WO1991018975A1

WO1991018975A1 - Method for purifying hiv reverse transcriptase

Info

Publication number: WO1991018975A1
Application number: PCT/US1991/003764
Authority: WO
Inventors: Michael Louis Mellini; Patrick Kevin Clark; Gary Mathew Muschik; Stephen Hill Hughes
Original assignee: The United States Of America, Represented By The Secretary, United States Department Of Commerce
Priority date: 1990-05-31
Filing date: 1991-05-30
Publication date: 1991-12-12
Also published as: AU8001291A

Abstract

Relating to the production and purification of recombinant proteins, a process for purifying recombinant reverse transcriptase from human immunodeficiency virus (HIV-RT) is provided. E. coli lysate containing HIV-RT is contacted with polyethyleneimine to precipitate cell debris and nucleic acids; the resulting supernatant is contacted with anion exchange resin at low salt, pH 6-9; the eluate is contacted with a cation exchange resin at pH 6-8; the second eluate is contacted with a second anion exchange resin to remove an E. coli protease; and the third eluate is contacted with a third anion exchange resin at a pH above 8.8 to yield highly purified, protease-free HIV-RT.

Description

Method for Purifying HIV Reverse Transcriptase

BACKGROUND OF THE INVENTION Field of the Invention The present invention is directed to the isolation and purification of basic proteins such as reverse tran¬ scriptase (RT) .

Description of Related Art The development of drugs that specifically inhibit viral replication without harming the host has been difficult. All viruses depend on the host for replicative functions, and, as a consequence, provide relatively few targets for drugs. This problem is especially true for viruses eg. human immunodeficiency virus-type l (HIV-l) , that have small genomes and rely extensively on the host for their replication. Drugs are usually screened for antiviral activity by monitoring their effects on viral replication in cultured cells. As a complementary approach, individual viral components can be chosen as specific targets and studied in detail.

There are several HIV-l gene products that are potential targets for directed drug screening and drug design. The viral enzyme reverse transcriptase is one of these targets. Retroviral replication is absolutely dependent on both the RNase H and DNA polymerase functions of RT and, so far as is now known, RT does not play a direct role in the life cycle of a normal cell. Cur¬ rently, the drug most commonly used against HIV, 3'-azido- 3 'deoxythymidine (AZT) , is believed to interfere, either directly or indirectly, with reverse transcription. Since copying the RNA genome into DNA is the first step in the viral life cycle following absorption and penetration of the virus, a drug that interferes with either RNase H or the DNA polymerase functions of RT could block the estab- lishment of the provirus in the genome of the host.

Purified viral components, such as RT, can be used directly in drug screens that are simpler, faster and safer than from drug screens which employ live virus. However, the quantity of RT found in virions is relatively small. Thus, it is impractical to purify enough RT from virions for structural or biochemical studies.

Further, a better understanding of the structure and biochemical properties of the replicative machinery of human immunodeficiency virus-type 1 (HIV-l) may be useful in the screening and design of drugs that could be used to treat AIDS.

The purified 66-kDa RT has all of the appropriate enzymatic functions and properties. Thus, the recombinant protein can be substituted for the viral enzyme in struc¬ tural and biochemical studies and used in screens for drugs that could inhibit HIV replication.

Currently, known methods of RT purification require monoclonal antibody production, purification and preparation; all of which are time consuming and costly.

Also the products produced by current method degrade and thus, result in impure RT.

SUMMARY OF THE INVENTION The present invention is directed to an improved method for purification of basic proteins and highly purified basic proteins.

In one aspect, the invention relates to a method for purifying a non-naturally occurring basic protein from cells, which comprises the steps of removing cell debris and nucleic acids from a cell lysate containing said basic protein to produce a solution containing said basic protein; contacting said solution with a first anion exchange resin; and selectively recovering said basic protein from said first anion exchange resin in a first eluate thereby separating said basic protein from contami¬ nating cellular proteins.

In another aspect, the invention relates to a method for purifying human immunodeficiency virus reverse transcriptase (HIV-RT) which comprises the steps of lysing E_i. coli cells which contain HIV-RT therein; contacting said lysed cells with polyethyleneimine to precipitate E. coli cell debris, DNA and RNA to thereby produce a solu¬ tion containing partially purified human immunodeficiency virus reverse transcriptase; lowering the salt concentra¬ tion of said solution to a concentration which is low enough to allow contaminating E coli proteins to bind to an anion exchange resin but high enough to allow said HIV-RT to remain in solution; contacting said solution having a low salt concentration with an anion exchange resin which binds contaminating J . coli proteins but which does not bind HIV-RT at a pH of about 6 to 9 to obtain a first eluate; contacting said first eluate with a cation exchange resin which selectively binds degraded reverse transcriptase present in said first eluate to thereby recover a second eluate from said cation exchange resin which contains undegraded reverse HIV-RT at a pH of about 6 to 8; contacting said second eluate with an ion exchange resin at a pH of about 6 to 9 to selectively bind E. coli protease to said anion exchange resin to thereby produce a third eluate; and contacting said third eluate with an anion exchange resin at a pH of greater than about 8.8 to produce a highly purified and substantially prote- ase-free reverse transcriptase.

