Shibuya K, Kaji H, Ohyama Y, Tate S, Kainosho M, Inagaki F, Samejima T
Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Tokyo.
J Biochem. 1995 Sep;118(3):635-42. doi: 10.1093/oxfordjournals.jbchem.a124957.
The expression system for human recombinant cystatin A has already been established to be a fusion protein with porcine adenylate kinase in Escherichia coli [Kaji et al. (1990) Biol. Chem. Hoppe-Seyler 371, Suppl., 145-150]. After cyanogen bromide cleavage of the fused protein expressed in E. coli, the cystatin portion could be readily isolated. The inhibitory activity of the obtained variant (Cyst A (2-98)) was found to be almost identical with that of the wild type, and thereafter a mutation was introduced into this variant (Ctst A(2-98)), called the standard variant. To elucidate the role of the Gly-4 residue, which is completely conserved in all cystatin species, this residue was substituted with 17 other amino acids by means of cassette mutagenesis. Thus 17 variants (Cyst A(2-98)[G4X]) obtained were examined as to their inhibitory activity towards papain. As the side chain of the substituted amino acid residue became more bulky, the inhibitory activity of the variant markedly decreased. Variants whose side chains were bulkier than a Val residue showed almost no inhibitory effect towards papain. Consequently, it was deduced that the large side chain of a substituted amino acid may cause steric hindrance, which may be responsible for the decrease in inhibitory activity. Thus, we could conclude that the 4th (Gly) residue on cystatin A must be small, because amino acids which existed on the N-terminal side of this residue could interact with a papain molecule.
人重组胱抑素A的表达系统已被确立为在大肠杆菌中与猪腺苷酸激酶形成的融合蛋白[Kaji等人（1990年），《生物化学与霍普-赛勒》371，增刊，145 - 150页]。在对大肠杆菌中表达的融合蛋白进行溴化氰切割后，胱抑素部分能够很容易地分离出来。发现所获得的变体（胱抑素A（2 - 98））的抑制活性与野生型几乎相同，此后对这个变体（Ctst A(2 - 98)）进行了突变，该变体被称为标准变体。为了阐明在所有胱抑素物种中完全保守的甘氨酸 - 4残基的作用，通过盒式诱变将该残基替换为其他17种氨基酸。因此，对所获得的17个变体（胱抑素A(2 - 98)[G4X]）针对木瓜蛋白酶的抑制活性进行了检测。随着被取代氨基酸残基的侧链变得更大，该变体的抑制活性显著降低。侧链比缬氨酸残基更大的变体对木瓜蛋白酶几乎没有抑制作用。因此，推断被取代氨基酸的大侧链可能会造成空间位阻，这可能是抑制活性降低的原因。因此，我们可以得出结论，胱抑素A上的第4位（甘氨酸）残基必须很小，因为该残基N端一侧存在的氨基酸可能会与木瓜蛋白酶分子相互作用。
