hMYH蛋白於大腸桿菌表現量與可溶性之改善

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Please use this identifier to cite or link to this item: http://140.128.103.80:8080/handle/310901/2187

Title:	hMYH蛋白於大腸桿菌表現量與可溶性之改善
Other Titles:	Improving the overexpression of hMYH and its solubility in E.coli.
Authors:	施賓豪 Shih, Pin-Hao
Contributors:	顧野松 Gu, Ye-Song 東海大學化學工程與材料工程學系
Keywords:	hMYH基因表達;稀有密碼子;包涵體;可溶性蛋白質 hMYH gene expression;rare codon;inclusion body;soluble protein
Date:	2008
Issue Date:	2011-03-09T08:01:00Z (UTC)
Abstract:	DNA修復途徑的缺陷可能會導致基因的不穩定和癌症的發生。對於DNA氧化損傷來說，DNA的鹼基切除修復(base excision repair)為最重要的保護機制。從文獻得知，特殊的DNA醣基解?，可專一性的切除損傷的鹼基。其中與MutY同源之人類修復蛋白hMYH醣基解?是修復途徑中不可或缺的酵素，其主要功能為移除與鳥糞嘌呤(G)或7,8-雙羥基-8-氧化鳥糞嘌呤(8-oxoG)錯配中的腺嘌呤(A)，從而避免G:C配對轉置成為T:A配對的突變。hMYH在DNA修復途徑中的詳細機制尚未十分清楚，其中一個原因就是hMYH在大腸桿菌裡面的表達程度不高，因此無較純的蛋白質可供研究使用。分析hMYH基因序列，發現hMYH中含有許多鮮少被大腸桿菌使用的密碼子，推測此為導致hMYH基因在大腸桿菌中難以表達的原因之一。在此研究中，我們利用定點式突變(site-directed mutagenesis)技術，寂靜式突變了已建構在pET21a(+)載體上的hMYH基因，希望藉由改變hMYH基因的稀有密碼子為大腸桿菌常用的密碼子來增進其表達。同時將重組質體轉型入經由argU、ileY和leuW tRNAs基因修飾的BL21-CodonPlus系統，接著加入IPTG進行誘導。實驗結果表明在大腸桿菌中大量表達重組hMYH蛋白質會形成不溶性的包涵體(inclusion body)，其原因可能是大腸桿菌無法正確折疊重組蛋白質，造成其不溶於水且失去活性。因此本研究的重要目的為提升重組hMYH蛋白質的表達及其可溶性。研究顯示在30℃下添加1mM IPTG，可溶性hMYH比37℃時提升約6%左右。透過改變細胞破碎儲存緩衝液，可進一步提升可溶性hMYH約24%左右。另外在表現時作Heat shock處理以及添加助劑如乙醇、阿拉伯醣、甘油等，也有助於提升可溶性hMYH。本研究提供了穩定且大量表現生產可溶性重組hMYH蛋白質的方法，另外透過調整表達條件，以及選擇合適的緩衝溶液及添加助劑，可有效的提升可溶性蛋白質的產量。本實驗結果可以為未來更深入的研究，及大量表達其他重組蛋白質提供有用的資訊。 It is well-known that the defectiveness in DNA repair pathways may lead to genetic insatiability and tumorogenesis. Base excision repair has been shown to effectively repair the oxidative damages of the DNA, which are important in the prevention of genetic mutations. It is reported that DNA glycosylases, which among the most critical enzymes in the pathway, can excise the damaged nucleosides specifically. Human MutY glycosylase homolog (hMYH), which is homologous to MutY in bacteria, is vital for the base excision pathway. The enzymatic function of hMYH is to remove adenine mismatched to guanine (G) or 7,8 dihydro- 8 –oxoguanine (8-oxoG) and thereby prevent the formation of G:C to T:A transversion. The detail of hMYH involved DNA repair mechanism is unclear. One of the restrictions is that the expression level of hMYH in E. coli is limited. It is found that the codon usage of E. coli may be responsible for the low expression level of hMYH, since there are number of codons in hMYH which are rarely used by bacteria. In this study, we performed site-directed mutagenesis to introduce silent mutations in hMYH that had been constructed in the pET21a(+) based expressing plasmid, and the hMYH-containing expressing plasmid was therefore adapted to the codon usage of E. coli to increase its expression level. Meanwhile, the recombinant plasmids were further transformed into the BL21-CodonPlus system, in which was complement with extra copies of argU, ileW, leuY and proL tRNAs The expressing of hMYH was induced by the addition of IPTG. We found that the overexpression of hMYH in E. coli resulted in the formation of inclusion body (IB). The recombinant misfolded hMYH was insoluble and did not have enzymatic activity. We therefore designed experiments to improve the solubility of recombinant hMYH. By incubating the transformed E. coli under 30℃instead of 37oC with IPTG induction at a final concentration of 1 mM, the soluble portion of recombinant hMYH was increased by 6%. By modifying the cell extraction buffer, the recovery of soluble recombinant hMYH was efficiently increased by 24%. Furthermore, the addition of supplements in culture medium, such as ethanol, arabinose, and glycerol, could all improve the solubility of recombinant hMYH.Our study provided a method to express recombinant hMYH in a large scale by modifying the expression condition and extraction buffer. These results may also provide useful information for further applications of our study in large scale expressions of other recombinant proteins in bacteria.
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