Institute of Biological Chemistry, Academia Sinica and Core Facility of Recombinant Protein Production, Taipei, Taiwan.
Biotechniques. 2009 Dec;47(6):1029-32. doi: 10.2144/000113303.
A 3C-like protease (3CLpro) from the severe acute respiratory syndrome-coronavirus (SARS-CoV) is required for viral replication, cleaving the replicase polyproteins at 11 sites with the conserved Gln [downward arrow](Ser, Ala, Gly) sequences. In this study, we developed a mutant 3CLpro (T25G) with an expanded S1' space that demonstrates 43.5-fold better k(cat)/K(m) compared with wild-type in cleaving substrates with a larger Met at P1' and is suitable for tag removal from recombinant fusion proteins. Two vectors for expressing fusion proteins with the T25G recognition site (Ala-Val-Leu-Gln [downward arrow]Met) in Escherichia coli and yeast were constructed. Identical cloning sites were used in these vectors for parallel cloning. PstI was chosen as a 5' cloning site because it overlapped the nucleotide sequence encoding the protease site and avoided addition of extra amino acids at the N terminus of recombinant proteins. 3CL(pro) (T25G) was found to have a 3-fold improvement over TEV(pro) in tag cleavage at each respective preferred cleavage site.
一种来自严重急性呼吸系统综合征冠状病毒（SARS-CoV）的 3C 样蛋白酶（3CLpro）对于病毒复制是必需的，它可以在 11 个位点切割复制酶多蛋白，其保守的 Gln[↓]（Ser、Ala、Gly）序列。在这项研究中，我们开发了一种突变的 3CLpro（T25G），其 S1'空间扩大，与野生型相比，在切割 P1'位带有更大 Met 的底物时，k(cat)/K(m)提高了 43.5 倍，并且适合从重组融合蛋白中去除标签。构建了两个用于在大肠杆菌和酵母中表达带有 T25G 识别位点（Ala-Val-Leu-Gln[↓]Met）的融合蛋白的载体。这些载体使用相同的克隆位点进行平行克隆。选择 PstI 作为 5'克隆位点，因为它与编码蛋白酶位点的核苷酸序列重叠，并且避免在重组蛋白的 N 末端添加额外的氨基酸。在各自的首选切割位点，3CL(pro)（T25G）在标签切割方面比 TEV(pro)提高了 3 倍。
