Escherichia coli and analysis of mutants

We have constructed a plasmid that, when introduced into Escherichia coil, induces the synthesis of large quantities of a protein with an apparent molecular mass of 66 kDa that differs from human immunodeficiency virus (HIV) RNA-dependent DNA polymerase (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase or reverse transcriptase, EC 2.7.7.49) only in that it has two additional aminoterminal amino acids. This protein is soluble in E. coli extracts, is active in reverse transcriptase assays, and shows inhibition profiles with dideoxy-TTP and dideoxy-GTP that are indistinguishable from the viral enzyme. The deletion of 23 amino-terminal or carboxyl-terminal amino acids or the insertion of 5 amino acids at position 143 substantially decreases the polymerizing activity of the HIV reverse transcriptase made in E. coli. The properties of a 51-kDa reverse transcriptaserelated protein made in E. coli suggests that the p51 found in the virion probably does not have substantial polymerizing activity. The full-length HIV reverse transcriptase and the various mutant proteins produced in E. coli should be quite useful for structural and biochemical analyses as well as for the production of antibodies. Retroviruses encode an enzyme, RNA-dependent DNA polymerase (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase or reverse transcriptase, EC 2.7.7.49), that is responsible for copying the RNA genome found in the virion into DNA. This copying of viral RNA into DNA is an obligate step in the viral life cycle, and the DNA copy is subsequently inserted into the genome of the infected cell (1). No cellular homolog of viral reverse transcriptases has been discovered, although many normal cells carry endogenous retroviruses and retrotransposons that encode reverse transcriptases. The viral reverse transcriptase is, for this reason, a potential target for drug therapies designed to interfere with the life cycle of retroviruses, most notably the human immunodeficiency virus (HIV) (2-9). The drug 3'azido-3'deoxythymidine (also called "AZT") is an effective anti viral agent presumably because the derivative triphosphate is recognized as thymidine triphosphate by HIV reverse transcriptase (4-6). Only small amounts of reverse transcriptase are found in virions, and the search for drugs that would interrupt the HIV viral life cycle by interfering with reverse transcription would be facilitated by a better source of the HIV enzyme. In the virion, the reverse transcriptase is released from a larger polypeptide precursor by proteolytic cleavage (1). To permit the expression of the free HIV reverse transcriptase, we have taken the viral segment encoding the reverse transcriptase and modified the region at the ends of this segment so that there are initiation and termination codons at the positions where proteolytic cleavage takes place. The modified segment was introduced into an Escherichia coli expression plasmid. When introduced into E. coli, the plasmid with the HIV reverse transcriptase insert induces the synthesis of a large amount (several percent of the total E. coli protein) of soluble, enzymatically active, HIV reverse transcriptase. We show here that the ability of the HIV reverse transcriptase produced in E. coli to be inhibited by the dideoxy analogs of thymidine and guanosine is identical to the inhibition of virion reverse transcriptase. To study the catalytic domains of the HIV reverse transcriptase, we have begun a mutagenic analysis of this enzyme using the E. coli system. The E. coli-produced HIV polymerase can be used to search for inhibitors, to facilitate structural and biochemical studies, and to produce both monoclonal and polyclonal antibodies. MATERIALS AND METHODS Bacteria and Plasmids. E. coli strain DH5 (a derivative of DH-1, see ref. 10) was used as a recipient for DNA transformations (frozen competent cells were purchased from Bethesda Research Laboratories). The HIV DNA polymerase gene constructs were derived from the HIV proviral clone BH10 (11). The construction of the plasmid is given in detail in Fig. 1. E. coli DH5 cells transformed with the various plasmids were grown in NZYM liquid broth (25) supplemented with 100 Ag of ampicillin per ml. Virus. Twice-banded HIV-H9 was obtained from Program Resources, Frederick, MD (batch p2627). Bacterial and Viral Lysis. The bacteria containing either plasmids that express HIV reverse transcriptase or the parental plasmid pUC12N were grown for 12-16 hr with shaking at 370C. Bacteria were collected by centrifugation in a Microfuge for 2 min at 10,000 rpm, and the pellet was washed once with cold 100 mM NaCl/20 mM Tris chloride/i mM EDTA, final pH 7.4 (TSE buffer). Bacteria were then disrupted in 0.2 M NaCl/20% (vol/vol) glycerol/1% Triton X-100/1 mM EDTA/2 mM dithiothreitol/25 mM Tris chloride, pH 8.0 (lysis buffer). The lysates were kept at 4°C for 15 min. The insoluble material was removed by centrifugation at 10,000 rpm in a Microfuge. The supernatant was collected and stored at 20°C. No significant decrease in DNA polymerase activity was observed after several freezings and thawings. Usually a bacterial pellet from 1 volume of stationary culture was disrupted in 1/4 volume of the lysis buffer. The protein concentration in the supernatants of the lysed cells (as measured by the Bio-Rad protein assay) was 1-1.5 mg/ml. The insoluble material must be removed after lysis by centrifugation before the RNA-dependent DNA polymerase activity can be specifically assayed in the disrupted bacteria. Abbreviations: HIV, human immunodeficiency virus; Mo-MuLV, Moloney murine leukemia virus; RSV, Rous sarcoma virus. *Permanent address: Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. 1218 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 85 (1988) 1219 BclBal Kpn Kln Bal Nde