Cloning, expression, purification, and biological activity of recombinant native and variant human alpha 1-antichymotrypsins.
Cloning, expression, purification, and biological activity of recombinant native and variant human alpha 1-antichymotrypsins.
复制标题
DOI：
10.1016/s0021-9258(19)40178-6
复制
发表时间：
1990-01
期刊：
影响因子：
--
通讯作者：
H. Rubin;Z. M. Wang;E. Nickbarg;S. Mclarney;N. Naidoo;O. Schoenberger;J. Johnson;B. Cooperman
中科院分区：
文献类型：
--
作者：
H. Rubin;Z. M. Wang;E. Nickbarg;S. Mclarney;N. Naidoo;O. Schoenberger;J. Johnson;B. Cooperman
人α 1-抗糜蛋白酶已被克隆，测序和表达在大肠杆菌和重组蛋白以及点特异性突变体已被纯化和表征。校正的基因推导的氨基酸序列与α 1-蛋白酶抑制剂具有45%的总体同一性，这高于先前报道的42%（Chandra，T.，斯塔克豪斯河基德，V. J.，罗布森，J.H.，和Woo，S. L. C.（1983）Biochemistry 22，5055-5060）。重组抗胰凝乳蛋白酶（rACT）在其与蛋白酶的相互作用的特异性方面类似于天然抗胰凝乳蛋白酶。其与牛胰凝乳蛋白酶结合的二级速率常数为6-8 × 10（5）M-1 s-1，与血清衍生抑制剂相同。定点突变已用于产生rACT的两种变体，其中P1位置已从亮氨酸改变为甲硫氨酸（L358 M-rACT）或精氨酸（L358 R-rACT）。L358 M-rACT对丝氨酸蛋白酶的抑制活性的特异性与天然rACT非常相似。相比之下，L358 R-rACT的特异性与天然rACT的特异性完全不同，最显著的是有效抑制胰蛋白酶和人凝血酶，同时显示出降低的抑制胰凝乳蛋白酶的能力。
Human alpha 1-antichymotrypsin has been cloned, sequenced and expressed in Escherichia coli and recombinant protein as well as point-specific mutants have been purified and characterized. The corrected gene-deduced amino acid sequence has 45% overall identity with alpha 1-protease inhibitor, which is higher than the 42% previously reported (Chandra, T., Stackhouse, R., Kidd, V. J., Robson, J. H., and Woo, S. L. C. (1983) Biochemistry 22, 5055-5060). Recombinant antichymotrypsin (rACT) is similar to natural antichymotrypsin with respect to the specificity of its interactions with proteases. Its second-order rate constant for association with bovine chymotrypsin is 6-8 x 10(5) M-1 s-1, which is identical to that of the serum-derived inhibitor. Site-specific mutagenesis has been used to produce two variants of rACT in which the P1 position has been changed from leucine to either methionine (L358M-rACT) or arginine (L358R-rACT). L358M-rACT has a specificity of inhibitory activity toward serine proteases closely similar to that of native rACT. By contrast, the specificity of L358R-rACT is quite different from that of native rACT, most notably in efficiently inhibiting trypsin and human thrombin while showing a decreased ability to inhibit chymotrypsin.