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

Production of Bioactive Porcine Antibacterial Peptide PR-39 in Escherichia coli Using SUMO Fusion Technology

10.6578/TJACFS.2016.002

陳正文(Zeng-Weng Chen)；宣詩玲(Shih-Ling Hsuan)；林俊宏(Jiunn-Horng Lin)；黃文正(Weng-Zeng Huang)；林慧傑(Hui-Jei Lin)；王志鵬(Jyh-Perng Wang)

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

臺灣農業化學與食品科學

54卷1期（2016 / 02 / 01）

9 - 19

繁體中文

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.