文献类型：期刊文献

英文题名：Peroxisome-targeted and tandem repeat multimer expressions of human antimicrobial peptide LL37 in Pichia pastoris

作者：Xiao, Siwei[1];Gao, Yanyun[1];Wang, Xiaolong[1];Shen, Wei[1];Wang, Jinjia[1];Zhou, Xiangshan[1];Cai, Menghao[1];Zhang, Yuanxing[1,2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]Shanghai Collaborat Innovat Ctr Biomfg Technol, Shanghai, Peoples R China

年份：2017

卷号：47

期号：3

起止页码：229

外文期刊名：PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000396051000002)】；

基金：This work was supported by the Chinese National High Technology Research and Development Program (2014AA093501), Fundamental Research Funds for the Central Universities (22A201514040), and Grants of Young and Middle-aged Leading Science and Technology Innovation Talents from Ministry of Science and Technology of China.

语种：英文

外文关键词：Antimicrobial peptide; AOX1 promotor; LL37; Pichia pastoris; peroxisome-targeted expression; tandem expression

摘要：Although the human antimicrobial peptide LL37 has a broad spectrum of antimicrobial activities, it easily damages host cells following heterologous expressions. This study attempted two strategies to alleviate its damage to host cells when expressed in Pichia pastoris using the AOX1 promoter. Tandem repeat multimers of LL37 were first designed, and secretion expression strains GS115-9K-(DPLL37DP)(n) (n=2, 4, 6 and 8) containing different copies of the LL37 gene were constructed. However, LL37 tandems still killed the cells after 96hr of induction. Subsequently, peroxisome-targeted expression was performed by adding a peroxisomal targeting signal 1 (SKL) at the C-terminus of LL37. The LL37 expression strain GS115-3.5K-LL37-SKL showed no significant inhibition in the cells after induction. Antibacterial activity assays showed that the recombinant LL37 expressed in peroxisomes had good antimicrobial activities. Then, a strain GS115-3.5K-LL37-GFP-SKL producing LL37, green fluorescent protein, and SKL fusion proteins was constructed, and the fusion protein was confirmed to be targeting the peroxisomes. However, protein extraction analysis indicated that most of the fusion proteins were still located in the cell debris after cell disruption, and further studies are required to extract more proteins from the peroxisome membrane.