Renaturation, purification, and characterization of human truncated macrophage colony-stimulating factor expressed in Escherichia coli.
A human truncated macrophage colony-stimulating factor (M-CSF) encoding the amino acid residues from 3 to 153 of the native M-CSF was expressed by using a two-cistron expression system in Escherichia coli. The truncated M-CSF found in inclusion bodies was renatured and had CSF activity. Purification, which included a QAE-ZeTa preparative cartridge concentration step followed sequentially by HPLC on TSK-gel Phenyl-5PW and TSK-gel DEAE-5PW columns, gave an overall yield of 63.8%. The purified truncated M-CSF had a specific activity of 4 x 10(7) units/mg of protein. Peptide mapping of a lysylendopeptidase digest by reversed-phase HPLC confirmed the amino acid sequence predicted from the cDNA sequence. SDS-PAGE of the purified truncated M-CSF gave a single band at 17 kDa under reducing conditions and at 32 kDa under non-reducing conditions. Activated Thiol-Sepharose 6B column chromatography and other experiments failed to detect any free cysteine residue in spite of the existence of 7 cysteine residues in the truncated M-CSF subunit. These results indicate that it is a dimeric structure linked by one or more intermolecular disulfide bonds.
Yamanishi
K
K
Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima.
Takahashi
M
M
Nishida
T
T
Ohmoto
Y
Y
Takano
M
M
Nakai
S
S
Hirai
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014162:Transfection
D013438:Sulfhydryl Compounds; D016173:Macrophage Colony-Stimulating Factor