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EP0517790A1 - Procede d'amelioration de la recuperation de proteines recombinees - Google Patents

Procede d'amelioration de la recuperation de proteines recombinees

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Publication number
EP0517790A1
EP0517790A1 EP91905560A EP91905560A EP0517790A1 EP 0517790 A1 EP0517790 A1 EP 0517790A1 EP 91905560 A EP91905560 A EP 91905560A EP 91905560 A EP91905560 A EP 91905560A EP 0517790 A1 EP0517790 A1 EP 0517790A1
Authority
EP
European Patent Office
Prior art keywords
protein
bacterial
cells
medium
recombinant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91905560A
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German (de)
English (en)
Inventor
J. A. Peterson
S. S. Boddupalli
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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Publication of EP0517790A1 publication Critical patent/EP0517790A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • 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
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a method for enhancing the recovery of recombinant protein from cultured cells.
  • the invention concerns a method for enhancing recombinant protein recovery from cultured bacterial cells containing such proteins on a plifiable DNA vectors.
  • the invention applies to a method for enhancing recovery of bacterial he e proteins such as cytochrome P450 BM - 3 , and the related electron transfer proteins such as putidaredoxin reductase and putidaredoxin.
  • Heme enzymes and related proteins represent a superfamily of proteins which are known to catalyze a variety of oxidative reactions on diverse substrates including endogenous prostaglandins, leukotrienes, steroids and fatty acids as well as exogenous drugs, carcinogens, and pesticides.
  • endogenous prostaglandins include endogenous prostaglandins, leukotrienes, steroids and fatty acids as well as exogenous drugs, carcinogens, and pesticides.
  • the ability of this family of proteins to insert an oxygen atom into various organic compounds has been, therefore, the focus of intense investigation.
  • the production of fatty acids from hydrocarbons, the detoxification of pollutants and new routes to pharmacologically-active compounds are but a few of the possibilities arising from reactions catalyzed by these proteins.
  • Fatty acid monooxygenase represents one member of this class of commercially important enzymes. These monooxygenases have been purified from a variety of mammalian tissues (Kupfer 1980), plants (Kolattukudy 1969; Croteau and Kolattukudy 1975a; Croteau and Kolattukudy 1975b) , yeasts (Tulloch et al. 1962; Heinz et al. 1969), and bacteria (Peterson et al. 1966; Miura and Fulco 1974) .
  • putida is very similar to the one functional in higher organisms - in particular, it appears similar to the one active in the adrenal cortex mitochondrial metabolism of steroids or the steroid oxidizing enzymes in other endocrine tissues.
  • research into the mechanism of electron transfer from NADH to P450 CAM in the metabolism of commercially important substrates has been significantly hindered by the limited availability of the proteins responsible for the transfer of electrons to the cytochrome (Geren et al. 1986) .
  • Only low level expression of putidaredoxin reductase has been feasible to date [Roome et al. (1983); Unger et al. (1986)].
  • putidaredoxin reductase may be purified from the wild type strain of P. putida.
  • enzymes such as putidaredoxin reductase may be purified from the wild type strain of P. putida.
  • drawbacks to this approach (Roome and Peterson 1988) . It is, for example, difficult to separate putidaredoxin reductase from P450 CAM due to the similarity in size and ionic charge and, the level of putidaredoxin reductase in the wild type cell is significantly below that of P450 CAM .
  • the cloning and expression of these proteins distinct from one another in a non-pseudomonad is necessary.
  • the present invention provides a surprisingly simple method for enhancing recovery of recombinant proteins from cultured bacterial cells.
  • the method is not limited to induction by exogenously added inducers such as barbiturates or synthetic inducers of bacterial gene promoters (e.g., inducers for the ,9-galactosidase gene of the lac operon in E. coli) .
  • inducers such as barbiturates or synthetic inducers of bacterial gene promoters (e.g., inducers for the ,9-galactosidase gene of the lac operon in E. coli) .
  • the method allows for the enhanced expression of heterologous proteins in E. coli. the workhorse standard of the fermentation industry and research laboratories.
  • a method of enhancing the expression of a recombinant protein is described which is surprisingly simple and straightforward.
  • the cells are then cultured in the protein expression enhancing medium to at least the late stationary phase of cell growth in order to allow the cells to express the desired recombinant protein and to allow recovery of the recombinant protein with high efficiency.
  • a protein expression enhancing medium is a medium which when used in combination with the methods of the invention allows comparable levels of recovery.
  • the method allows the expression of recombinant proteins to significantly higher levels than heretofore possible without specific genetic manipulation of the regulatory genes encoded along with the recombinant protein.
  • Using the method of the invention it is possible to obtain recovery of a desired recombinant protein at levels which represent at least fifteen percent of the recombinant protein by wet weight of the total soluble protein from the bacterial host cells.
