Holwerda B C, Wittwer A J, Duffin K L, Smith C, Toth M V, Carr L S, Wiegand R C, Bryant M L
Searle Research and Development, St. Louis, Missouri 63198.
J Biol Chem. 1994 Oct 14;269(41):25911-5.
The human cytomegalovirus UL80 protease was expressed in Escherichia coli and purified by metal-chelate chromatography using a histidine tag engineered at the amino terminus. Cleavage of the 30-kDa protease at an internal site, VEA/A144, resulted in the recovery of 16- plus 14-kDa two-chain protease. The amino-terminal 16-kDa chain and the carboxyl-terminal 14-kDa chain remained associated as an active enzyme that was modified specifically at Ser132 on the 16-kDa chain by [3H]diisopropyl fluorophosphate. Disruption of the cleavage site by mutation from VEA/A to AEA/A facilitated the recovery of active 30-kDa one-chain enzyme that could be similarly modified at Ser132 by [3H]diisopropyl fluorophosphate. Both one- and two-chain enzymes cleaved recombinant assembly protein at the maturation site, VNA/S, and a peptide, GVVNASARL, mimicking this site. Internal processing does not inactivate the protease but forms a two-chain enzyme that retains activity.
人巨细胞病毒UL80蛋白酶在大肠杆菌中表达，并利用在氨基末端设计的组氨酸标签通过金属螯合层析进行纯化。30 kDa蛋白酶在内部位点VEA/A144处切割，得到了16 kDa加14 kDa的双链蛋白酶。氨基末端的16 kDa链和羧基末端的14 kDa链保持结合状态，形成一种活性酶，该酶在16 kDa链上的Ser132处被[3H]二异丙基氟磷酸特异性修饰。通过将切割位点从VEA/A突变为AEA/A来破坏该位点，有助于回收活性30 kDa单链酶，该酶同样可以被[3H]二异丙基氟磷酸在Ser132处修饰。单链酶和双链酶都在成熟位点VNA/S处切割重组装配蛋白，以及切割模拟该位点的肽GVVNASARL。内部加工不会使蛋白酶失活，而是形成一种保留活性的双链酶。
