Cj. Beall et al., EXPRESSION OF HUMAN MONOCYTE CHEMOATTRACTANT PROTEIN-1 IN THE YEAST PICHIA-PASTORIS, Protein expression and purification, 12(2), 1998, pp. 145-150
Citations number
15
Categorie Soggetti
Biochemical Research Methods",Biology,"Biothechnology & Applied Migrobiology
The human monocyte chemoattractant protein-1 (MCP-1) was expressed at
high levels in Pichia pastoris with the alcohol oxidase promoter. It w
as secreted from the yeast when either its natural signal sequence or
the Saccharomyces cerevisiae alpha-factor signal peptide was used, SDS
-PAGE and Western blot revealed two immunoreactive MCP-1 species at 15
and 8.5 kDa designated MCP-1H and MCP-1L, respectively; both were pur
ified by cation-exchange chromatography. MCP-1H could be converted to
MCP-1L by treatment with peptide N-glycosidase F, showing that the for
mer is an N-glycosylated form of the latter, Laser desorption mass spe
ctrometry showed that MCP-1L actually consisted of a mixture of three
polypeptides of 8449, 8614, and 8780 Da and MCP-1H showed a broad peak
at 11,134 Da. N-terminal peptide sequencing indicated that nearly hal
f of MCP-1L lacked the two N-terminal amino acids found in the native
protein. Both MCP-1H and MCP-IL could induce monocyte migration and ca
lcium influx in THP-1 monocytic leukemia cells, although these activit
ies were about 10- to 100 fold lower than those of MCP-1 produced in i
nsect cells. (C) 1998 Academic Press.