LABORATORY-SCALE PRODUCTION AND PURIFICATION OF RECOMBINANT HIV-1 REVERSE-TRANSCRIPTASE

HIV-1 reverse transcriptase from the HIV-1 strain WMF 1.13 was express ed in Escherichia coli JM 105 using a pKK233-2 vector. The bacteria we re cultivated in a 20-1 fermenter with 14-1 net volume using M9ZB medi um containing bactotryptone and yeast extract. After induction of reve rse transcriptase (RT) expression by addition of isopropyl-beta-D-thio galactopyranoside the enzyme concentration was monitored. Both soluble and inclusion-body deposited RT were detected by Western blots. Inclu sion-body formation was confirmed by transmission electron microscopy. Further purification of soluble and insoluble RT was investigated. Af ter cell desintegration by enzymatic treatment combined with osmotic s hock and centrifugation, the supernatant was desalted by size-exclusio n chromatography and further purified by DEAE-Sepharose FF, AF-Heparin Toyopearl 650 M and Fractogel EMD TMAE 650 (S). The results of the pu rification steps were monitored by SDS-PAGE with silver staining, non- radioactive RT assay and protein determination with Coomassie Blue. Th e sediment was extracted with 6 M GuHCl and after clarification and co nventional refolding, treated in the same manner as soluble RT. This m ethod is well suited for studying fermentation conditions as well as p urification conditions. The RT is expressed in approximately equal amo unts as soluble and insoluble enzyme.