EXPRESSION OF HUMAN MONOCYTE CHEMOATTRACTANT PROTEIN-1 IN THE YEAST PICHIA-PASTORIS

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.