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荞麦胰蛋白酶抑制剂(BTI)属于丝氨酸蛋白酶抑制剂Potato I家族,典型构象中有1段暴露在分子外侧的结合区,该区内P1’、P2、P6’与P8’位的氨基酸残基具有高度保守性.本文依据近期解析的rBTI晶体结构以及rBTI与胰蛋白酶复合物晶体结构信息,对rBTI中的P2和P8’位氨基酸进行突变,构建了pExSecI-Bti-P44T和pExSecI-Bti-W53R重组质粒,转入大肠杆菌BL21(DE3)中进行表达,通过Resource Q阴离子交换层析和Superdex G 75 HR 10/300凝胶柱进行分离纯化后,测定了rBTI及其突变体对胰蛋白酶的抑制常数,以及它们对HepG2细胞内的蛋白酶体和细胞增殖的抑制作用.实验结果显示,rBTI的44和53位分别突变为Thr和Arg后,Ki分别为2.91×10-9mol/L和2.97×10-7mol/L,前者较rBTI(Ki=3.56×10-8 mol/L)降低1个数量级,而后者较rBTI升高1个数量级.功能分析显示,rBTI及2种突变体对HepG2细胞内的蛋白酶体基本没有抑制作用,但是它们都保留了对HepG2细胞增殖的抑制活性.这些结果揭示,作为一种特异的胰蛋白酶抑制剂,rBTI分子中的保守区域氨基酸残基虽然对胰蛋白酶的抑制作用有显著影响,但并不影响其抑制肿瘤细胞的增殖,仍能发挥其原有的生物学功能.
Buckwheat trypsin inhibitor (BTI) belongs to the Potato I family of serine protease inhibitors. In the typical conformation, there is a segment exposed at the outer side of the molecule. The amino acid residues at P1 ’, P2, P6’ and P8 ’ Highly conserved.This paper based on the recent analysis of the rBTI crystal structure and rBTI and trypsin complex crystal structure information, the rBTI P2 and P8 ’amino acids were mutated to construct pExSecI-Bti-P44T and pExSecI-Bti-W53R recombination The plasmid was transformed into E. coli BL21 (DE3) for expression. After purification by Resource Q anion exchange chromatography and Superdex G 75 HR 10/300 gel column, the inhibitory constant of trypsin by rBTI and its mutants , And their inhibitory effects on the proteasome and cell proliferation in HepG2 cells.The results showed that the mutation rates of Thr and Arg at positions 44 and 53 of rBTI were 2.91 × 10-9 mol / L and 2.97 × 10- 7mol / L, the former is one order of magnitude lower than that of rBTI (Ki = 3.56 × 10-8 mol / L), while the latter is one order of magnitude higher than that of rBTI.Functional analysis showed that rBTI and two kinds of mutants could inhibit protease activity in HepG2 cells The body has basically no inhibitory effect, but they all retain the right HepG2 cell proliferation. These results revealed that, as a specific trypsin inhibitor, the conserved region of amino acid residues in rBTI molecules have a significant effect on the inhibitory effect of trypsin, but does not affect the inhibition of tumor cells Proliferation, still able to play its original biological function.