Denaturation/refolding of purified recombinant HIV reverse transcriptase yields monomeric enzyme with high enzymatic activity.
We engineered a prokaryotic expression vector encoding the HIV reverse transcriptase (RT). We grew Escherichia coli JM109 carrying the vector in a 250-liter stirred tank fermentor and purified RT (p66) under native conditions to apparent homogeneity. Purified p66 (greater than or equal to 5 mg/ml) was not stable, and was rapidly processed to its 51 kD derivative (p51), until p66:p51 levels were approximately 1:1. These latter RT preparations were chromatographed as heterodimers and had approximately fivefold higher specific RT enzymatic activities compared with those containing predominantly p66. P66 purified under dilute concentrations (less than or equal to 0.5 mg/ml) was monomeric in solution, resistant to p51 processing for weeks at 4 degrees C, but also had low specific RT enzymatic activities. To attempt the preparation of homogeneous p66 with specific RT enzymatic activities equivalent to p66:p51 heterodimers, purified heterodimers were denatured and p66 was purified and refolded during extensive dialysis (refolded p66). Refolded p66 (less than or equal to 0.5 mg/ml) was monomeric in solution and had identical specific RT enzymatic activities, Km for dTTP, and inhibition by 3'-azido-3'-deoxythymidine triphosphate compared with heterodimeric p66:p51 RT. The data indicates that HIV RT obtained from recombinant E. coli under native conditions is extensively processed at concentrations promoting dimerization. Moreover, RT denaturation and refolding yields apparently homogeneous monomeric p66, with specific RT enzymatic activities equivalent to heterodimeric RT.
Division of Biopolymer Chemistry, Upjohn Company, Kalamazoo, MI 49001.
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Deibel
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TJ
McQuade
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Brunner
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Tarpley
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D006678:HIV / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase