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

A. Hizi, C. McGill, S. H. Hughes

Research output: Contribution to journalArticlepeer-review

Abstract

We have constructed a plasmid that, when introduced into Escherichia coli, 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 amino-terminal 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 transcriptase-related 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.

Original languageEnglish
Pages (from-to)1218-1222
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number4
DOIs
StatePublished - 1988
Externally publishedYes

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