利用大腸桿菌及嗜甲醇酵母菌生產具有抗菌活性之重組LsGRP1^C

Production of LsGRP1^C recombinants with antimicrobial activity in Escherichia coli and Pichia pastoris

10.6649/PPB.201512_24(3_4).0008

王瑜蕙(Y. H. Wang)；張明偉(M. W. Chang)；林家華(C. H. Lin)；陳昭瑩(C. Y. Chen)

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 ； LsGRP1^C ； antimicrobial peptide ； Escherichia coli protein expression system ； Pichia pastoris protein expression system

植物病理學會刊

24卷3&4期（2015 / 12 / 01）

267 - 278

繁體中文

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。

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.