High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris.
Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a approximately 6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use.
Li
Lei
L
School of Life Sciences, Shandong University, Jinan 250100, China.
Wang
Jin-Xing
JX
Zhao
Xiao-Fan
XF
Kang
Cui-Jie
CJ
Liu
Ning
N
Xiang
Jian-Hai
JH
Li
Fu-Hua
FH
Sueda
Shinji
S
Kondo
Hiroki
H
D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005658:Fungi / Q000187:drug effects; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006434:Hemocytes / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D033561:Penaeidae / Q000737:chemistry* / Q000235:genetics*; D010843:Pichia / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D012995:Solubility; D014170:Transformation, Genetic
D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins