Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants.

Abstract

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. 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.

Michellamine B, a novel plant alkaloid, inhibits human immunodeficiency virus-induced cell killing by at least two distinct mechanisms

“…X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) was also purchased from Sigma. Polymerase assays utilized the reagents poly(rAdT) [12][13][14][15][16][17][18] , poly(rC-dG) [12][13][14][15][16][17][18] , and poly(dT-dA) [12][13][14][15][16][17][18] (Pharmacia LKB, Piscataway, N.J.) and 16S-23S rRNA (Boehringer Mannheim, Indianapolis, Ind.) and random nonamer primers (Synthetic Genetics, San Diego, Calif.).…”

“…All RTs used in this study were recombinant enzymes expressed in Escherichia coli. The HIV-1 and HIV-2 RT expression plasmids have been described elsewhere (16,17). The two variant HIV RTs used in this study were a Tyr-181 3 Ile RT (⌬181) and a Tyr-188 3 Leu RT (⌬188).…”

“…The preparation, enzymatic activities, and drug sensitivities of these two variant RTs have been previously described (5, 6). The three template-primer systems used to evaluate the inhibition of the various RT preparations by michellamine B consisted of poly(rA-dT) [12][13][14][15][16][17][18] , poly(rC-dG) [12][13][14][15][16][17][18] , and rRNA-primer. Recombinantderived DNA polymerases ␣ and ␤ were the kind gift of Sam Wilson (University of Texas Medical Branch, Center for Molecular Sciences).…”

A possible role for cysteine residues in the fidelity of DNA synthesis exhibited by the reverse transcriptases of human immunodeficiency viruses type 1 and type 2

“…All RTs wild type and mutant used in this study were recombinant enzymes expressed by us in E. coll[l [3][4][5][6][7][8][9][10][11][12][13][14][15] and purified to homogeneity according to Clark et al [16]. The specific activities of the different RTs used were 4,000-5,000 units per/.t$.…”

Mutational studies of human immunodeficiency virus type 1 reverse transcriptase : the involvement of residues 183 and 184 in the fidelity of DNA synthesis

“…HIV-1 RT were recombinant enzymes of the BH-10 isolate of H1V-1 RT [23]. The purified proteins were kindly provided by Drs.…”