  • the present invention allows for the production of certain heme -1-
  • the invention may successfully enhance the expression of a bacterial cytochrome P450.
  • enhanced expression of the cytochrome P450 BM . 3 derived from B. meqaterium may be realized. Since this particular cytochrome P450 represents a commercially useful enzyme and since expression of this enzyme in commercially useful quantities has, heretofore, been extremely limited, the methods of the invention provide a means of achieving a long-felt need in the area of expression of cytochrome P450 p . This is particularly true in the instance of the enhanced expression of the cytochrome P 50 BM - 3 derived from B. meqaterium since this enzyme exhibits considerable similarity to certain human cytochrome P450s.
  • the methods of the invention may also be applied to obtain enhanced expression of bacterial electron transfer proteins.
  • the expression of bacterial electron transfer proteins may be realized.
  • a bacterial redoxin reductase may be recovered in significantly higher amounts than was possible using prior art techniques.
  • the methods of the invention were applied to the bacterial redoxin reductase, putidaredoxin reductase, derived from £. putida.
  • the bacterial redoxin, putidaredoxin which is derived from P. putida. was demonstrated to be amenable to the expression enhancement method of the invention.
  • the protein expression enhancing medium of the invention is very rich in nutrients compared to the media typically used in the prior art.
  • the protein enhancing medium should possess several important characteristics.
  • the medium should be rich enough in energy source molecules, protein synthesis substrates, and other nutrients to achieve normal logrythmic growth of bacterial cells.
  • Such a medium should also provide for significant increases in the copy number of the vectors encoding the recombinant protein in order to achieve a low ratio of lac repressor molecules in relation to the copies of vector molecules.
  • the medium should be rich enough so that following completion of logrythmic growth and amplification of vector molecules, when the bacterial cells have entered the stationary phase of growth or when the cells are otherwise stopped from growing, that at least enough of the energy source, molecules, protein synthesis substrates and other nutrients remain unused in order that unrestricted protein synthesis is possible.
  • the energy source molecule should be a molecule like glycerol such that, while providing ample reserve energy following logrythmic growth for unrestricted protein synthesis, the molecule will not prevent the required release from inhibition by lac repressor of the lac operon providing regulation for the recombinant protein's transcription.
  • lac repressor of the lac operon providing regulation for the recombinant protein's transcription.
  • the principal energy source molecule glucose, sucrose or lactose.
  • the expression enhancing medium is at least three times richer than Luria-Bertani (LB) medium.
  • the richness of the medium refers to the fact that the protein expression enhancing medium contains much more Bactotryptone and Bacto-yeast extract than encountered in the prior art media.
  • LB medium would typically contain 10 grams of Bactotryptone and 5 grams of Bacto-yeast extract in a liter of final volume
  • the protein expression enhancing medium of the invention may contain as much as 12 grams or more of Bactotryptone and as much as 24 grams or more of Bacto-yeast extract.
  • other sources of tryptone and of yeast extract may be substituted in place of the industry standards, Bactotryptone and Bacto-yeast extract.
  • the richness of the medium may be duplicated in a variety of ways including some of the media described in the catalog of the American Type
  • the protein expression enhancing medium may contain buffering agents.
  • the buffering agents used are KH 2 P0 and K 2 HP0 4 . It will be appreciated by those of skill in the art that a variety of buffering systems and a variety of different buffering capacities may be used successfully in combination with the methods of the invention. However, the addition of buffering agents in combination with the methods of the invention are disclosed herein and encompass any such addition to achieve a buffering of the protein expression enhancing medium in excess of the buffering capacity inherent in the other ingredients of the medium.
  • glycerol is added at a concentration of at least 0.2% volume of glycerol per volume of medium.
  • Glycerol may be added at a concentration of at least 0.7% volume of glycerol per volume of medium in other embodiments. It is assumed here that one of skill in the art will maximize the concentration of glycerol in the protein expression enhancing medium in combination with the other teachings of the invention.
  • TB medium has been shown to work effectively.
  • TB medium is routinely prepared by adding 100 milliliters of a sterile solution of 0.17 molar KH 2 P0 4 and 0.72 molar K 2 HP0 4 to a sterile solution containing 12 grams of Bacto-tryptone, 24 grams of Bacto- yeast extract, 4.0 milliliters of glycerol and water to a final volume of 900 milliliters.
  • the methods of the invention may be applied to any bacterial cells capable of enhanced protein expression.
  • the bacterial cells will be those which carry a mutation of the recA gene.
  • the bacterial cells used in combination with the methods of the invention designed to enhance expression of a desired recombinant protein will be cells of the industrial standard E. coli.
