Rubin H, Wang Z M, Nickbarg E B, McLarney S, Naidoo N, Schoenberger O L, Johnson J L, Cooperman B S
Department of Medicine, University of Pennsylvania, Philadelphia 19104-6073.
J Biol Chem. 1990 Jan 15;265(2):1199-207.
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.
人α1-抗糜蛋白酶已被克隆、测序，并在大肠杆菌中表达，重组蛋白以及位点特异性突变体已被纯化和鉴定。经校正的基因推导氨基酸序列与α1-蛋白酶抑制剂的总体一致性为45%，高于先前报道的42%（钱德拉，T.，斯塔克豪斯，R.，基德，V. J.，罗布森，J. H.，和吴，S. L. C.（1983年）《生物化学》22，5055 - 5060）。重组抗糜蛋白酶（rACT）在与蛋白酶相互作用的特异性方面与天然抗糜蛋白酶相似。它与牛胰凝乳蛋白酶结合的二级速率常数为6 - 8×10⁵ M⁻¹ s⁻¹，与血清来源的抑制剂相同。位点特异性诱变已用于产生rACT的两种变体，其中P1位从亮氨酸变为甲硫氨酸（L358M - rACT）或精氨酸（L358R - rACT）。L358M - rACT对丝氨酸蛋白酶的抑制活性特异性与天然rACT非常相似。相比之下，L358R - rACT的特异性与天然rACT有很大不同，最显著的是它能有效抑制胰蛋白酶和人凝血酶，而抑制胰凝乳蛋白酶的能力下降。
