Recombinant expression of human cathelicidin (hCAP18/LL-37) in Pichia pastoris.
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.
Hong
In-Pyo
IP
Department of Bioengineering and Technology, Kangwon National University, Chuncheon, Korea.
Lee
Sung-Jae
SJ
Kim
Yong-Seok
YS
Choi
Shin-Geon
SG
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D016982:Micrococcus luteus / Q000166:cytology* / Q000187:drug effects*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D054804:Cathelicidins
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D011994:Recombinant Proteins; D054804:Cathelicidins