Overproduction of human immunodeficiency virus type I reverse transcriptase in Escherichia coli and purification of the enzyme.
Overexpression of the reverse transcriptase was designed in E. coli. For a high level of expression, HIV protein was expressed as a protein fusion with beta-galactosidase. When the proviral DNA fragment covering the 3' half of the gag gene and the entire pol gene was ligated to the 3' end of the lacZ gene to fuse the truncated gag to lacZ in frame, a small quantity of reverse transcriptase was produced, indicating that frameshifting and post-translational processing have occurred. Much more reverse transcriptase was produced when the entire pol region was directly fused to the lacZ gene. From a one liter culture of bacteria, 1 mg of highly purified reverse transcriptase consisting of approximately equimolar amounts of two species (p64 and p51) was obtained. These proteins had identical N-termini consistent with the deduced amino acid sequence and therefore, might be correctly processed from the fusion protein in E. coli by the protease encoded by the pol region. The purified reverse transcriptase was enzymatically as active as the enzyme purified from the virus particles, and immunoreactive to the sera of HIV carriers with high sensitivity and specificity.
Department of Experimental Chemotherapy, Osaka University.
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Saitoh
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Iwasaki
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Nakata
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Adachi
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Shinagawa
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015483:HIV Antibodies / Q000276:immunology; D015658:HIV Infections / Q000175:diagnosis; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D010957:Plasmids; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D001616:beta-Galactosidase / Q000235:genetics
D015483:HIV Antibodies; D011993:Recombinant Fusion Proteins; D012194:RNA-Directed DNA Polymerase; D001616:beta-Galactosidase; D010450:Endopeptidases