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標題: Cloning of the Transglutaminase Gene from Streptomyces and Production of Active Recombinant Transglutaminase in E. coli
作者: 陳裕成
Chen, Yu-Cheng
關鍵字: 轉麩氨醯胺酶;transglutaminase;大腸桿菌
出版社: 分子生物學研究所
轉麩氨醯胺酶 (transglutaminase, TGase) 為一種催化蛋白分子間或分子內醯基轉移的酵素,可以形成共價鍵結。而目前大部分的TGase 由 Streptomyces 菌種中被純化出來或於其他微生物進行重組蛋白的表現。本研究目的主要為利用E. coli生產具活性的 TGase,此外亦針對 S. ladakanum TGase 基因進行選殖分析,期望更了解不同菌種間 TGase 基因的差異對酵素活性的影響。本研究利用 uneven PCR 從S. ladakanum 菌株中選殖出完整 TGase 基因,其ORF有 1,233-bp,可轉譯出 410 個胺基酸,分子量為 45.8 kDa,蛋白等電點 (pI) 為 6.2,其核苷酸與胺基酸序列同質性與S. mobaraense 最高。為了增加 E. coli體內可溶性 TGase的產量,在本研究中做了許多的嘗試,利用E. coli CspA cold shock promoter 於低溫誘導表現 S. kentuckense proTGase 蛋白,然而結果顯示並無增加可溶性 TGase 的表現。而為了獲得具活性的 mature TGase,我們於 E. coli體內藉由共同表現 proTGase 蛋白與專一性辨識切割的蛋白酶 (TVMV 或 TEV protease) 的方式進行實驗,結果發現蛋白產率約為 1.3 ~ 1.5 mg / 50 ml,比活性約為 35 ~ 39 U/mg。另人驚訝的是從 SDS-PAGE 的分析中指出,未含 His-tag 的 pro-peptide 卻與 mature TGase 一起被分離出。進一步利用陽離子交換樹脂管柱分離,可部份回收單獨的mature TGase,其比活性可達 77.5 U/mg,這顯示了被切割出的pro-peptide 可能影響 mature TGase 的活性。另一方面,為了達到不經蛋白酶切割即獲得具活性之 mature TGase 酵素的目的,將Trx-pro peptide 與 mature TGase 基因構築於同一質體上,且以 polycistronic mRNA 的方式進行共同表現,結果發現雖可獲得具活性的 mature TGase,但其比活性及產量則較 Trx-proTGase 基因的融合構築低。另外以不同啟動子分別表現 Trx-pro peptide 與 mature TGase 基因的實驗顯示產量與比活性亦沒有明顯提升。而為了使重組 TGase 分泌至胞外,於 E. coli進行S. kentuckense preproTGase 的蛋白表現,結果顯示重組 TGase只能分泌至細胞間質,而胞外培養液中則無法觀察到。此外,將 Bacillus subtilis YaB signal peptide 融合至 proTGase 基因前端,於B. subtilis進行表現,但胞外培養液中並無 TGase 的存在。

Transglutaminases (amine γ-glutaminyl transferase, EC ; TGase) are enzymes that catalyze the acyl transfer reaction between inter- or intramolecules of proteins, resulting in the formation of covalently cross-linkage and polymerization of proteins. To date, most of the TGases are isolated from Streptoverticillium spp. or expressed as recombinant proteins in other bacterial species. The object of this study was to produce the active form TGase in E. coli. To better understand the effect of sequence diversity on the TGase activity, we also embarked on cloning of Streptoverticillium TGase gene. The S. ladakanum TGase gene was cloned in this study by Uneven PCR and found to contain an ORF of 1233 bp encoding a 410 amino acids protein. The calculated molecular weight of the deduced ORF is 45.8 kDa and with a pI value of 6.2. The nucleotide and amino acid sequence of S. ladakanum TGase gene has the highest identity with that of the S. mobaraense. To enhance the production of soluble TGase in E. coli, several attempts have been conducted in this study. Expression of SkproTGase was directed by CspA cold shock promoter and induced by low temperature. However, no increase in the soluble TGase expression was observed. Active form of mature TGase could be obtained with protease digestion by coexpression of the proTGase and the specific protease (TVMV or TEV protease) in E. coli. The result revealed that the mature TGase was produced at 1.3~1.5 mg per 50 ml LB and the specific activity of the enzyme was about 35~39 U/mg. To our surprise, SDS-PAGE analysis indicated that the cleaved Trx-pro peptide was co-purified with the His-tagged mature SkTGase. The Resource S cation exchange column was then applied to purify the mature SkTGase from the cleaved Trx-pro peptide. The specific activity of the purified SkTGase was found to be 77.5 U/mg, indicating that the cleaved pro-peptide might affect the activity of the mature TGase. To obtain active form mature TGase without protease digestion, Trx-pro peptide and mature TGase genes were constructed in the same plasmid and co-expressed as a polycistronic mRNA. The result showed that active form of mature TGase was produced, however, with lower specific activity and yield than the fusion construct. Expression of Trx-pro peptide and mature TGase by two individual promoters displayed no significant increase in enzyme activity and production. To enhance the secretion of recombinant TGase into culture medium, expression of S. kentuckense preproTGase in E. coli was carried out. Our result indicated that recombinant TGase located in the periplasm but not in the culture broth. Moreover, no TGase was found in the culture broth when the YaB signal peptide of Bacillus subtilis was fused to the proTGase gene and expressed in B. subtilis.
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