  • a mutation of the recA gene will be present in such E. coli cells.
  • the methods of the invention used in combination with E. coli cells carrying a mutation of the recA gene will " be practiced using a DH5 ⁇ strain of E. coli. A variety of such strains will be known to those of skill in the art to possess certain advantages in specific instances.
  • the mutation of the recA may include any such mutation of the recA gene locus as long as a recA " phenotype is achieved.
  • the surprising and unexpected results realized using the method of the invention to enhance expression of protein is obtained by culturing of the bacterial cells to at least the late stationary phase of cell growth. In certain embodiments, it will be found advantageous to extend the growth for at least 16 hours. In another specific embodiment, the growth period will be extended for at least 24 hours. It is assumed here that one of skill in the art will maximize the extended growth period in the protein expression enhancing medium in combination with the other teachings of the invention.
  • the method of the invention may be ,viewed as a significant improvement over standard methods for expressing recombinant proteins.
  • the prior art methods relied on culturing bacterial cells containing DNA segments encoding such a protein in a rich medium under conditions appropriate to express that protein.
  • maximal protein production not dependent on addition of inducers to the medium is typically attained prior to the late stationary phase of cell growth. This procedure results in a significant reduction in the cell mass recovered from the growth medium even though the yield of enzyme per gram of cells is increased.
  • the surprising improvement realized by applying the method of the invention consists of culturing the bacterial cells in a protein expression enhancing medium and allowing the cells to grow to at least the late stationary phase of cell growth in order to allow the bacterial cells to express the protein.
  • the method of the invention may be successfully applied using TB medium.
  • expression of the recombinant protein may be routinely realized to levels representing at least fifteen percent of the recombinant protein by wet weight of the total soluble protein from the bacterial cells.
  • FIG. 1 Restriction Map of the BamHl to StuI Fragment Containing the Genes Encoding Putidaredoxin Reductase (PdR) and Putidaredoxin (Pd) .
  • the region encoding PdR is indicated by the longer of the two heavy bars while the short heavy bar represents the region coding for Pd.
  • the fragments which were subcloned for sequencing are represented by the light bars from BamHl to Sail. Sail to Nrul, and Nrul to StuI. The actual segments which were sequenced and the direction of sequencing are represented by the light bars with arrows.
  • Figure 2 Nucleotide and Deduced Amino Acid Sequence of Putidaredoxin Reductase and Putidaredoxin.
  • PdR is the first protein sequence and Pd is the second. Other regions of the sequence are described in the text.
  • Figure 3 Subcloning Strategy for the Expression of Putidaredoxin Reductase.
  • the relative position of the lacZ promoter is indicated by the heavy arrow on the circumference of the plasmid.
  • a surprisingly simple method of enhancing the recovery of cloned proteins is described in the present invention which includes inoculating bacterial cells which contain a recombinant protein encoded in DNA vector such as a plasmid into a medium designed for enhanced recovery of vector DNA (e.g., TB medium).
  • the culture is allowed to grow for at least 16 hours or to at least the late stationary phase of typical logorithmic bacterial growth. Following the extended growth in TB medium, it is possible to obtain recoveries of the recombinant protein in excess of 20% of the total soluble protein of the cells.
  • the discovery of the enhancement in protein expression by the inventors is illustrative of the surprising nature of the invention.
  • the inventors were interested in the expression of recombinant bacterial heme proteins, proteins which had presented significant problems in expression studies in the past.
  • the prior art existing at the time of the discovery suggested many ways of enhancing expression of such proteins including, in the instance of certain bacterial cytochrome P450s, the use of barbiturate additives to the medium.
  • the inventors had applied these techniques with only limited success and certainly anticipated difficulty in commercializing any such recombinant proteins due to the inability of recovering commercially feasible amounts of such proteins.
  • the method of the invention will routinely provide recovery of the cloned protein of at least fifteen percent total soluble protein from the cultured cells.
  • the amount of aeration of the culture and the concentration of glycerol in the medium may maximize the amount of the recovered protein for each individual case.
  • the invention has been found to have particular utility when applied to the cloning and expression of bacterial heme proteins such as bacterial cytochrome P450.
  • the method of the invention was applied to enhance the recovery of cytochrome P450 BM - 3 derived from B. meqaterium.
  • the invention methods allowed, for the first time, recovery of commercially significant quantities of this important monooxygenase in amounts exceeding 1 g purified protein/300 g wet weight of cells.
  • Bacterial redoxin reductase and bacterial redoxin are electron transfer proteins which are used to transfer electrons from an electron donor molecule such as NADH to the oxidizing cytochrome P450 cam .
  • the method of the invention was applied to the bacterial redoxin reductase derived from P. putida. putidaredoxin reductase.
