抗菌肽magaininⅡ的密码子优化及在大肠杆菌和毕赤酵母中的表达

Optimization of coding sequences and expression of antimicrobial peptide magaininⅡ in Escherichia coli and Pichia pastoris

摘要:

MagaininⅡ是非洲爪蟾皮肤分泌的一种抗菌肽，在低浓度下即能够对细菌、真菌、原虫、肿瘤等都具有杀伤作用，并且与同是来源于非洲爪蟾皮肤的另一种抗菌肽PGLa在杀菌、抗肿瘤等方面具有很好的协同效应。文中分别按照大肠杆菌和毕赤酵母的密码子利用频率，对抗菌肽magaininⅡ以及杂合抗菌肽magainin Ⅱ-PGLa，进行了密码子优化。构建了高效表达抗菌肽magaininⅡ的原核表达载体，经过蛋白酶切割获得纯化的抗菌肽magaininⅡ。同时进一步构建了杂合抗菌肽magaininⅡ-PGLa的分泌型毕赤酵母表达载体，实现了杂合抗菌肽在毕赤酵母中的分泌表达。本研究为magaininⅡ和magaininⅡ-PGLa的生产与应用奠定了基础。

关键词:抗菌肽，magaininⅡ，大肠杆菌，毕赤酵母，密码子利用频率

Abstract:

The antimicrobial peptide magaininⅡ is expressed in the skin of the African clawed frog, Xenopus laevis, and exhibits a broad spectrum of antimicrobial activity as well as tumoricidal properties at low concentrations. In addition, magaininⅡ plays a synergistic role during antimicrobial and tumoricidal processes with another antimicrobial peptide PGLa that is also expressed in Xenopus laevis. The optimized cDNA sequence of magaininⅡ and magainin Ⅱ-PGLa hybrid peptide according to E. coli or Pichia pastoris codon usage frequency were synthesized and sub-cloned into prokaryotic expression vector pGEX and Pichia pastoris secreted expression vector pPIC9k. The resulting recombinant plasmids were named as pGEX-magaininⅡ and pPIC9k-magainin Ⅱ-PGLa. The GST-magainin Ⅱ fusion protein was highly expressed in E. coli. Furthermore, magaininⅡ was successfully purified by digestion with PreScission Protease to cleave the GST tag. Additionally, our data obtained from the ELISA revealed that magainin Ⅱ-PGLa hybrid peptide was successfully expressed in Pichia pastoris. These experiments establish a useful system for further studies of these antimicrobial peptides.

Key words:antimicrobial peptide, magaininⅡ, Escherichia coli, Pichia pastoris, codon usage frequency