Ivanoff L A, Towatari T, Ray J, Korant B D, Petteway S R
Proc Natl Acad Sci U S A. 1986 Aug;83(15):5392-6. doi: 10.1073/pnas.83.15.5392.
We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector. The 3C region is a virus-specific protease, which, when expressed in E. coli, is shown to be active and autocatalytic. In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C). Mutants in the 3C region have been constructed by oligonucleotide-directed mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease. The mutation of highly conserved residues (cysteine-47 or histidine-161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity.
我们已将脊髓灰质炎病毒基因组的一段序列（核苷酸5438 - 6061）构建到一个大肠杆菌表达载体中，该序列编码病毒多聚蛋白3C区域的183个氨基酸残基和3D区域的25个氨基酸残基。3C区域是一种病毒特异性蛋白酶，在大肠杆菌中表达时显示具有活性且能自我催化。在我们的系统中，产生了三种脊髓灰质炎病毒特异性蛋白：一种前体多聚蛋白（3C - 3D）、一种内部起始产物和成熟蛋白酶（3C）。通过寡核苷酸定向诱变构建了3C区域的突变体，并通过成熟蛋白酶的体内产生来测定它们对蛋白水解活性的影响。高度保守残基（半胱氨酸 - 47或组氨酸 - 161）的突变产生了无活性的酶，而非保守残基（半胱氨酸 - 153）的突变对蛋白水解活性的影响可忽略不计。