  • the inventors additionally sought to separate the associated bacterial electron transfer protein, putidaredoxin also derived from P. putida. from the putidaredoxin reductase closely linked on the P. putida chromosome.
  • the heme protein of this electron transfer system could be expressed in high levels in the preferred expression system of the invention. In this manner, and using the methods of the invention, these bacterial electron transfer proteins could be expressed at commercially feasible levels.
  • the studies which gave the impetus for the current invention arose from a pressing need for commercially feasible quantities of the heme proteins briefly described above.
  • the activation of molecular oxygen for incorporation into drugs, steroids, and carcinogens is the focus of intense interest since the discovery of the role of cytochrome P450 in this diverse set of reactions.
  • the application of molecular cloning techniques to the studies of these enzymes has resulted in considerable detailed information including the sequence determination of more than 100 cytochrome P450s.
  • the study of the electron transfer reactions required to provide the cytochrome with the necessary redox potential to affect these reactions has also been intensely pursued.
  • P450 BM _ 3 being a soluble, catalytically self-sufficient enzyme with an extremely high turnover number in the w- hydroxylation of fatty acids should offer an excellent opportunity to enzymatically manipulate a wide variety of commercially important fatty acids.
  • study of these reactions with ample supply of the enzyme permitted by the invention provide a potential means for extrapolating the bacterial enzyme results to the medically important human w-hydroxylases. For instance, studies using the enzymes in the multiple oxidation reactions of long chain fatty acids will likely illuminate mechanisms of carbon-carbon cleavage reactions such as cholesterol side chain cleavage.
  • the over-production of proteins described in the present invention is suggested to result from a combination of factors: (1) significant increase in the copy number of the plasmid during late log phase; (2) decrease in synthesis of cellular protein and with it the lac repressor which accompanies late log phase of growth; (3) release of the inhibition of the lac operon because the ratio of repressor molecules to plasmid molecules is reduced; and, (4) enhanced protein synthesis from the lac operon which contains the genes for the desired proteins.
  • Putidaredoxin reductase Table II The Effect of Glycerol Content of the Medium on % Recombinant Cloned Protein in Total Soluble Protein Harvested at 24 Hours Incubation.
  • Table III The Effect of Aeration on % Recombinant Protein in Total Soluble Protein When 5 Cells are Grown in a 250 ml Flask and Harvested at 24 Hours
  • P450 BM - 3 is contained on an 14 Kbp plasmid which contains significant coding and noncoding regions for 0 other proteins (Ruettinger et al. 1989).
  • the plasmid was moved to a commercial recA " strain of E. coli (DH5 ⁇ ) which was shown to provide stable, high level expression of P-450 BM - 3 when used in conjunction with TB medium.
  • the expression of P-450 ⁇ - 3 under these conditions is not under the control of lacZ nor is it induced by barbiturates and yet realization of substantial increases in protein recovery may be had.
  • DE-52 anion exchange resin was purchased from Whatman BioSystems Ltd, Maidstone, Kent, England. All other reagents used were of purest grades available.
  • the E. coli strain DH5 ⁇ (F-endAl. hsdR17(r k " ,m k + ) , supE44. thi-1. recAl. qyrA96. relAl, A (arqF-laczya)U169. ⁇ 80dlacZAM15) was obtained from Bethesda Research Laboratories, Life Technologies, Inc., P.O. Box 6009, Gaithersberg, MD 20877, U.S.A. (BRL) as competent cells.
  • BM3-2A containing the 9.2 kbp plasmid encoding P450 BM . 3 was obtained from Dr. A.J. Fulco, Department of Biological Chemistry, University of California, Los Angeles, California.
  • the plasmid was isolated by conventional techniques (Birnboim and Doly 1979) and used to transform competent cells of E. coli strain DH5 ⁇ .
  • the transformed cells were isolated and the plasmid DNA of selected clones was examined by restriction analysis to demonstrate that the original plasmid was unchanged (Sambrook et al. 1989) . Cultures of cells were maintained in 2xYT media containing 50 ⁇ g per mL ampicillin.
  • the media was made 7.5% in glycerol and the cells were stored at - 70 * C.
  • MOPS buffer morpholinopropane sulfonate
  • the P450 BM _ 3 content of the whole cells was determined by difference absorbance spectrophotometry using 91 mM "1 cm “1 as the molar absorptivity (O'Keeffe et al. 1978) .
  • P450 BM - 3 is expressed in normal growth media without the addition of IPTG (isopropyl- ⁇ -D-thiogalactoside) to relieve the repression of the lacZ gene promoter (Wen and Fulco 1987) .
