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The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using …


Biology Articles » Biotechnology » Red Biotechnology » Recombinant expression of human cathelicidin (hCAP18/LL-37) in Pichia pastoris

Abstract
- Recombinant expression of human cathelicidin (hCAP18/LL-37) in Pichia pastoris

Recombinant expression of human cathelicidin (hCAP18/LL-37) in Pichia pastoris

In-Pyo Hong1, Sung-Jae Lee1, Yong-Seok Kim2 and Shin-Geon Choi1

(1)  Department of Bioengineering and Technology, Kangwon National University, 192-1, Hyoja2-dong, Chuncheon, 200-701, Korea
(2)  Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, Korea

Biotechnology Letters 10.1007/s10529-006-9202-8.

 

Abstract 

The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.

Keywords  Cathelicidin - LL-37 - Heterologous expression -  Pichia pastoris

 


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