在大腸桿菌胞外表現 Streptomyces 轉麩氨醯胺酶之探討

Extracellular Expression of Recombinant Streptomyces Transglutaminase in E. coli

10.6845/NCHU.2015.00464

陳楷婷

轉麩氨醯胺酶 ； 角質酶 ； 胞外表現 ； Transglutaminase ； Cutinase ； Extracellular Expression

中興大學分子生物學研究所學位論文

2015年

碩士

楊明德

繁體中文

轉麩氨醯胺酶 (transglutaminase, TGase) 為一醯基轉移酵素，可催化蛋白質間的醯基轉移反應，廣泛應用在食品加工、紡織及醫藥工業。過去研究主要聚焦在胞內表現具有酵素活性之 TGase 蛋白，但胞內蛋白在表現上須利用蛋白酶進行切割且在純化上較為不易。為克服上述困難，我們曾將 Streptomyces netropsis propeptide 及 mature TGase 基因以各別並多基因之形式在 E. coli 進行表現，並可於胞內獲得具活性之 mature TGase。然而，將目標重組蛋白表現於 E. coli 胞外，可減少純化上的繁複手續。本研究利用改造後之 S. netropsis TGase，以受同一 T7 啟動子調控但分別表現融合有 pelB 訊號胜肽之 propeptide 及 mature TGase 基因，並觀察改變基因表現順序及改變啟動子對於 TGase 分泌至胞外的影響。結果發現只有 propeptide 在前 mature TGase 在後之構築形式，在胞外才可以偵測到 mature TGase，而讓 propeptide 與 mature TGase 兩基因同時誘導表現或在 propeptide 前端融合 thioredoxin 對於 TGase 蛋白表現及分泌至胞外並無顯著差異。為了促進胞外蛋白的分泌量，進一步由 Thermobifida fusca 染色體 DNA 中增幅出具有水解磷脂質功能的 cutinase 基因，與不同構築形式之 Streptomyces TGase 基因在 E. coli BL21(DE3) 中進行共表現，尋找最適合 TGase 與 cutinase 基因共表現之條件，結果顯示 cutinase 可分泌至胞外且具有幫助胞內蛋白分泌至胞外之能力。利用具 cutinase 基因質體與含改造之 Shkpro- SkTGA 與 S. netropsis 金屬蛋白酶基因之質體在 E. coli 中進行共表現時，利用親合性管柱可由 50 ml 胞外培養液純化得到 0.35 mg 之 mature TGases，純化酵素之比活性為 18.9 U/mg。

Transglutaminase (protein-glutamine:amine γ- glutamyl transferase, EC 2.3.2.13;TGase) is an acyl-transferase enzyme that catalyzes crosslinking between glutamine residue and a variety of primary amines in proteins. It is currently widely used in food processing, textile and pharmaceutical industry. An efficient production of intracellular soluble recombinant pro-TGases in E. coli have been demonstrated by processing the TGases with exogenous protease. Moreover, the strategy of applying co-expression of individual propeptide and mature TGase genes as polycistronic mRNA in E. coli, active form mature TGase could be obtained without protease digestion. However, efficient secretion of TGase into culture medium without tedious purification steps was consider to be more cost-effective. In this study, the modified Streptomyces netropsis TGase propeptide and its mature TGase were individually fused with PelB signal peptide and coexpressed as individual polypeptide under a single T7 promoter control. The amount of extracellular production of mature TGase by the order of gene expression, fusion of thioredoxin to propeptide, and genes regulated by different promoters, were investigated in this study. Results showed that extracellular production of mature TGase was detected only when the genes were expressed in the order of pro-peptide and mature TGase, indicating that the order of gene expression has impact on the mature TGase secretion. However, no significant increase in extracellular TGase production by simultaneous expression of propeptide and mature TGase and those constructs with thioredoxin fused to the propeptide was observed. To facilitate extracellular production of mature TGase, cutinase gene from Thermobifida fusca was cloned and co-expressed with the recombinant TGase genes in E. coli BL21(DE3). Results revealed that T. fusca cutinase as well as the recombinant TGases could be successfully secreted into culture medium. By introducing of exogenous protease genes from S. netropsis into the above host strains, mature S. netropsis TGases could be recovered from intracellular and culture supernatant. After purification by Ni2+-NTA affinity column, 0.35 mg of mature TGase could be obtained from 50 ml culture supernatant and have a specific activity of 18.9 U / mg.

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