  • the level of expression of P450 ⁇ . 3 which was obtained in DH5 ⁇ with either LB or 2xYT media was 2-4 fold higher than that reported by Narhi et al. (1988) and repeated in our laboratory for similar growth conditions in E. coli strain JM109.
  • Extension of the time of incubation of the cells to late stationary phase in TB media resulted in a remarkable increase in the level of expression of P450 EM - 3 with this enzyme representing approximately 20% of the soluble protein of these cells.
  • °Cells when grown overnight (14 hrs.) d Cells when grown for 24 hours
  • the cells were grown for 24 hrs in TB media in a 10 L or 150L fer enter.
  • the cells were harvested by centrif gation, washed and resuspended in Buffer A.
  • the cell suspension containing a ratio of 1 g of cell paste to 4 iL of buffer, was subjected to four freeze-thaw cycles followed by lysozyme treatment (0.5 mg per mL) at 4'C for 1 hr, to lyse the cells.
  • DNase A (1 g per mL) and magnesium chloride (8mM) were added to the suspension to hydrolyze the DNA and decrease the viscosity of the solution.
  • the suspension was incubated at 4'C for an additional hour.
  • the lysate was centrifuged at 20,000 RPM for 1 hour and the supernatant solution was subjected to ammonium sulfate precipitation.
  • the fraction which precipitated between 40-80% of saturated ammonium sulfate contained P450 BM - 3 .
  • the precipitate was dialyzed, diluted to a protein concentration of 60 mg per mL, and loaded on a DE-52 ion exchange column which had been equilibrated with 20 mM MOPS and 2mM DTT (dithiothreitol), pH 7.4 buffer (bed volume of 1 L) .
  • the column was washed with 3 L of the equilibration buffer and the enzyme was eluted with a linear gradient of potassium chloride (0.1 to 0.5 M in equilibration buffer) . Most of the extraneous protein was removed by the initial wash and the P450 BM _ 3 was eluted by the gradient as a single peak.
  • Oxygen Uptake Determinations Oxygen consumption was measured with a Clark type oxygen electrode immersed in a sealed reaction chamber containing 1.6 mL of 50 mM MOPS, pH 7.4, and 0.4 ⁇ M P450 ⁇ - 3 . The desired concentration of fatty acid in 50 mM potassium carbonate was added to the chamber and the solution preincubated at 25'C for about 5 min before the addition of NADPH. The oxygen concentration in the reaction solution was determined and the recorder calibrated using beef heart electron transport particles and NADH as has been previously described (Estabrook 1967) .
  • the protein was estimated by the Warburg and Christian (1941) method for the impure fractions while the amount of protein in the pure P450 BM - 3 was estimated by the Lowry method (Lowry et al, 1951) .
  • the plasmids pIBI24 and pIBI25 which are derived from pEMBL plasmids, were obtained from International Biotechnologies, Inc. Restriction enzymes and bacteriophage M13mpl8 and M13mpl9 were obtained from BRL.
  • P. putida (ATCC17453) was obtained from the American Type Culture Collection. The cell line was stored in media containing 7.5% glycerol at -80 * C.
  • the nucleotide sequencing kit Sequenase Ver. 1.0 was obtained from U.S. Biochemicals, Corp., P.O. Box 22400, Cleveland, OH 44122, U.S.A.. Ampicillin was obtained from Sigma Chemical Co., P.O. Box 14508, St.
  • Mutagenic oligonucleotides and DNA sequence primers were synthesized on an Applied Biosystems 380A oligonucleotide synthesizer and purified by Sep-Pak C18 (Waters Associates, Milford, MA 01757, U.S.A.) column chromatography.
  • Bacterial Growth Stock cultures of E. coli strain DH5 ⁇ which harbored the appropriate plasmids were grown in 2xYT media containing 50 ⁇ g per mL ampicillin. For long term storage of the cell lines, the cell suspension was made 7.5% in glycerol, frozen in a dry ice/acetone bath, and stored at -80 * C until used.
  • the desired cell line was grown for 16 to 24 hours in TB media Tartof and Hobbs (1987) containing 50 ⁇ g per mL ampicillin.
  • the cells were harvested by centrifugation at 8,000 rpm in a Beck an J21 centrifuge (Beckman Instruments, Inc., Mail Station E-06-A, 2500 Harbor Boulevard., Box 3100, Fullerton, CA 92634-9989, U.S.A.).
  • the cells were broken by gentle sonication in a Branson Sonifier, Eagle Road, Danbury, CO 06810, U.S.A. for 60 sec and the cell debris and broken cells were removed by centrifugation.
  • the amount of enzyme present in the extract was determined as described below.
  • the cells were resuspended in 100 mL of 10 mM Tris buffer, pH 8.0, containing 1.0 mM EDTA, and 20% sucrose and stored on ice.