In still another aspect, the invention relates to a method for purifying a recombinant basic protein which comprises the steps of lysing cells which contain said basic protein therein; contacting said lysed cells with a material which selectively binds to cell debris, DNA and RNA to thereby produce a solution containing partially purified basic protein; lowering the salt concentration of said solution to a concentration which is low enough to allow contaminating proteins to bind to an anion exchange resin but high enough to allow said basic protein to remain in solution; contacting said solution having a low salt concentration with an anion exchange resin which binds contaminating proteins but which does not bind said basic protein at a pH of about 6 to 9 to obtain a first eluate; contacting said first eluate with a cation exchange resin which selectively binds degraded basic protein present in said first eluate to thereby recover a second eluate from said cation exchange resin which contains undegraded basic protein at a pH of about 6 to 8; contacting said second eluate with an ion exchange resin at a pH of about 6 to 9 to selectively bind protease to said anion exchange resin to thereby produce a third eluate; and contacting said third eluate with an anion exchange resin at a pH of greater than about 8.8 to produce a highly purified and substantially protease-free basic protein.

The process of the present invention unexpectedly produces highly purified HIV-RT which is stable to proteo- lytic degradation. That is, the invention allows for removal of E__t. coli proteases which degrade the 66-kDa HIV-RT protein to the 51 kDa form, and overcomes disadvan¬ tages associated with the prior art in obtaining a puri- fied product.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the time course of the synthesis of HIV-l RT in JL. coli. The JL. coli cells were grown in a 14-liter or in a 30-gallon fermentor on Superbroth without glycerol as described in detail hereinbelow. Samples were removed at various intervals, the cells collected by centrifugation, and lysed directly in sample buffer according to the procedure described in Proc. Natl. Acad. Sci. USA (1988) . The proteins were fractionated on a 9% SDS-PAGE, fixed, and stained with Coomassie brilliant blue. Molecular weight markers were included on the gel; the positions of migration of the markers and their molecular weights are indicated on the left side of the figure. The arrow on the right side of the figure indi- cates the position of migration of HIV-l RT.

Lane 1. Proteins from E__s_ coli containing the parental pUC12N plasmid without an HIV-l RT insert.

Lane 2. Proteins from E_j_ coli containing the pUC12N-HIV-l RT grown to late log phase in a 14-liter fermentor.

Lane 3. Proteins from ΕL. coli containing pUC12N HIV-l RT grown in the 30-gallon fermentor and harvested at 3 hours after inoculation. Lane 4. Harvested at 4 hours. Lane 5. Harvested at 5 hours. Lane 6. Harvested at 6 hours. Lane 7. Harvested at 6.25 hours. Fig. 2 shows the elution profile of the CM-

Trisacryl column. The chromatography conditions are described in detail hereinbelow.

Fig. 2A shows the elution of protein, monitored spectrophotometrically at 280 n . The protein was loaded on the column and washed until the absorbance of the eluate returned to baseline. A salt gradient was applied (5-150 mM NaCl) . The conductivity of the fractions was measured (indicated by the points in the graph) and the concentration of NaCl was determined relative to known standards.

Fig. 2B indicates column fractions assayed for HIV-l RT by SDS-PAGE. HIV-l RT elutes in two peaks: the first peak contains the undegraded 66-kDa form; and the second is a mixture of the 66-kDa form and several proteo- lytic breakdown products. The arrow marks the position of migration of the 66-kDa HIV-l RT.

Fig. 3 shows Q-Sepharose chromatography at pH 9.3. The chromatography conditions are described in the Material and Methods Section of Aids Research and Human Retroviruses, Volume 6, November 6, 1990, pp. 753-764. The HIV-l RT elutes as a single symmetrical peak, measured by absorbance at 280 nm. The protein-containing fractions were pooled. A Coomassie-stained gel of the pooled fractions is shown in FIG. 4. Fig. 4 shows the purification of HIV-l RT.

Samples from various steps in the purification were fractionated by SDS-PAGE, fixed, and visualized by stain¬ ing with Coomassie brilliant blue. To demonstrate that the purified HIV-l RT is essentially free of proteases that degrade the 66-kDa form, purified protein was incu¬ bated at 25°C for 24 hours. Lane: 1, Post PEI superna¬ tant (25 μg) ; 2, Pellicon concentrate (25 μg) ; 3, Q- Sepharose (pH 8.0) flowthrough (25 μg) ; 4, CM-Trisacryl pooled fractions (25 μg) ; 5, Amicon concentrate (25 μg) ; 6, Q-Sepharose, pH 8.0 pooled fractions (25 μg) ; 7, Q-Sepharose, pH 9.3 pooled fractions (25 μg) ; 8, molecular weight markers: Phosphorylase b - 94,000, BSA - 67,000, Ovalbumin - 43,000, Carbonic Anhydrase - 30,000, Soybean Trypsin Inhibitor - 20,100; 9, Q-Sepharose, pH 9.3 pooled fractions (50 μg) ; 10, Q-Sepharose, pH 9.3 pooled frac¬ tions, 25°C for 24 hours (50 μg) .

Fig. 5 discloses glycerol gradient sedimentation of HIV-l RT. Linear glycerol gradients (20-40%) were prepared in Beckman SW40 tubes. The gradients contained 25 M Tris-Cl, 0.05% Triton X-100, 0.3 M NaCl, 2 mM DTT, 1 mM EDTA, pH 7.6. 50 μg of purified HIV-l RT was loaded on to the gradient, together with molecular size markers, bovine serum albumin (BSA) , alkaline phosphatase (AP) and myoglobin (Myo) . The gradient was subjected to centrifu- gation for 40 hours at 38,000 rpm. 20-drop fractions were collected from the bottom of the tube. The position of the markers is shown in the figure. RNase H activity and RNA-dependent DNA polymerase activity were determined for the various fractions according to published procedures (Hizi A. et al, J. Biol. Chem.. 252. 2281-2289 (1975)). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All percentages below are by weight unless other- wise indicated. Ideally, all steps of the process of the present invention are conducted under conditions (pH, temperature, etc.) wherein the desired basic protein remains stable and is not degraded or denatured. Cell Lysis Cell lysis can be accomplished by any conventional procedures. The particular type of procedure utilized to lyse the cells will depend upon the particular types of cells being lysed, the equipment available to the researcher, etc. For example, lysis can be effectuated by freezing and thawing the cells, by treating the cells with chemicals such as enzymes, by ultrasonic methods or by combinations of these procedures. Removal of Cell Debris and Nucleic Acids

The next step is removal of cell debris and nucleic acids from the bacterial lysate. This can be accomplished by contacting the cell lysate with a weak anion exchange material such as polyethyleneimine (PEI) which selectively binds to nucleic acids, e.g., DNA and RNA, and which also binds to cell debris and contaminating cell proteins. Ammonium sulfate precipitation can also be used to precipitate nucleic acids and contaminating proteins. Other materials can also be used which perform this function. The thus treated material can then be separated from the solution, e.g., by centrifugation whereby the PEI precipitates together with the materials which are bound thereto to produce a partially purified solution containing the basic protein. The cell lysate PEI mixture can also be filtered either before or after centrifugation to remove cell debris and other insoluble materials. The material which is used in this step should be one which binds the nucleic acids and cell debris but which does not bind to the basic protein or reverse transcriptase to be recovered under the conditions employed. Alternatively, a newer form of anion-exchange system consisting of stacks of thin, non-compressible, microporous poly(vinyl chloride)-silica sheets (FASTCHROM, Kontes, Life-Sciences Products, Vineland, NJ) can be coated with PEI to yield a positively charged hydrophilic surface for separation of proteins and DNA, Piotrowski et al, BioChromatoσraphy. 2, 161 (1988) . If bound PEI is used, it would probably be necessary to centrifuge the lysate before contacting the supernatant with bound PEI to avoid clogging. Thus, the PEI can be suspended in the solution and thereafter removed or can be bound to a solid support and the cell lysate can be passed thereover or contacted therewith to remove the undesired proteins and nucleic acids.

The PEI should be suspended in a solution which has a conductivity equivalent to a NaCl concentration of about 1.0 M or less, preferably about 0.5 M. The pH of the solution should be about 6 to 9, preferably 7 to 8. The conductivity of the solution should be one which allows the RT to remain in solution, i.e., equivalent to a NaCl concentration of about 0.25 to 1.0 mM. Desalting of Cell Lvsate

The cell lysate obtained from the previous step is desalted in order to lower the conductivity of the cell- free supernatant for the first chromatography step. Desalting can be accomplished by dialyzing the supernatant. of the previous step, by diluting the supernatant or by any other procedure. The object of this step is to lower the salt concentration of the supernatant to a concentra¬ tion which is low enough to allow contaminating E_j_ coli proteins to bind to the resins in the next step. The salt concentration should be high enough for the basic protein or reverse transcriptase to stay in solution. A suitable conductivity is thought to be equivalent to a NaCl concen¬ tration of 25 to 75 mM, preferably 40 to 60 mM, more preferably about 40 mM. The solution may then be filtered and/or centrifuged to remove further insoluble materials. First Chromatography Step

The purpose of the first chromatography step is to remove contaminating cellular proteins while allowing the basic proteins to elute in the flowthrough. Thus, the anion exchange resin used in this step should be one which binds contaminating cellular proteins but which does not bind basic proteins such as RT. .An example of a suitable anion exchange resin is Elude Q-Sepharose. The functional group on this resin is a quaternary a inoethyl group. Other anion exchange resins which may be used have the following functional groups: DEAE (diethylaminoethyl) , AE (Amino-ethyl) , quaternary ammonium, and PEI (polyethyl- eneimine) which may be bound to a number of different column supports such as Sepharose®, sephadex®, various celluloses, dextran, polystyrene, polyvinylchloride, agarose and various silicons. The pH of the eluant should be one which allows the desired basic protein or reverse transcriptase to pass through the column. The pH should also be one at which the protein is stable. Preferably, the pH of the solution is below the pi of the basic protein. For reverse transcriptase a pH of about 8 is useful. A pH of about 9 or less, preferably about 6 to 9 may be suitable. The conductivity of the solution should be equivalent to a NaCl concentration of about 25 to 75 mM, preferably 40 to 60 mM, more preferably about 40 mM. Second Chromatography Step

The purpose of the second chromatography step is to separate undegraded RT from a mixture of undegraded and degraded forms. This step also removes small amounts of contaminating cell proteins such as contaminating E_j_ coli proteins. In order to accomplish this objective, a cation exchange resin should be used which has a different affinity for the undegraded and degraded forms of RT. Preferably, the cation exchange resin has a higher affinity for the degraded form. Examples of suitable cation exchange resins include CM-Trisacryl. The func¬ tional group on this resin is a carboxy methyl. Examples of functional groups on the cation exchange resins are not limited to but include carboxyl methyl, sulphopropyl, phospho and carboxy-sulfone groups. These groups can be bound to the same column supports as described in connection with the anion exchange resins. The degraded and undegraded forms of RT can be sequen¬ tially eluted by passing a buffer having a linear gradient salt concentration across a column containing the cation exchange resin. The linear gradient should preferably have an increasing salt (e.g., NaCl) concentration whereby undegraded RT is eluted first at a conductivity, of about 50 mM NaCl and degraded RT is optionally thereafter eluted at a higher salt concentration of about 65 mM NaCl. Thus, the conductivity of the eluant should be initially at least about 20 mM NaCl and should be raised to selectively elute the undegraded RT. The pH of the buffer is below the pi of the protein to be eluted, preferably below about 8, more preferably about 6 to 8. Third Chromatography Step

The purpose of the third chromatography step is to stabilize the basic protein such as RT and remove high molecular weight contaminants, e.g., proteins having a molecular weight of about 70,000 daltons or more, along with residual degraded basic protein. The anion exchange resin useful for this step can be, but is not necessarily, the same anion exchange resin as used in the first chroma¬ tography step. Although a buffer having a linear salt gradient concentration of increasing concentration can be passed through the column to elute the various materials sequentially, such a linear gradient is not necessary unless it is desired to recover the degraded protein. If such a linear gradient is used, undegraded RT elutes first followed by the above-described undesired proteins. The pH of the buffer is below the pi of the protein and is preferably about 6 to 9, more preferably about 7 to 8.5. The pH of the buffer used in this step should be high enough to bind high molecular weight contaminants and degraded basic protein, but low enough to allow the basic protein to elute in the flow through. Preferably, the salt concentration is less than about 50 mM, preferably 20 to 50 mM. This third chromatography step should be conducted within 48 hours of the second chromatography step, preferably within 48 hours of cell lysis because this step removes E^ coli proteases which degrade reverse transcriptase. The material contacted with the column in this step should have a concentration of degraded RT which is low enough so that the undegraded form of RT will not bind to the column. It is thought that the concentration of degraded RT should be less than 25% of the total RT, more preferably less than 20% of the total RT. If too much degraded RT is present, both the degraded and undegraded forms of RT will bind to the column. Fourth Chromatography Step

The purpose of the final chromatography step is to remove any small amounts of remaining contaminating proteins. The conditions of this chromatography step can be the same as those of the third step except that the pH of the solution is higher, i.e., above the pi of the protein, preferably greater than about 8.8, more prefera¬ bly 9 to 10. The concentration of RT after this step may be at least 0.5 mg/ml, e.g., 0.5 to 2 mg/ml or about 0.8 to 1.2 mg/ml. The Final Product

The final product is a highly purified basic protein. In the preferred embodiment, the final product is a substantially protease-free reverse transcriptase. The reverse transcriptase is at least about 90% pure, preferably at least about 95% pure, more preferably about 97% pure. The purified substantially protease-free reverse transcriptase is very stable due to the absence of significant amounts of proteases. Definitions

The term "basic protein" means protein having a pi of greater than 8, preferably a pi between 8 and 12, more preferably between about 9 and 11. Basic proteins include the reverse transcriptase listed below as well as, for example, HIV-1-rev.

The term "reverse transcriptase" includes any reverse transcriptase such as HIV-l RT, HIV-2 RT, HTLV RT and MuLV RT. The term "non-naturally occurring" basic protein includes any protein which does not naturally occur in the cell from which it is obtained. Non-naturally occurring basic proteins include proteins which are obtained from lysed bacterial cells, such as J . coli, transformed with a vector containing a gene for the basic protein or. infected with a virus which produces the basic protein, lysed tissue culture transformed, transfected or infected with a gene, virus or vector which expresses the basic protein, tissue culture infected with virus or plasmids which produce basic proteins e.g., reverse transcriptase, lysed yeast cells transformed with a vector which expresses the basic protein, etc. Non-naturally occurring proteins include all proteins expressed by recombinant DNA technology and all proteins made by a virus which has infected a host cell. .An example of a virus infected tissue culture is an insect cell line infected with Bacculovirus having an RT gene inserted therein. An example is Autographica californica nuclear polyhedrosis virus (AcNPV) which can be grown in the clonal tissue culture line Sf9 derived from Spodopetra frugiperda cells, See, Methods in Enzvmology. Vol. 182, 117-119, Murray P. Deutscher, Ed. (1990) . The term "chromatography" includes all types of chromatography such as open column chromatography, as described in the Examples, high pressure liquid chroma¬ tography (HPLC) , batch chromatography wherein the station¬ ary phase is used in the form of a slurry, etc. Bacterial strains

The plasmid used to express HIV-l RT is described in Farmerie WG, et al: Expression and processing of the AIDS virus reverse transcriptase in Escherichia coli. Science; 236:305-308 (1987); and Hizi A, et al: Expres- sion of soluble, enzymatically active, human immunodefi¬ ciency virus reverse transcriptase in Escherichia coli and analysis of mutants, Proc. Natl. Acad. Sci. USA: 85:1218- 1222 (1988) .

The ends of the region encoding HIV-l RT were modified using synthetic DNA segments to introduce an initiation and a termination codon at the sites in the HIV-l genome that encode the amino acids where HIV-l RT is normally cleaved from the polyprotein precursor. This modified segment was inserted into the E_j_ coli expression plasmid pUC12N. The plasmid was introduced into |__ coli strain DH-5. When compared with the HIV-l RT isolated from virions, the recombinant HIV-l RT (rHIV-l RT) expressed by transformed E^ coli has two additional N-terminal amino acids i.e., methionine and valine. Fermentation

For the evaluation of growth conditions on a small scale, bacteria were picked from a single colony grown on NZYM plates as described in Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, and supplemented with 100 μg/ml of ampicillin. The colony was dispersed in broth and used to inoculate 25 ml cultures in 125 ml flasks. Cells were incubated for varying times under varying conditions to determine optimal conditions for the produc¬ tion of rHIV-1 RT. Pilot scale studies were reevaluated when fermentation was scaled up to 10 liters in 14-liter New Brunswick Scientific fermentors. Several growth media, varied percent inoculation, rate of aeration, agitation, and time of harvest were compared. The follow¬ ing conditions provide a reproducible production scheme that has been successfully scaled up to 30 gallons in a New Brunswick Scientific stainless steel bacterial fermen¬ tor. Frozen stocks of the rHIV-1 RT expression strain were prepared by picking a single colony from an NZYM plate supplemented with 100 μg/ml ampicillin and inoculat¬ ing NZYM broth supplemented with 100 μg/ml of ampicillin. NZYM is a standard growth medium containing Sodium Chlo- ride (NaCl) 5 g/liter. Magnesium Chloride (MgC1.6 H₂0) 2.18 g/liter, NZ-Amine-E (Humko Scheffield) 10 g/liter. Yeast Extract 5 g/litsr and Ampicillin 100 μg/ml. When growth was in early log fhase, sterile glycerol was added to a final concentration of 10% and the mixture was aliquoted, frozen and stored at -70°C.

A vial of frozen E^. coli stock was thawed and used to inoculate an NZYM/amp plate. After overnight growth at 37°C, a single colony was used to inoculate a flask containing Superbroth without glycerol and containing ampicillin [Bactotryptone (Difco Labs) , 12 g/L; yeast extract (Difco Labs), 24 g/L; KH₂P0₄, 2.31 g/L; K₂HP0₄, 12.54 g/L; ampicillin, 50 μg/ml] and the flask was incu¬ bated on a rotary shaker at 150 revolutions per minute at 37°C. The overnight culture was used to provide a 1% inoculum for each fermentor.

Fermentation was routinely carried out in 14-liter New Brunswick Scientific fermentors. Each fermentor contained 10 liters of Superbroth without glycerol. Phosphates and ampicillin were filter sterilized and added to the fermentor after the medium had been autoclaved and allowed to cool. Optimum production parameters are defined as follows: agitation, 350 rpm; aeration, 0.5 wm; temperature, 37°C. Growth was monitored and when it reached the early stationary phase, the culture was harvested by continuous centrifugation.

Fermentation under these conditions routinely produces 50 grams wet cell weight per 10-liter fermenta- tion. 50 gram aliquots can be stored at -70°C for several months before being used for the purification of rHIV-1 RT. Ouantitation of rHIV-1 RT

The amount of rHIV-1 RT produced by the recombi- nant E. coli was determined by SDS-PAGE after each fermen¬ tation. The yield of rHIV-1 RT relative to E^. coli proteins was determined by staining with Coomassie bril¬ liant blue (Biorad Laboratories) . The results with the direct staining are more reproducible than direct enzy- matic assays of the RNA-dependent DNA polymerase activity found in the extracts. Protein concentrations were measured using the Bio-Rad protein assay with bovine 7-globulin standards as described by Bradford M. , Anal. Biochem. , 72., 248 (1976) . Example 1

Purification of rHIV-1 RT

Materials. Q-Sepharose was obtained from Pharmacia LKB. CM Trisacryl was obtained from IBF Biotechnics. Triton X-100 was obtained from Bio-Rad Laboratories and the diethanolamine hydrochloride from Aldrich Chemical Company. Chemicals for electrophoresis were obtained from Bio-Rad Laboratories with the exception of the Pharmacia LKB low-molecular-weight standards. Radiochemicals were obtained from Amersham. All other chemicals and biochemicals were obtained from Sigma Chemical Company. The Diaflo ultrafiltration membrane (YM-30, 150 mM) and the Pellicon cassette (PTCC, 10,000 m.w. cut-off) were obtained from Amicon and Millipore, respectively.

Buffers. The following buffers were used: Buffer A - 10 mM Tris-Cl, 1 mM EDTA, 25% sucrose, pH 8.0. Buffer B - 50 mM Tris-Cl, 6.25 mM EDTA, 0.1%

Triton X-100, 50 mM NaCl, 0.2 mM PMSF, pH 8.0. The PMSF solution was dissolved in ethanol at a concentration of 17.4 mg/ml (100 mM) and added to the buffer 15 minutes before the buffer was to be used. Buffer C - 50 mM Tris-Cl, 6.25 mM EDTA, 0.1%

Triton X-100, 950 mM NaCl, 0.2 mM PMSF, pH 8.0.

Buffer D -5 mM HEPES, 2 mM DTT, 0.2 mM EDTA, 0.2 mM PMSF, 10% glycerol, pH 8.0.

Buffer E - 20 mM HEPES, 2 mM DTT, 0.2 mM EDTA, 5 mM NaCl, 0.2 mM PMSF, 10% glycerol, pH 8.0.

