利用合成基因方法研究心臟毒蛋白II：表現毒素的結構功能鑑定

Abstract

我們利用聚合?鏈鎖反應（PCR）合成出一段稱為mdctxII的基因，在這個核酸片段上除了有能夠轉譯出心臟毒蛋白II的核酸密碼之外，它的5'端還有一段可以在表現毒素的N-terminus轉譯出 (Asp)4-Lys的核酸序列。在這篇論文中，我們選擇pQE30為載體，這個系統可以在表現蛋白的N-terminus製造一個His-tag因而能簡化純化表現毒素的工作。人工合成的mdctxII與pQE30接合後轉形到E. coli.中，經過多次篩選與基因改造，最後我們得到一個符合最初設計的菌株Y1來進行蛋白質的表現。表現蛋白先經過親和層析的初步分離，然後在特定的條件下進行重新摺疊（refolding）並進一步以HPLC純化，之後我們以enterokinase去除N-terminus的His-tag及(Asp)4-Lys，如此所得到的蛋白質在電泳上呈現的分子量與天然的心臟毒蛋白II相仿，並且經蛋白質定序分析可確認其前15個胺基酸序列是正確的。不過目前這個利用人工合成基因製造蛋白質的研究遭遇了幾個實驗操作上的瓶頸，以致我們無法得到大量具有正確摺疊型式且又去除His-tag及(Asp)4-Lys的表現毒素，這些問題都值得我們日後再做更詳細的研究以找到最適合大量製造具有活性的心臟毒蛋白II的方法。

The mdctxII gene, which encodes cardiotoxin II (CTX II) and an N-terminal extension of (Asp)4-Lys, was constructed by means of polymerase chain reaction (PCR). We chose pQE30 expression system, which could additionally produce a His-tag at N-terminus of expressed protein, to express CTX II in this study. The synthetic mdctxII gene was ligated into pQE30 vector and then transformed into E. coli. A positive clone, named Y1 whose nucleotide sequence corresponds what we designed, was obtained after multiple screening steps and used for the expression of 6×His-mCTX II. The expressed protein was first purified by affinity chromatography, followed by refolding under defined conditions and further purification of the refolded product on HPLC. Enterokinase was then used to cut off the His-tag and (Asp)4-Lys attached at the N-terminus of the expressed toxin. The final expressed product was confirmed to possess a similar molecular mass by gel electrophoresis to that of native CTX II isolated and purified from crude venom. Sequencing analysis of the first N-terminal 15 residues of the expressed toxin also corroborates the identity of the product. However, the yield of fully refolded CTX II obtained after the cleavage of His-tag and (Asp)4-Lys was very low. This pilot study using synthetic gene poses an operational problem for large-scale expression of the synthetic toxin gene product, which deserves a detailed mechanistic study on the in vitro refolding process of this toxin in the future.