  • the cell suspension was made (TE buffer) and recentrifuged.
  • the cells were finally resuspended in 100 L of lysis buffer containing 50 mM Trischloride, pH8.0, 50mM EDTA, made 2 mg per mL in lysozyme and the incubation on ice continued for 30 min.
  • the cells were lysed by the addition of SDS (sodium dodecyl sulfate) to a final concentration of 4% and the mixture was heated to 70 * C and mixed gently for 30 min.
  • SDS sodium dodecyl sulfate
  • Proteinase K was added to a final concentration of 0.1 mg per mL and the heating continued for an additional 60 min.
  • Potassium acetate was added to a final concentration of 0.5M and the incubation at 70'C continued for 15 min.
  • the DNA was precipitated from the supernatant solution by the addition of PEG8000 to a final concentration of 10%.
  • the solutions were mixed by gentle inversion and stored at 4'C overnight.
  • the precipitate was collected by centrifugation at 12,000 rpm in the JA20 rotor for 15 min at 4'C, rinsed with cold 95% ethanol, and resuspended in 8 ml of TE buffer.
  • the suspension was made 0.1 mg per mL in RNase A and heated at 60'C for 30 min. The sample was cooled and extracted with: 1) 1 volume of phenol; 2) 1 volume of phenol, 1/2 volume of chloroform:isoamyl alcohol (24:1); and, 3) 1 volume of chloroform:isoamyl alcohol.
  • the DNA was precipitated with 1/2 volume of 7.5 M ammonium acetate and 2 volumes of ethanol. The precipitate was collected by centrifugation and dissolved in 1 volume of TE buffer and the ethanol-ammonium acetate precipitation was repeated. The precipitate was washed with a small volume of 70% ethanol and dried in a vacuum desiccator. The DNA was resuspended in 2 mL of TE buffer.
  • Plasmid DNA was isolated for restriction enzyme analysis by the alkaline-lysis method of Birnboim and Doly 1979. Selected restriction fragments were fractionated on a 1.5% low melting point agarose gel, the desired fragment was excised, melted at 65'C in a final volume of 0.4 mL of TE containing 100 mM NaCl, and extracted two times with TE-saturated phenol. The extracted fragment was precipitated twice with ethanol, and analyzed by agarose gel electrophoresis. Purified fragments were ligated into similarly cleaved pIBI24 or pIBI25, and transformed into DH5 ⁇ competent cells according to procedures recommended by the manufacturer.
  • Recombinant clones were identified as white colonies in the presence of the chromogenic substrate X-gal, and screened by restriction enzyme analysis. In most cases, the orientation of the insert in the ligation reaction was controlled by the nonidentical cohesive ends of the insert which matched the vector.
  • the procedure used for the ligation of target DNA into either the plasmid vector or into the replicative form of the M13mpl8 or M13mpl9 bacteriophage was identical and has been described previously (Sambrook et al. 1989) .
  • Nucleotide Sequence Determination Selected restriction fragments were purified from low melting point agarose, ligated into similarly cleaved M13mpl8 and M13mpl9, and transformed into DH5 ⁇ F' competent cells. Recombinant clones were identified as clear plaques in the presence of X-gal, and screened by restriction enzyme analysis of replicative form DNA isolated by the alkaline-lysis procedure. Single-strand phage DNA was isolated from infected cell cultures by polyethylene glycol precipitation, followed by SDS-Proteinase K digestion, phenol extraction and ethanol precipitation. The nucleotide sequence of the purified ssDNA was determined by the dideoxynucleotide chain termination method (Sanger et al.
  • USBiochemicals Sequenase kit U.S. Biochemical Corp., P.O. Box 22400, Cleveland, OH 44122, U.S.A.
  • a modified T7 DNA polymerase Tebor and Richardson 1987 version 1.0.
  • the determinations were repeated with deaza-dGTP to resolve band compression artifacts (Barnes et al. 1983; Gough and Murray 1983; Mizusawa et al. 1986). In each instance, the discrepancies were resolved by this procedure.
  • Oligonucleotide directed mutagenesis was performed using the two primer method of Zoller and Smith (1984) . In each case, the single base change was inserted in the middle of a 21mer which would hybridize with the single stranded DNA at the desired location. The newly synthesized double stranded DNA was used to transform DH5 ⁇ F* competent cells. Recombinant plaques were transferred and fixed to the filters as described (Sambrook et al. 1989) and mutants were identified by hybridization to the polynucleotide kinase-labeled mutagenic oligonucleotide.
  • tetramethylammonium chloride was used to accentuate the difference in melting temperature between the probe and the wild-type and changed sequences (Wood et al. 1985) .
  • the bacteriophage was purified until all of the plaques on a given plate would hybridize with the probe. To ascertain that only the desired base change occurred, each mutant was completely sequenced following plaque purification.
