Expression, purification, and characterization of an unstable lysozyme mutant in Pichia pastoris.
To investigate the expression and purification of an unstable heterologous protein in Pichia pastoris, the cDNA of H5-lysozyme, a hen egg lysozyme mutant with a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) fused to the carboxyl terminus, was integrated into the genome of P. pastoris. It was found that medium composition, induction time, and fermenter type were important factors for the expression of H5-lysozyme. Substantially active H5-lysozyme was secreted by induction with methanol when the prepro-sequence of alpha-factor was used as secretion signal sequence. The amount secreted was 422-fold greater than that observed with Saccharomyces cerevisiae. Recombinant H5-lysozyme was recovered and purified by cation-exchange chromatography directly from fermentation broth. The mutant lysozyme showed bactericidal activity against Gram-positive as well as Gram-negative bacteria.
Liu
Shu-Tao
ST
Institute of Biotechnology, Fuzhou University, PR China.
Saito
Akira
A
Azakami
Hiroyuki
H
Kato
Akio
A
D000818:Animals; D002412:Cations; D002645:Chickens; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D000432:Methanol / Q000494:pharmacology; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D009154:Mutation; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013997:Time Factors
D002412:Cations; D018076:DNA, Complementary; D009113:Muramidase; D000432:Methanol