ABSTRACT
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.
Subject(s)
Chemokine CCL2/biosynthesis , Pichia/metabolism , Amidohydrolases/metabolism , Amino Acid Sequence , Animals , Biological Assay , Blotting, Western , Calcium/metabolism , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Fungal , Glycosylation , Humans , Immune Sera/immunology , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Pichia/genetics , Protein Sorting Signals/chemistry , Protein Sorting Signals/genetics , Rabbits , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tumor Cells, CulturedABSTRACT
Extraction and derivatization of carbonyls to benzyloximes, pentafluorobenzyloximes or 2,4-dinitrophenylhydrazones is simplified and reaction times are substantially reduced by simultaneous sorption and derivatization from aqueous solution onto a solid phase. In this reaction a macroreticular polystyrene-divinylbenzene resin acts as a sorbent and catalyst to allow simultaneous extraction and derivatization of hydrophilic and lipophilic aldehydes and ketones from simple as well as complex matrices including plasma. Conversion to the 2,4-dinitrophenylhydrazones or pentafluorobenzyloximation at ambient temperature requires 10 and 20 min, respectively. These reaction conditions correspond to at least a 6-fold reduction in reaction times for derivatization of the reactive aldelhydes and a 36-72-fold reduction for preparation of derivatives for the slower reacting ketones.
