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Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants. 1988

A Hizi, and C McGill, and S H Hughes
National Cancer Institute-Frederick Cancer Research Facility, Bionetics Research Inc.-Basic Research Program, MD 21701.

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.

UI MeSH Term Description Entries
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D004790 Enzyme Induction An increase in the rate of synthesis of an enzyme due to the presence of an inducer which acts to derepress the gene responsible for enzyme synthesis. Induction, Enzyme
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006678 HIV Human immunodeficiency virus. A non-taxonomic and historical term referring to any of two species, specifically HIV-1 and/or HIV-2. Prior to 1986, this was called human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV). From 1986-1990, it was an official species called HIV. Since 1991, HIV was no longer considered an official species name; the two species were designated HIV-1 and HIV-2. AIDS Virus,HTLV-III,Human Immunodeficiency Viruses,Human T-Cell Lymphotropic Virus Type III,Human T-Lymphotropic Virus Type III,LAV-HTLV-III,Lymphadenopathy-Associated Virus,Acquired Immune Deficiency Syndrome Virus,Acquired Immunodeficiency Syndrome Virus,Human Immunodeficiency Virus,Human T Cell Lymphotropic Virus Type III,Human T Lymphotropic Virus Type III,Human T-Cell Leukemia Virus Type III,Immunodeficiency Virus, Human,Immunodeficiency Viruses, Human,Virus, Human Immunodeficiency,Viruses, Human Immunodeficiency,AIDS Viruses,Human T Cell Leukemia Virus Type III,Lymphadenopathy Associated Virus,Lymphadenopathy-Associated Viruses,Virus, AIDS,Virus, Lymphadenopathy-Associated,Viruses, AIDS,Viruses, Lymphadenopathy-Associated
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D012191 Retroviridae Proteins Proteins from the family Retroviridae. The most frequently encountered member of this family is the Rous sarcoma virus protein. Leukovirus Proteins,Retrovirus Proteins,Proteins, Leukovirus,Proteins, Retroviridae,Proteins, Retrovirus
D012194 RNA-Directed DNA Polymerase An enzyme that synthesizes DNA on an RNA template. It is encoded by the pol gene of retroviruses and by certain retrovirus-like elements. EC 2.7.7.49. DNA Polymerase, RNA-Directed,RNA-Dependent DNA Polymerase,Reverse Transcriptase,RNA Transcriptase,Revertase,DNA Polymerase, RNA Directed,DNA Polymerase, RNA-Dependent,RNA Dependent DNA Polymerase,RNA Directed DNA Polymerase
D018894 Reverse Transcriptase Inhibitors Inhibitors of reverse transcriptase (RNA-DIRECTED DNA POLYMERASE), an enzyme that synthesizes DNA on an RNA template. Reverse Transcriptase Inhibitor,Inhibitors, Reverse Transcriptase,Inhibitor, Reverse Transcriptase,Transcriptase Inhibitor, Reverse

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