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 was 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 purified by cation-exchange chromatography. MCP-1H could be converted to MCP-1L by treatment with peptide N-glycosidase F, showing that the former is an N-glycosylated form of the latter. Laser desorption mass spectrometry 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 half of MCP-1L lacked the two N-terminal amino acids found in the native protein. Both MCP-1H and MCP-1L could induce monocyte migration and calcium influx in THP-1 monocytic leukemia cells, although these activities were about 10- to 100-fold lower than those of MCP-1 produced in insect cells.
Beall
C J
CJ
Neurobiotechnology Center, Ohio State University, Columbus, Ohio 43210, USA.
Breckenridge
S M
SM
Chakravarty
L
L
Kolattukudy
P E
PE
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D018932:Chemokine CCL2 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D006031:Glycosylation; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018932:Chemokine CCL2; D007106:Immune Sera; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D002118:Calcium