Parks T D, Howard E D, Wolpert T J, Arp D J, Dougherty W G
Department of Microbiology, Oregon State University, Corvallis 97331-3804, USA.
Virology. 1995 Jun 20;210(1):194-201. doi: 10.1006/viro.1995.1331.
The tobacco etch virus 27-kDa nuclear inclusion a (NIa) proteinase was expressed in Escherichia coli as a recombinant fusion protein containing a seven-histidine tag at the amino-terminus. Catalytically active and inactive (by virtue of a single amino acid change) forms of the proteinase were purified to homogeneity in a two-column chromatographic procedure. The active form of the proteinase was slowly converted to a lower molecular weight form, while the inactive form was not. This conversion was dilution independent and thought to be intramolecular. Isolation of the approximately 2-kDa peptide cleavage product and determination of its N-terminal amino acid sequence positioned the cleavage site 24 amino acids from the carboxy-terminus of the proteinase. A recombinant NIa proteinase lacking the C-terminal 24 amino acids was shown to possess limited activity. Kinetic analyses of cleavage of a synthetic peptide by the full-length or truncated proteinase were conducted and indicated that the Km of the truncated proteinase was approximately fourfold higher than that of the full-length form. The truncated proteinase was approximately one-twentieth as efficient in proteolysis of the test peptide substrate as the full-length form.
烟草蚀纹病毒27 kDa核内含物a（NIa）蛋白酶在大肠杆菌中作为一种在氨基末端含有七组氨酸标签的重组融合蛋白表达。通过两步柱层析法将具有催化活性和无活性（由于单个氨基酸变化）的蛋白酶形式纯化至同质。蛋白酶的活性形式缓慢转化为较低分子量形式，而无活性形式则不会。这种转化与稀释无关，被认为是分子内的。分离出约2 kDa的肽裂解产物并确定其N端氨基酸序列，将裂解位点定位在蛋白酶羧基末端的24个氨基酸处。缺失C端24个氨基酸的重组NIa蛋白酶显示出有限的活性。对全长或截短蛋白酶切割合成肽的动力学分析表明，截短蛋白酶的Km约为全长形式的四倍。截短蛋白酶对测试肽底物的蛋白水解效率约为全长形式的二十分之一。
