篇名 | 利用SUMO融合技術於大腸桿菌中生產具生物活性之豬隻抗菌胜肽PR-39 |
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卷期 | 54:1 |
並列篇名 | Production of Bioactive Porcine Antibacterial Peptide PR-39 in Escherichia coli Using SUMO Fusion Technology |
作者 | 陳正文 、 宣詩玲 、 林俊宏 、 黃文正 、 林慧傑 、 王志鵬 |
頁次 | 009-019 |
關鍵字 | 抗菌胜肽 、 小分子類泛素修飾蛋白質 、 融合蛋白質 、 Antibacterial peptide 、 Small ubiquitin-like modifier 、 Fusion protein 、 Scopus 、 TSCI |
出刊日期 | 201602 |
PR-39是一種分離自豬小腸、嗜中性白血球及脾臟之抗菌胜肽。此胜肽由39個胺基酸所組成,富含脯胺 酸與精胺酸。過去之研究顯示,此胜肽除可抑制數種革蘭氏陰性菌與陽性菌之生長外,亦具有調控血管生 成、促進傷口癒合及抑制癌細胞之侵入與轉移等生理活性。本研究之主要目的在於建立利用大腸桿菌生產重 組PR-39之方法,以期獲得重組PR-39進行醫藥與飼料添加劑用途之可行性評估。研究中,依據大腸桿菌偏好 之密碼子設計PR-39基因,並利用重疊延展聚合酶連鎖反應合成該基因。將密碼子最適化基因嵌入帶有小分子 類泛素修飾蛋白質 (small ubiquitin-like modifier, SUMO) 基因之表現載體中,建構融合表現載體,並轉形入大腸 桿菌中進行融合蛋白質之表現。帶有組胺酸親和性標籤之重組SUMO-PR-39融合蛋白質可於大腸桿菌中以可溶 型式進行表現,且可利用固定化金屬離子親和性層析法進行純化。純化之融合蛋白質以SUMO蛋白酶進行剪 切後,再利用凝膠過濾層析法進行重組PR-39之純化。由1 L之培養菌體中可獲得55 μg之重組PR-39,純度高於 95%以上。重組PR-39對大腸桿菌具有抑菌效果,最小抑制濃度為0.2 mg/mL。本研究已建立重組PR-39之生產 技術。未來將進一步探討生產重組PR-39之最適條件。
PR-39 is an antibacterial peptide that could be isolated from porcine small intestine, neutrophils, and spleen. The peptide is a proline-arginine-rich peptide with 39 amino acid residues. Previous studies showed that PR-39 exhibits antibacterial activity against several Gram-positive and Gram-negative bacteria. In addition to its antibacterial properties, PR-39 also exerted other biological activities such as regulation of angiogenesis, promotion of wound repair, and inhibition of cancer cell invasion and metastasis. The objective of this study is to establish a method for production of recombinant RP-39 (rPR-39) using Escherichia coli in order to obtain rPR-39 for further evaluating its potential applications as therapeutic agent and feed additive. The DNA sequence encoding the PR-39 was designed according to E. coli preferred codons and synthesized through overlapping extension polymerase chain reaction. The codon-optimized PR-39 gene was inserted into an expression vector which contains the small ubiquitin-like modifier (SUMO) gene for construction of fusion expression vector, and then this vector was transformed into E. coli. Recombinant His-tagged SUMO-PR-39 (rSUMO-PR-39) fusion protein was expressed in soluble form in E. coli and could be purified by immobilized metal ion affinity chromatography. After the rSUMO-PR39 fusion protein was cleaved by the SUMO protease, released rPR-39 was further purified by gel filtration. The typical yield of rPR-39 was 55 μg with purity above 95% from 1 L culture. The rPR-39 showed antibacterial activity against E. coli with a minimum inhibitory concentration of 0.2 mg/mL. This study has established a method for production of rPR-39. The optimal conditions for production of rPR-39 will be further determined.