Le Grice S F, Grüninger-Leitch F
Central Research Units, F. Hoffman La-Roche & Co. Ltd, Switzerland.
Eur J Biochem. 1990 Jan 26;187(2):307-14. doi: 10.1111/j.1432-1033.1990.tb15306.x.
We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.
我们对一个表达66 kDa HIV-1逆转录酶（p66）的大肠杆菌载体进行了改造，使其能同时表达该逆转录酶和由pol基因编码的蛋白酶。这个双表达盒能产生大量的逆转录酶和蛋白酶；然而，在这些条件下，50%的过表达p66逆转录酶会被加工，导致大量p66/p51酶的积累。此外，在逆转录酶编码序列的氨基末端添加一个聚组氨酸亲和标签（His-p66），使得通过金属螯合亲和层析能够从粗裂解物中简单、快速地纯化出毫克量的p66或p66/p51酶。纯化后的His-p66和His-p66/His-p51逆转录酶同时具有逆转录酶和核糖核酸酶H活性。通过金属螯合层析对仅p66组分被标记的p66/p51酶进行纯化，进一步支持了异二聚体存在的观点。