Buffer F - same as Buffer D, except pH 7.0. Buffer G - same as Buffer E, except pH 7.0. Buffer H - 20 mM Tris-Cl, pH 8.0. Buffer I - 37 mM DEA, 10% glycerol, pH 9.3. Buffers H, I, and the gradient buffers used in the second Q-Sepharose (pH 8.0) and in the third Q-Sepharose (pH 9.3) columns were prepared at room temperature with the pH compensation set at 4°C. The rest of the buffers were prepared at room temperature and the pH adjusted without temperature compensation. All purification procedures were carried out at 0-4°C. SDS-polyacrylamide gels were used to follow the purification through the chromato- graphic steps.

Bacterial lvsis and polyetheyleneimine (PEI) precipitation Frozen cells (100 g) were homogenized in .220 ml of

Buffer A using a Brinkmann Polytron with a PT 35/4 genera¬ tor head at a setting of 5-6 (3 bursts, 10 seconds each) . Following homogenization, 45 ml of 0.5 M EDTA was added and the suspension was treated with lysozyme (45 ml of a 5 mg/ml stock solution) for 15 minutes. After the lysozyme treatment, the cells were disrupted in 350 ml of Buffer B for 15 minutes. After disruption, 700 ml of Buffer C was added to the suspension, followed immediately by the addition of polyethyleneimine (10%_? pH 7.5, 40 ml). The lysate was then allowed to stir for an additional 15 minutes. Insoluble material was removed by centrifugation at 5000 x g for 30 minutes at 4°C using a GSA rotor. The resulting supernatant (1,400 ml) was then filtered through glass wool. Desalting of cell lysate

The cell-free supernatant was concentrated to 1000 ml using a Millipore Pellicon ultrafiltration unit equipped with a 10,000 m.w. cut-off polysulfone cassette. After concentration, the retentate was desalted with 2,500 ml of Buffer D to a conductivity equivalent to 40 mM NaCl using the Pellicon in the constant-volume diafiltration mode. After desalting, the retentate was concentrated to 700 ml and an additional 700 ml of Buffer D was flushed through the Pellicon to clear the system of protein. Adjustments to the solution conductivity and pH were made with cold distilled water, NaCl or dilute HC1 as needed. Final conductivity was equivalent to 40 mM NaCl and the final pH was 8.0. The solution was then subjected to centrifugation at 5,000 x g for 30 minutes and the result¬ ing supernatant filtered through glass wool. O-Sepharose chromatography (PH 8.0)

The above supernatant (1400 ml) was loaded onto a 5.0 cm x 15.0 cm Q-Sepharose column pre-equilibrated with buffer E. After the sample was loaded (20 ml/minute) , the column was washed with equilibrating buffer (Buffer E) until the absorbance at 280 nm returned to baseline. The fractions were analyzed by SDS-PAGE for HIV-l RT. Most of the HIV-l RT was eluted in the wash fraction. CM-Trisacryl chromatography

The breakthrough fractions from the Q-Sepharose containing the HIV-l RT were pooled (1500 ml) and diluted with Buffer F until the conductivity was equivalent to 20 mM NaCl (1500 to 3000 ml) . Concentrated HCl was added to give a final pH of 7.0. The fraction was then loaded onto a 5.0 cm x 15.0 cm CM-Trisacryl column at a flow rate of 15 ml/minute and washed with 900 ml of equilibrating buffer (Buffer G) . The column was then eluted at 10 ml/minute with a linear gradient of NaCl in Buffer G from 5 to 150 mM NaCl with a total volume of 1400 ml collected in 25 ml fractions. Two protein peaks were obtained. The first peak contained relatively undegraded (66 kDa) HIV-l RT. The column fractions were analyzed by SDS PAGE and the fractions containing the bulk of the RT pooled. O-Sepharose chromatography (pH 8.0)

The pooled sample from the CM-Trisacryl column (300 ml) was concentrated to 150 ml using a 2500 ml Amicon stir cell equipped with a 150-mm YM-30 membrane. The sample was diluted to 550 ml with Buffer H and concentrat¬ ed to 150 ml, then diluted to 550 ml with Buffer H and concentrated to 150 ml a second time. The membrane was washed with 150 ml of Buffer H and added to the 150 ml concentrate. The above sample was then loaded (4 ml/minute) onto a 2.6 x 14.0 cm Q-Sepharose column and the column was washed with 300 ml equilibrating buffer (Buffer H) . The bound proteins were eluted with a linear gradient (250 ml) of NaCl in Buffer I from 0-300 mM NaCl. The relatively undegraded HIV-l RT eluted in the breakthrough fraction and in the first 40-50 ml of the gradient. O-Sepharose chromatography fpH 9.3)

The undegraded fractions were diluted with four volumes of Buffer I. The conductivity and pH were adjusted with 2 M NaCl or NaOH to give a final conduct¬ ivity and pH equivalent to Buffer I. The sample was then loaded onto a Q-Sepharose column 2.6 x 14.0 cm at a flow rate of 2 ml/minute, and the column was washed with 100 ml of equilibrating buffer I at a flow rate of 4 ml/minute. The HIV-l RT was eluted with a linear 250-ml gradient of NaCl from 0-0.5 M NaCl in Buffer I at 4 ml/minute. Discussion

In the first step of the purification scheme, PEI precipitation was used to remove cell debris and nucleic acids from the crude cell lysate. When the precipitation was done with 0.3% PEI and 0.5 M NaCl, the majority of the nucleic acids were precipitated and the HIV-l RT was left in the soluble fraction. Cell debris and nucleic acids were removed by low speed centrifugation (5,000 x g). In the second step, the conductivity of the cell-free super¬ natant was decreased to a level equivalent to 40 mM NaCl. It was important to keep the conductivity at about 40 mM to avoid the precipitation of protein which can clog the Pellicon membrane. During concentration and desalting, the solution turned cloudy but this did not appear to affect the yield of rHIV-1 RT. The first chromatographic step utilized a pH 8.0

Q-Sepharose column. The majority of the rHIV-1 RT does not bind to this resin under these conditions, but many of the contaminating JL. coli proteins do bind tightly.

The second chromatographic step utilized CM- Trisacryl as the column. The RT was eluted by a linear salt gradient from 5 to 150 mM NaCl as seen in Fig. 2. The first protein peak, which contained undegraded RT, eluted at approximately 50 mM NaCl. The second peak, eluting at approximately 65 mM NaCl, contained a mixture of the undegraded and degraded forms. The degraded forms appear as a series of bands migrating between 51 and 60kDa

(see Fig. 2) and that these are HIV-l RT related products.

In the third purification step, a pH 8.0

Q-Sepharose column was used. As with the first Q-Sepharose column, rHIV-1 RT did not bind to the resin; however, high-molecular-weight contaminants along with residual degraded RT bound tightly. It should be noted that, if the material loaded onto the column contained a high proportion of the degraded form of rHIV-1 RT, virtu- ally all the RT bound to the resin.

In the final step in the purification procedure, the flow-through from the first Q-Sepharose was loaded onto a second Q-Sepharose column at pH 9.3. The RT bound under the conditions used for the second column and was eluted with a linear 0-0.5 M NaCl gradient. The elution profile from this column showed a single protein peak that contains highly purified rHIV-1 RT. SDS-PAGE of aliquots from various stages of the purification procedures is shown in Fig. 4. A small amount of degraded RT is seen in the 50-μg load (Lane 1) along with a high molecular weight band that appears to be aggregated HIV-l RT. All of the bands in the final product react with monoclonal anti- bodies against HIV-l RT (data not shown) . The average yield of rHIV-1 RT obtained from using this procedure eight times was 90 mg with a range of 47-185 mg.

Properties of the purified HIV-l RT. The biochem¬ ical properties of the HIV-l RT purified from extracts of the JL. coli expression strain have been investigated.

DNA polymerase. The DNA polymerase activity has been measured with several synthetic primers and tem¬ plates, with activated salmon sperm DNA described by Aposhian, H.V. et al, J. Biol. Chem.. 237. 519-525 (1962) and with mRNA using oligo(dT) primers (see Table I) .

Table I.

Substrate Specificities of Purified E coli-Synthesized

HIV Reverse Transcriptase

DNA polymerase activity*

1. Poly(rA) .oligo(dT) 1106

2. Poly(dA) .oligo(dT) 0.43

3. Poly(rC) .oligo(dG) 97

4. Poly(2'-0-methyl rC) .oligo(dG) 3.1 5. Poly(dC) .oligo(dG) 223

6. Activated DNA^b 48.2

7. mRNA.oligo(dT)^c 16.6

* Expressed in pMoles dNMP incorporated into DNA Iri 30 minutes at 37°C. Each assay contained 10 ng of purified HIV-l RT. The concentration of dNTPs was 20 uM, the concentration of template/primers was 10 μg/ml. Assays were done in 25 mM Tris-Cl, 50 mM KC1, 10 mM MgCl₂, 2 mM DTT, pH 8.0, in a final volume of 100 μl. Activated DNA was prepared by the protocol of Aposhian et al, J. Biol. Chem.. 237_f 519-525 (1962), and the particular DNA of the example was a gift from S. Youngren. ^c The template was polyA⁺ RNA isolated from chicken muscle and the particular poly A+ RNA of the example was a gift from C. Gruber. The purified enzyme has a marked preference for poly(rA) as a template, and is more than a thousand-fold more active using poly(rA)-oligo(dT) than poly(dA)-oligo- (dT) . However, the enzyme prefers poly(dC)-oligo(dG) to poly(rC)-oligo(dG) . The enzyme copies random DNA segments at least as well as it copies random RNA segments. The purified enzyme is more efficient in copying poly(rA) than poly(rC) .

RNase H. As expected, the purified HIV-l RT has intrinsic RNase H activity. Since the strain of E_j_ coli used to purify the rHIV-1 RT has high levels of endogenous RNase H, the purified rHIV-1 RT was used to show that the RNase H activity co-sediments with the DNA polymer¬ ase activity in glycerol gradients (Fig. 5) . The gradi- ents were run in 0.3 M NaCl to diminish possible adven¬ titious interactions between unrelated proteins.

Size of rHIV-1 RT. The 66-kDa form of HIV-l RT behaves as a monomer on glycerol gradients in 0.3 M NaCl (Fig. 5) . The size of the 66-kDa form of rHIV-1 RT by column chromatography was also evaluated. At lower salt (100 mM) , the behavior of the protein in gel filtration chromatography suggests that it is an approximately equal mixture of monomeric and dimeric forms (data not shown) .

