with _E_. coli DNA polymerase I large fragment.
  • 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 * P > - lactamase by pLEI103-l shows that T7 RNA polymerase can easily transcribe adjacent genes into polycistronic mRNAs (i.e. mRNAs containing more than one gene) and thereby direct the expression of two different polypeptide chains.
  • This allows the construction of artificial "operons” (i.e. groups of adjacent genes con ⁇ trolled by a common regulatory sequence) in T7 expression vectors for the expression of dimeric or multimeric pro- teins in _E_. coli.
  • Immunoglobulins are a class of multimeric proteins of enormous biological and medical interest that could be expressed in JS_. coli using the system of the present invention. This is accomplished by cloning cDNAs for an immunoglobulin light chain and heavy chain into the T7 vector of the present invention with a synthetic "spacer" sequence containing a prokaryotic RBS. Furthermore, genetic manipulations in plasmids could then allow the construction of mutant antibodies of altered antigen specificity, both for practical uses and for detailed studies of the molecular basis of antibody specificity.
  • pLDlOl is a novel T7 expression vector with three cloning sites: Ncol, PstI and Hindlll.
  • the complete sequence of the synthetic regulatory element containing the T7 «510 promoter and the gene 10 leader region and RBS is also shown in Fig. 7. This figure shows the main features of pLDlOl. It is a circular double stranded DNA with a molecular length of 4406 base pairs (bp) .
  • T7 gene 10 non-translated region and RBS This whole regu- latory region, shown in Fig. 7, is 89 bp long and it is followed by the three cloning sites Ncol, PstI and Hindlll. 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 pLDlOl provides the advantages of both T7-promoter vectors and of ATG vectors in a single system.
  • the pre-existing translation vectors using the T7 promoter have one restriction site (Ndel) 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 Ndel site contains the start codon ATG.
  • Ndel is a rare site on eukaryotic DNA
  • the restriction enzyme Ndel is a very unstable enzyme (half life of 15 min at 37°C, according to manu- facturer specifications) and does not work well unless the substrate DNA is thoroughly purified (see New England Biolabs catalog, 1987);
  • Ndel leaves a 2 bp overhang (AT) which is more difficult to ligate than 4 bp overhangs (in fact, ligation of Ndel ends relies on the formation of an AT-TA base pairing, which is held together by only 4 hydrogen bonds, and is rather unstable);
  • Ndel ends are made "blunt" by treatment with the "Klenow" fragment of DNA polymerase, the ATG is not reconstituted.
  • the Ncol site present in the novel construct of the present invention allows a greater ver ⁇ satility of cloning, for the following reasons: (i) the Ncol cite (CCATGG) is frequently present in eudaryotic translational starts.
  • the consensus sequence derived from 211 mRNAs is in fact CC,CCATG(G) (Kosak, Nucleic Acid Res. 12, 857-872, 1984).
  • Ncol is a stable enzyme, and it also works properly on partially purified DNA ⁇ such as plasmid "minipreps;" (iii) after cut, Ncol leaves a 4 bp overhang, which allows easier ligations; (iv) when a cut Ncol site is "filled in” with Klenow fragment, the ATG is reconstituted.
  • This allows "Blunt"-ended DNAs to be cloned in-frame directly by attaching them to a "filled-in” Ncol site.
  • This situa ⁇ tion also results in production of an unfused recombinant protein, provided that the inserted "blunt-ended" frag ⁇ ment is in the correct reading frame (see, for example, construction of pLE102).
  • PstI and Hindlll sites allow "forced” or “directional” cloning (i.e. using an insert with an Ncol or blunt end and a PstI orHindlll end, so that it can be inserted into the plasmid only in one orientation) .
  • Cloning inserts into the PstI or Hindlll results in the insertion of only two (PstI), or three (Hindlll) alanine residues after N-terminal methionine (see for example construction of pLElOl and pLel03-l) .
  • constructing cDNA libraries in pLDlOl 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 PLA 2 inhibitors. Both proteins were tested in the range of concentrations which have been reported to be optimal for the PLA 2 inhibitory activity. It is evident that the two curves are essen- tially identical. This indicates that purified recombi ⁇ nant UG is as potent a PLA 2 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 PLA 2 source and batch.
  • 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 Ncol site; (ii) the PstI site; (iii) the Hindlll 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.
  • plasmids constructed in accordance with the present invention can be utilized for expression in any strain of E ⁇ . coli of a suitable geno ⁇ type. Screening of cDNA expression libraries with anti ⁇ body can be performed by any standard methodology well known in the art and described in such texts as Davis et al, 1986, Basic Methods in Molecular Biology, Elsevier publication.
  • a deposit of pLElOl, 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 5, 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.

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Abstract

De nouvelles structures de plasmides permettant la production à haut niveau de protéines d'eucaryotes dans leur forme naturelle sont décrites.
PCT/US1989/004427 1988-10-11 1989-10-10 Nouvelles structures de plasmide pour la production a haut niveau de proteines d'eucaryotes WO1990004026A1 (fr)

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Publication number Priority date Publication date Assignee Title
EP0618977A1 (fr) * 1991-12-18 1994-10-12 The University of Calgary Vecteurs d'expression-secretion servant a produire des fragments fv biologiquement actifs
US5874299A (en) * 1990-08-29 1999-02-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US10208113B2 (en) 2014-06-23 2019-02-19 Janssen Biotech, Inc. Interferon α and ω antibody antagonists

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US5135855A (en) * 1986-09-03 1992-08-04 The United States Of America As Represented By The Department Of Health And Human Services Rapid, versatile and simple system for expressing genes in eukaryotic cells

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US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (fr) * 1986-01-30 1990-11-27 Cetus Corp

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BIOCHEMISTRY, Volume 25(22), issued 4 November 1986, BEGLEY, "Bacterial organomercurial lyase: overproduction, isolation, and characterization". See entire document. *
BIOCHEMISTRY, Volume 26(17), issued 25 August 1987, LIN, "Expression of human factor IX and its subfragments in Escherishia coli and generation of antibodies to the subfragments". See entire document. *
DNA, Volume 1(1), issued 1981, CHANDRA, "Hormonally regulated mammalian gene expression: steady-state level and nucleotide sequence of rabbit uteroglobin mRNA". See entire document. *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5874299A (en) * 1990-08-29 1999-02-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
EP0618977A1 (fr) * 1991-12-18 1994-10-12 The University of Calgary Vecteurs d'expression-secretion servant a produire des fragments fv biologiquement actifs
EP0618977A4 (en) * 1991-12-18 1997-02-12 Univ Calgary Expression-secretion vectors for the production of biologically active fv fragments.
US10208113B2 (en) 2014-06-23 2019-02-19 Janssen Biotech, Inc. Interferon α and ω antibody antagonists
US10358491B2 (en) 2014-06-23 2019-07-23 Janssen Biotech, Inc. Interferon alpha and omega antibody antagonists
US10759854B2 (en) 2014-06-23 2020-09-01 Janssen Biotech, Inc. Interferon alpha and omega antibody antagonists

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