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題名 | 利用大腸桿菌及嗜甲醇酵母菌生產具有抗菌活性之重組LsGRP1C=Production of LsGRP1C Recombinants with Antimicrobial Activity in Escherichia Coli and Pichia Pastoris |
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作者 | 王瑜蕙; 張明偉; 林家華; 陳昭瑩; | 書刊名 | 植物病理學會刊 |
卷期 | 24:3/4 2015.12[民104.12] |
頁次 | 頁267-278 |
分類號 | 368.6 |
關鍵詞 | 抗菌胜肽; 大腸桿菌蛋白質表現系統; 嗜甲醇酵母菌蛋白質表現系統; LsGRP1C; Antimicrobial peptide; Escherichia coli protein expression system; Pichia pastoris protein expression system; |
語文 | 中文(Chinese) |
中文摘要 | 葵百合防禦相關蛋白LsGRP1(Lilium 'Star Gazer' glycine-rich protein 1)的C端富含半胱胺酸區段LsGRP1^C具有優異的抗細菌與抗真菌活性,可發展應用為抗菌胜肽。本研究嘗試以大腸桿菌BL21(DE3)及嗜甲醇酵母菌X-33分別表現重組之SUMO-LsGRP1^C與LsGRP1^C-c-myc,二者抑制大腸桿菌DH5α生長之效率相當,但略遜於化學合成之LsGRP1C 胜肽;表現量則以前者較高。進一步進行大腸桿菌BL21(DE3)表現SUMO-LsGRP1^C之最適化條件測試,證實以1 mM isopropyl β-D-1-thiogalactopyranoside(IPTG)誘導時,培養於37℃的大腸桿菌BL21(DE3)表現SUMO-LsGRP1^C的產率高於培養於28℃與20℃者,且其表現量於誘導後5-7 小時達到高峰;於37℃下測試IPTG之合適誘導濃度,以0.30-0.75 mM濃度區間能誘導較高量的SUMO-LsGRP1^C表現。續以SUMO-LsGRP1^C進行抗菌活性之熱穩定性及蛋白酶耐受性試驗,確認以100℃處理1小時、60℃處理18小時或1 mg/ml蛋白酶K處理1.5小時,均不影響SUMO-LsGRP1C 抑制大腸桿菌DH5α 生長的效率,但以1 mg/ml胰蛋白酶處理90分鐘卻會使SUMO-LsGRP1^C的抗菌活性減弱。經由本研究,得知以大腸桿菌生產SUMO-LsGRP1^C之合適條件及SUMOLsGRP1^C抗菌活性之熱穩定性、蛋白酶耐受性,且其生產量較高,故可用以開發抗菌胜肽LsGRP1^C之相關產品,以利進一步之應用發展。 |
英文摘要 | LsGRP1 (Lilium 'Star Gazer' glycine-rich protein 1) is a defense-related protein with C-terminal cysteinerich region (LsGRP1^C) exhibiting good antibacterial and antifungal activities, and promisingly acting as an antimicrobial peptide for practical use. In this study, LsGRP1^C recombinants of SUMO-LsGRP1^C and LsGRP1^C- c-myc were expressed in Escherichia coli BL21 (DE3) and Pichia pastoris X-33, respectively, and higher yield of SUMO-LsGRP1^C was achieved; meanwhile, two LsGRP1^C recombinants showed comparable efficiencies for the growth inhibition of E. coli DH5α, which were lower than that of synthetic LsGRP1^C. The optimized conditions for SUMO-LsGRP1^C expression were further examined. As induced by 1 mM isopropyl β-D-1- thiogalactopyranoside (IPTG), E. coli BL21(DE3) carrying pET SUMO-LsGRP1^C incubated at 37℃ but not 28℃ or 20℃ conducted the most productive expression of SUMO-LsGRP1^C which reached a peak during 5-7 hr post IPTG induction. The IPTG concentration for induction was accordingly assayed at 37℃ and resulted in the range of 0.30-0.75 mM. Then, thermal and proteinase resistance of antimicrobial activity conferred by SUMOLsGRP1^C were examined and the results showed that the inhibitory ability of SUMO-LsGRP1^C on E. coli DH5α growth was not altered after incubated at 100℃ for 1 hr, at 60℃ for 18 hr, or with 1 mg/ml proteinase K for 1.5 hr, but decreased after incubated with 1 mg/ml trypsin for 1.5 hr. In this study, the optimized conditions for SUMO-LsGRP1^C production in E. coli and the traits of heat tolerance and proteinase resistance of SUMOLsGRP1^C antimicrobial activity were concluded. Since the yield of LsGRP1^C recombinant produced by E. coli was higher than that from methylotrophic yeast P. pastoris, LsGRP1^C-related products could be developed from SUMO-LsGRP1^C for further application. |
本系統之摘要資訊系依該期刊論文摘要之資訊為主。