[The study on purification and characterization of HIV-1 reverse transcriptase from a recombinant strain of E. coli].
Of the HIV proteins, reverse transcriptase(RT) has been probably the most useful target protein for screening and designing of its specific inhibitors. Because retroviral replication is absolutely dependent on both the RNase H and the polymerase function of RT and, so far as is now known, RT does not play a direct role in the life cycle of a normal cell. Under suitable fermentation conditions in our experiments, HIV-1 RT was highly expressed in E. coli JM109(pKRT-2)* by inducing the trc promoter with isopropyl-beta-Dthiogalactopyranoside(IPTG). 1. 1 mg of purified RT was obtained from one liter culture of bacteria by DEAE-cellulose and phosphaellulose chromatography. SDS-PAGE analysis of the purified RT showed two major protein bands of 66 kD and 51 kD, indicating that the purified RT was a heterodimer composed of two subunits. Results of enzyme assay showed that the purified RT had high activity(1.4 x 10(4) umit/mg). We also improved the reaction system of enzyme assay. The effect of PFA on HIV-1 RT was determined with the improved enzyme assay and the mechanism of inhibition was non-competitive with respect to substrate consistent with the reports of Dr. Bo Oberg. This suggests that the purified HIV-1 RT by this simple method can be applied to the anti HIV-1-drug screening. (*E. coli JM109(pKRT2) was obtained from NIAID, NIH; pKRT2 from Dr. Richard D'Aquila and Dr. William C. Summers.)
Jia
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Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100050.
Tao
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D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase