Expression and purification of a small cytokine growth-blocking peptide from armyworm Pseudaletia separata by an optimized fermentation method using the methylotrophic yeast Pichia pastoris.
A small multifunctional cytokine, growth-blocking peptide (GBP), from the armyworm Pseudaletia separata larvae was expressed as a soluble and active recombinant peptide in the methylotrophic yeast Pichia pastoris. An expression vector for GBP secretion was constructed using vector pPIC9, and GBP was expressed under the control of the alcohol oxidase (AOX1) promoter. Although we first tried to cultivate GBP in shake flask cultures, the yield was low, probably due to proteolysis of the recombinant protein. To overcome this problem, we utilized a high-density fermentation method. The pH of the medium in the fermenter was kept at 3.0, and the medium was collected within 48h post methanol shift to minimize exposure of the target peptide to proteases. Recombinant GBP was purified through three reverse-phase HPLC columns. We characterized the 25 amino acid GBP by molecular mass spectrometry and amino acid sequencing. Plasmatocyte spreading, one of the activities of GBP, was similar between chemically synthesized GBP and purified recombinant GBP. Up to 50mg GBP was recovered per 1L of yeast culture supernatant.
Koganesawa
Nozomi
N
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.
Aizawa
Tomoyasu
T
Shimojo
Hiroshi
H
Miura
Kazunori
K
Ohnishi
Atsushi
A
Demura
Makoto
M
Hayakawa
Yoichi
Y
Nitta
Katsutoshi
K
Kawano
Keiichi
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005285:Fermentation; D006376:Helminths / Q000737:chemistry*; D019476:Insect Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D016207:Cytokines; D019476:Insect Proteins; D010455:Peptides; D011994:Recombinant Proteins; C068690:growth blocking peptide, Apanteles kariyai