  • Putidaredoxin Reductase and Putidaredoxin Determinations The standard assay for the amount of putidaredoxin reductase and putidaredoxin takes advantage of the ability of the enzymes to catalyze the reduction of cytochrome c. This reaction is dependent on the presence of the reducing agent NADH, and both enzyme components (Roome 1983) . In the typical assay either putidaredoxin reductase or putidaredoxin was in excess while the other component was limiting. Under these conditions, the rate of reduction of cytochrome c was linearly dependent on the concentration of the limiting component. Although there is a slight background of
  • the standard assay for putidaredoxin reductase contained the following components in 20 mM MOPS buffer, pH 7.4: 0.1 mM NADH, 10 ⁇ M cytochrome c and 5.5 ⁇ M putidaredoxin.
  • An appropriate dilution of the cell-free extract was added to the reaction mixture prior to the addition of the putidaredoxin.
  • the rate of reduction of cytochrome c was compared to a standard curve to determine the amount of putidaredoxin reductase present in the cell extract.
  • the standard assay for putidaredoxin contained the following components in 20 mM MOPS buffer, pH 7.4:0.1 mM NADH, 10 ⁇ M cytochrome c, and 1 nM putidaredoxin reductase.
  • the cell extract was added prior to the addition of putidaredoxin reductase.
  • the concentration of putidaredoxin in the whole cells was also determined by EPR spectroscopy and the signal compared to a known standard.
  • the concentration of cytochrome P450 cam in cell free extracts was determined by standard procedures (O'Keeffe et al. 1978).
  • Putidaredoxin Reductase and Putidaredoxin Cloning and Sequence Determination The cloning of PdR 2 and Pd was aided by the publication in 1986 of the nucleotide sequence of P450 c ⁇ which included the N-terminal 153 nucleotides of putidaredoxin reductase (Unger et al. 1986) . Publication of the preliminary restriction map for the 4.4 and 2.6 kbp Hindlll fragments which contained the regulatory region, an alcohol dehydrogenase, P450 cam and both of these proteins Unger et al. (1986) indicated their general position within these fragments.
  • an oligonucleotide was synthesized which was complimentary to a portion of the N-terminal sequence of putidaredoxin reductase between the BamHl and Hindlll sites.
  • the whole cell DNA from £. putida was cleaved sequentially with BamHl and StuI and the digested DNA was ligated into BamHI-Sma I cleaved pIBI25.
  • the resulting reaction mixture was used to transform E. coli strain DH5 ⁇ .
  • the oligonucleotide probe was used to select clones which contained the 2.2 kbp BamHl-StuI fragment.
  • the preliminary restriction map published by Unger et al. 1986 indicated that the coding sequence for putidaredoxin should span the StuI restriction site.
  • the 2.2 kbp BamHl-StuI fragment was subcloned into both M13mpl8 and M13mpl9 for sequence determination.
  • the BamHI-Sall. Sall-Nrul, and Nrul-StuI (the EcoR I site from the polylinker region of the plasmid vector was actually used to clone the 3' end of this piece of DNA) fragments shown in Fig. 1 were cloned into the appropriate bacteriophage. Either the universal primer or synthetic oligonucleotides were used to prime the synthesis of the complimentary strand of DNA for the sequence determination. As indicated in Fig. 1, essentially all of the DNA was sequenced on both strands. The restriction map shown in this figure was deduced from the determined nucleotide sequence.
  • the nucleotide sequence of the BamHl-StuI fragment is shown in Fig. 2.
  • the deduced amino acid sequence for both putidaredoxin reductase and putidaredoxin are also shown in this figure. There are several points to be made about these sequences: (1) the amino acid sequence of the N-terminus of putidaredoxin reductase is in perfect agreement with that reported (Unger et al. 1986a; Unger et al 1986b;Romeo et al. 1987) ; (2) the deduced amino acid sequence of putidaredoxin exactly matches the published sequence (Tsai et al. 1971; Tanaka et al.
  • putidaredoxin As would be expected for putidaredoxin, the differences between the deduced and reported sequences are minor and explainable on the basis of the removal of the initiating methionine residue in post-translational processing of the protein. There is one additional glutamic acid residue and one fewer glutamine residue. The calculated molecular weight of this protein including the two iron and two acid-labile sulfur atoms of the active site is 11,726 daltons. The deduced and reported composition of putidaredoxin reductase are similar enough to lead one to the conclusion that this is probably the same protein. The calculated molecular weight of 46,215 agrees well with the reported value of 48,500 (Roome et al. 1983).