Stability. During purification, the 66-kDa form present in the original E . coli lysate breaks down to a series of smaller forms that have apparent molecular weights ranging from 51-60 kDa. Analyses of these break¬ down products with a panel of monoclonal antibodies suggested that they had lost the C-terminal segment of rHIV-1 RT, and as such, they resembled to some degree the 51-kDa form found in virions. The highly purified RT of the present invention does not show significant breakdown, i.e., less than 1% proteolytic breakdown into the 51 kDa form, on prolonged incubation in Buffer I containing 150 mM NaCl at either 4°C or 25°C (2 weeks) or after shorter incubations at 37°C (24 hours) (Fig. 5).

As many different embodiments of this invention may be made without departing from the spirit and scope thereof, it is to be understood that this invention is not to be limited to the specific embodiments thereof.

Claims

WHAT IS CLAIMED IS:

1. A method for purifying a non-naturally occurring basic protein from cells, which comprises the steps of: removing cell debris and nucleic acids from a cell lysate containing said basic protein to produce a solution containing said basic protein; contacting said solution with a first anion exchange resin; and selectively recovering said basic protein from said first anion exchange resin in a first eluate thereby separating said basic protein from contaminating cellular proteins.

2. The method of claim 1, wherein said cell debris and nucleic acids are removed by contacting said solution with polyethyleneimine in a solution which has a conductivity equivalent to a NaCl concentration of about 1.0 M or less to thereby selectively bind nucleic acids and cell debris to said polyethyleneimine.

3. The method of claim 2, which further com¬ prises the steps of separating said polyethyleneimine from said solution to produce a partially purified solution containing said basic protein.

4. The method of claim 1, which further com- prises the steps of contacting said first eluate with a cation exchange resin to obtain a second eluate.

5. The method of claim 4, wherein said basic protein is reverse transcriptase, said cation exchange resin selectively binds degraded reverse transcriptase present in said first eluate and undegraded reverse transcriptase in said first eluate is recovered from said cation exchange resin to obtain a second eluate.

6. The method of claim 5, which further com¬ prises the step of contacting said second eluate with a second anion exchange resin which selectively binds proteases present in said second eluate to thereby obtain a third eluate containing reverse transcriptase and a low concentration of protease.

7. The method of claim 6, which further com¬ prises the step of concentrating said reverse transcrip¬ tase by contacting said third eluate with a third anion exchange resin to obtain reverse transcriptase having a concentration of at least 0.5 mg/ml.

8. The method of claim 1, wherein said basic protein is HIV-RT.

9. The method of claim 1, wherein said cell lysate is a bacterial cell lysate.

10. The method of claim 1, wherein said cell lysate is an J _ coli cell lysate containing reverse transcriptase.

11. A purified reverse transcriptase prepared by the method of claim 7.

12. Substantially protease-free reverse tran¬ scriptase.

13. The reverse transcriptase of claim 12, which does not show significant breakdown on incubation at 4°c or 25°C for two weeks or at 37°C for 24 hours.

14. The reverse transcriptase of claim 13, which is HIV reverse transcriptase.

15. A method for purifying recombinant man immunodeficiency virus reverse transcriptase (rHIV-RT) according to claim 1, which comprises the steps of: lysing E^. coli cells which contain rHIV-RT therein; contacting said lysed cells with poly¬ ethyleneimine to precipitate J_a. coli cell debris, DNA and RNA to thereby produce a solution containing partially purified rHIV-RT; lowering the salt concentration of said solution to a concentration which is low enough to allow contaminating E__s_ coli proteins to bind to an anion exchange resin but high enough to allow said HIV-RT to remain in solution; contacting said solution having a low salt concentration with an anion exchange resin which binds contaminating J_s. coli proteins but which does not bind rHIV-RT at a pH of about 6 to 9 to obtain a first eluate; contacting said first eluate with a cation exchange resin which selectively binds degraded reverse transcriptase present in said first eluate to thereby recover a second eluate from said cation exchange resin which contains undegraded reverse rHIV-RT at a pH of about 6 to 8; contacting said second eluate with an ion exchange resin at a pH of about 6 to 9 to selectively bind ILs. coli protease to said anion exchange resin to thereby produce a third eluate; and contacting said third eluate with an anion exchange resin at a pH of greater than about 8.8 to produce a highly purified and substantially protease-free rHIV-RT.

16. Highly purified and substantially protease- free HIV-RT produced by the process of claim 15.

17. A method for purifying a recombinant basic protein which comprises the steps of: lysing cells which contain said basic protein therein; contacting said lysed cells with a material which selectively binds to cell debris, DNA and RNA to thereby produce a solution containing partially purified basic protein; lowering the salt concentration of said solution to a concentration which is low enough to allow contaminating proteins to bind to an anion exchange resin but high enough to allow said basic protein to remain in solution; contacting said solution having a low salt concentration with an anion exchange resin which binds contaminating proteins but which does not bind said basic protein at a pH of about 6 to 9 to obtain a first eluate; contacting said first eluate with a cation exchange resin which selectively binds degraded basic protein present in said first eluate to thereby recover a second eluate from said cation exchange resin which contains undegraded basic protein at a pH of about 6 to 8; contacting said second eluate with an ion exchange resin at a pH of about 6 to 9 to selectively bind protease to said anion exchange resin to thereby produce a third eluate; and contacting said third eluate with an anion exchange resin at a pH of greater than about 8.8 to produce a highly purified and substantially protease-free basic protein.

18. The method of claim 17, wherein said material which selectively binds cell debris, DNA and RNA is polyethyleneimine.

19. The method of claim 17, wherein said basic protein is reverse transcriptase.