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標題: 鏈黴菌轉麩氨醯胺酶前胜肽之作用探討
Studies on the propeptides of Streptomyces transglutaminases
作者: 張文政
Chang, Wen-Cheng
Contributors: 楊明德
分子生物學研究所
關鍵字: 轉麩氨醯胺酶;大腸桿菌
Streptomyces;transglutaminase;TGase
日期: 2013
Issue Date: 2013-11-07 12:21:00 (UTC+8)
Publisher: 分子生物學研究所
摘要: 轉麩氨醯胺酶 (Transglutaminase,簡稱 TGase) 是一種醯基轉移的酵素,可以催化蛋白分子間或分子內的共價鍵形成,在生醫及食品加工上具有廣泛的應用性。實驗室過去的研究指出,在 E. coli 於 17℃ 環境下共同表現 Trx-proTGase 和 tobacco vein mottling virus (簡稱 TVMV) 蛋白酶可直接獲得具有活性的 Streptomyces mature TGase,但以Ni2+-NTA 親和性管柱進行純化時,被蛋白酶切割的 propeptide 會結合至 mature TGase,並和 mature TGase 一起被純化出來。為探討 propeptide 和 mature TGase 間的作用關係,分別將 propeptide 與 mature TGase 純化後進行 TGase 活性抑制分析。結果顯示 Streptomyces mobaraensis (簡稱 Sm ) 及 Streptomyces kentuckense (Sk or Sn) mature TGases 受其 Trx-propeptide 抑制皆屬於競爭形抑制,而 Trx-Smpropeptide 抑制其 mature TGase較 Trx-Skpropeptide 抑制 Sk mature TGase 強。為了能夠篩選到減少或不會對 mature TGase 活性的產生抑制,並且不影響其監護子(chaperone) 功能之 Sm propeptide 突變株,本研究針對 Sm 之 propeptide 以 Error prone PCR 方式進行隨機突變。藉由 TGase 活性篩選的方式得到 34 顆突變株,並將其分別表現且以Ni2+-NTA 親和性管柱進行純化,從中挑選出 6 顆突變株 #302、#672、#C12、#D50、#434、#767 和 #583 分別為 TGase 活性較高或以Ni2+-NTA 親和性管柱進行純化後,Trx-propeptide 和 mature TGase 結合量明顯減少之突變株。進一步以點突變在單一胺基酸上作不同置換進行分析。結果發現 Tyr12 突變為 Phe 會使得 Sm propeptide 活性抑制和監護子的功能都會減弱,若突變為 Ala 則失去此二功能。而 Asp22 和 Asn27 此二胺基酸分別突變為 Val 和 Ala,會使得 Trx-propeptide 結合至 Sm mature TGase 能力大為減弱。結果顯示 Tyr12、Asp22 及 Asn27 此三個胺基酸在 Sm propeptide 幫助 mature TGase 形成可溶性蛋白及抑制 mature TGase 活性上扮演著重要的角色。
Transglutaminase is an acyl-transferase enzyme, which catalyzes the formation of covalent bond between inter- or intra molecule of proteins. It has been widely used in biomedical and food processing. Previous studies in this laboratory have demonstrated that active form mature TGase can be obtained by co-expression of Trx-proTGase and tobacco vein mottling virus (TVMV) protease in E. coli at 17℃. During purification by Ni2+-NTA affinity column, the cleaved propeptide was found to be co-purified with the mature TGase. To study the interaction between the propeptide and mature TGase, these two proteins were purified individually and inhibition assay was conducted. Results showed that Streptomyces mobaraensis (Sm) and Streptomyces kentuckense (Sk or Sn) mature TGases interact with its Trx-propeptide by competitive type inhibition. The inhibition of Trx-Smpropeptide to its mature TGase was stronger than that of Sk TGase. Random mutagenesis was carried out on Sm propeptide by error-prone PCR to construct mutants which keep their chaperone functions and had less or no inhibitory activities to their mature TGases. A total of 34 mutants were obtained by TGase activity screening, and mature TGases from these mutants were expressed and purified by Ni2+-NTA column. Among these mutants, #302, #672, #C12, #D50, #434, #767, and #583, were characterized to have higher TGase activities or less binding abilities to their mature TGases. Furthermore, point mutations were generated on selected amino acids of Sm propeptide. Results showed that both of the inhibitory and chaperone functions of Sm propeptide are decreased in Y12F mutant and almost completely abolished in Y12A mutant. Moreover, the binding ability of Trx-propeptide to mature TGase was significantly decreased when Asp22 or Asn27 of the Sm propeptide was replaced by Val and Ala, respectively. The results indicated that Tyr12, Asp22 and Asn27 of the Sm propeptide play important roles in folding and inhibitory activity of Sm mature TGase.
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