γ-Echistatin 突變株種的探討

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（circula 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-irected mutagenesis）的技術得到六個突變株，其中一株表現出的蛋白質（R24K)，是將第24個胺基酸由精胺酸（arginine）變成了由離胺酸（lysine)，另外兩株表現的蛋白（NMA21.22.23 RPT and NMA21.22.23 RPS）是將第21,22和第23個胺基酸由天門冬素（asparagine）甲硫氨酸（methionine）氨基丙酸（alanine）分別變成了精胺酸(arginine）比胳氨酸（proline）丁氨酸（threonine）和精胺酸（arginine）比胳氨酸（proline)絲氨酸（serine)。 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由離胺酸，經本實驗結果證實，抗凝血的活性會降低3倍以上。將第21,22和第23個胺基酸由原本的天門冬素（asparagin）甲硫氨酸（methionine）氨基丙酸（alanine）分別變成了精胺酸（arginine）比胳氨酸（proline)丁氨酸（threonine）和精胺酸（arginine ）比胳氨酸（proline）絲氨酸（serine)，實驗的結果指出活性大約降低8-12倍。因此我們推論，第21,22和23個胺基酸可能和RGD loop有協同的作用，使得γ-echistatin 對於血小板上的細胞膜蛋白integrin αIIbβ3有較弱的親和力，而抑制血小板的凝集。 根據過去的實驗，將γ-echistatin的C端，從第46個胺基酸之後切除或再將（Cys8-Cys37）這對雙硫鍵都改成氨基丙酸（alanine)，活性都約降低1.7倍，並且我們從實驗結果及電腦模擬的γ-echistatin結構推測第7-32這對雙硫鍵（Cys7-cys32）或第8-37這對雙硫鍵（Cys8-Cys37）在γ-echistatin摺疊時只須一對存在即可。且從實驗結果可推測易最快形成的第2-11這對雙硫鍵會影響7-32這對雙硫鍵（Cys7-Cys32）或第8-37(Cys8-Cys37)這對雙硫鍵的形成。而且由實驗結果可推測20-39這對雙硫鍵（Cys20-Cys39）在蛋白質摺疊時可能扮演重要的角色。

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 R24N, C2A, C7A, C20A, NMA21.22.23 RPT and NMA21.22.23 RPS by site directed mutagenesis. The inhibitory potency of R24K mutant is only 3-fold less than that of the wild type. For the mutants, NMA21.22.23 RPT and NMA21.22.23 RPS, their inhibitory potency were decreased about 8-12 folds. Our previous research has pointed out that the disulfide bond (8-37) might not be necessary in platelet aggregation inhibition because the inhibitory potency of des(45-49)-γ-echistatin and des(45-49)-[Ala8,37]-γ-echistatin both decrease about 1.7- fold. In summary, according to our results and on the basis of the structural model of γ-echistatin we speculated there were needed three disulfide bonds (2-11, 7-32, 20-39) or (2-11, 8-37, 20-39) during the folding pathway of γ-echistatin.