γ-Echistatin的基因合成，表現、純化及結構、功能的探討

Abstract

我們利用PCR的技術，合成γ-echistatin的基因，並將此基因轉殖到pQE-30載體上，再將載體送入大腸桿菌（Escherichia coli）以表現所要探討的抗凝血蛇毒—γ-echistatin。轉殖後的菌落，以PCR篩選出含有γ-echistatin基因菌體，再利用核酸定序儀以確定送入的基因為正確。 篩選出的大腸桿菌，以IPTG誘導菌體，大量表現出C端含有γ-echistatin勝?鍵的蛋白。我們先利用親和性管柱（Ni-NTA column）將此蛋白純化出來，再經還原劑、透析處理之後，以蛋白?enterokinase將C端的γ-echistatin切下，在enterokinase緩衝液的條件下，切下的γ-echistatin將自行摺疊成具活性的結構。此摺疊完成的γ-echistatin以HPLC純化出來，經質譜儀（mass spectrometry）、圓偏光二色光譜（circular dichroism）、和抗凝血活性的測試，其結果皆與原先由毒蛇身上萃取出來的γ-echistatin具有相同的性質。 我們利用點突變（Site-directed mutagenesis）的技術得到兩個突變株，其中一株表現出的蛋白質（R24N），是將第24個胺基酸由精胺酸（arginine）變成了天門冬醯胺（asparagine），另外一株表現的蛋白（K45E）是將第45個胺基酸由離胺酸（lysine）變成了麩胺酸（glutamic acid）。 由於γ-echistatin是屬於含有精胺酸－甘胺酸－天門冬胺酸序列（RGD sequence） 的抗凝血蛇毒（disintegrin），若將其中的精胺酸改成天門冬醯胺，經本實驗結果證實，抗凝血的活性將降低250倍以上。根據過去的實驗，將γ-echistatin的C端，從第46個胺基酸之後切除，活性降低1．7倍，若從第45個胺基酸離胺酸之後切除，則活性降低15倍，並且我們從電腦模擬的γ-echistatin結構看出，此離胺酸與RGD sequence非常的靠近，因此推斷第45個胺基酸在活性上可能扮演重要的角色。將第45個胺基酸由原本的離胺酸變成了麩胺酸，實驗的結果指出活性大約降低2倍。因此我們推論，第45個胺基酸可能和其末端的胺基酸有協同的作用，使得γ-echistatin對於血小板上的細胞膜蛋白integrin αIlbβ3有較強的親和力，而抑制血小板的凝集。

A gene encoding an RGD-containing platelet aggregation inhibitor, γ-echistatin, has been synthesized through PCR method using four overlapping oligonucleotides. The synthetic gene has Hind III sites at both ends for cloning into pQE-30 expression vector and an (Asp)4-Lys coding sequence recognized by enterokinase to cleave the fusion protein. The recombinant expression vector was transferred into M15[pREP4] competent cells, the positive clones were identified by PCR and verified by DNA sequence analysis. After over-expression by inducing with IPTG, crude γ-echistatin fusion protein was purified through Ni-NTA column. The crude fusion protein was first denatured and reduced to prevent mis-linkage of disulfide bonds. Then γ-echistatin fusion protein was cleaved by enterokinase and refolded. The recombinant, mature γ-echistatin was purified to homogeneity by HPLC, and verified by CD spectrum and mass spectrometry. This recombinant γ-echistatin was also assayed for inhibiting platelet aggregation and found to be identical to that of native γ-echistatin. We also constructed the mutants of γ-echistatin, K45E and R24N, by site directed mutagenesis. As our previous research pointed out, Lys45 might play an important role in platelet aggregation inhibition because the inhibitory potency of des(46-49)-γ-echistatin decrease 1.7-fold whereas that of des(45-49)-γ-echistatin is 15-fold less than native γ-echistatin. Furthermore, on the basis of the structural model of γ-echistatin, Lys45 is situated near the RGD loop and facing the same side of the molecule. Nevertheless, the inhibitory potency of K45E mutant is only 2-fold less than the wild type. It implies that Lys45 need to cooperate with other C-terminal residues for the effect of platelet aggregation inhibition instead of acting alone. The R24N mutant, as expected, had little activity in inhibiting platelet aggregation.