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