  • Putidaredoxin Reductase To ascertain the validity of the identification of the coding region as putidaredoxin reductase, this fragment was subcloned into pIBI24 in the correct orientation for transcription directed by the lacZ promoter. To obtain a clone of E. coli which would express only putidaredoxin reductase, the BamHl-StuI/EcoRI fragment was digested with both BamHl and Mlul and the resulting 1.5 kbp fragment of DNA was purified by electrophoresis in low melting point agarose. The fragment was recovered and ligated into the plasmid pIBI24 as shown in Fig.
  • coli cell line might result in cell death due to leakage of electrons from NADH through the reductase to putidaredoxin which has a greater sensitivity to oxidation by molecular oxygen than do the other components of this electron transfer system.
  • putidaredoxin reductase and putidaredoxin were subcloned together in the correct orientation for transcription, no colonies were found (data not shown) .
  • the E. coli clone which was isolated following transformation with the plasmid construct, would express active putidaredoxin reductase.
  • This level of putidaredoxin reductase is similar to that observed in wild type P. putida (Roome et al. 1983) .
  • Effect of the Start Codon on the Level of Expression of Putidaredoxin Reductase is the rare initiation codon GTG (Unger et al. 1986a; Unger et al. 1986b). This codon has been presumed to be important in the post-transcriptional regulation of protein abundance.
  • the G at position 1 was changed to an A by site-directed mutagenesis in the single-stranded bacteriophage M13mpl8 which contained the BamHI-Sall fragment.
  • This mutant clone was isolated and completely sequenced to ascertain whether there were any changes other than the desired one.
  • the BamHl-Xhol fragment containing the desired nucleotide change was purified, from low melting point agarose, from the replicative form of the bacteriophage, and ligated into the plasmid described above for putidaredoxin reductase expression as shown in Fig. 3.
  • the wild type BamHl to Xhol fragment had been removed from this plasmid by restriction digestion and low melting point agarose gel electrophoresis.
  • the plasmid was transformed into E. coli.
  • DH5 ⁇ and clones which were able to grow in the ampicillin- containing media were selected.
  • Samples of the cells were grown up and the sequence of the double stranded DNA was determined with the expected change in nucleotide sequence present (data not shown) .
  • These cells were grown in TB media and their putidaredoxin reductase content was determined as shown in Table VII. The average of three determinations gave a content of 7.4 mg of putidaredoxin reductase per gram wet weight of cells which is approximately an 18 fold increase over the level in JC. coli cells containing the wild type gene.
  • Putidaredoxin To obtain the expression of putidaredoxin-independent of putidaredoxin reductase, the 856 bp Narl-S al fragment of the original BamHl-StuI clone was subcloned into the AccI and Smal sites in the polylinker region of M13mpl8. " A single base change of a G to a T resulted in the formation of a Hindlll site at about 1160 bp in the putidaredoxin reductase coding sequence (see Fig. 2) . The mutagenic oligonucleotide was used as a probe for the clones with the anticipated base change as described in supra Wood et al. 1985.
  • the 580 bp fragment of DNA from the new Hindlll site to the Smal site was purified from the replicative form of M13mpl8 by low melting point agarose gel electrophoresis and ligated into pIBI25 which had been cleaved with the same restriction enzymes.
  • the level of expression of putidaredoxin in this cell line and control cells is shown in Table VII.
  • N.D. means not determined. a The amount of each of these proteins is expressed as the number of mg of protein per g wet weight of cells.

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Abstract

Procédé d'amélioration de la quantité de protéine recombinée récupérée après fermentation de bactéries contenant des vecteurs codant la protéine recombinée. Le procédé n'est pas limité par l'induction requise du gène cloné par addition d'inducteurs exogènes. Grâce aux procédés de l'invention, la production de protéines recombinée bactérienne peut dépasser un niveau de 20 % de la protéine soluble totale exprimée. Les procédés de l'invention sont démontrés sur cytochrome P450 bactérien et sur des protéines de transfert d'électrons bactériennes habituellement difficiles à exprimer à des niveaux commercialement réalisables.
EP91905560A 1990-02-27 1991-02-27 Procede d'amelioration de la recuperation de proteines recombinees Withdrawn EP0517790A1 (fr)

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DE69231306T2 (de) * 1991-02-26 2001-01-11 Zeneca Ltd., London Fermentationsverfahren zur Herstellung von Peptiden durch einen Wirt
US5886157A (en) * 1994-02-10 1999-03-23 Vanderbilt University Expression and purification of human cytochrome P450
US5741894A (en) * 1995-09-22 1998-04-21 Baxter International, Inc. Preparation of pharmaceutical grade hemoglobins by heat treatment in partially oxygenated form
DE19726823A1 (de) * 1997-06-24 1999-01-07 Deutsches Krebsforsch Detoxifizierungsproteine

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