Purification of recombinant human granulocyte-macrophage colony-stimulating factor from the inclusion bodies produced by transformed Escherichia coli cells.
Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), produced as inclusion bodies in genetically transformed Escherichia coli cells was purified to homogeneity by a three-step chromatographic procedure involving hydrophobic interaction, ion exchange and gel filtration. Each purification step is reproducible and well suited for process-scale operations. The purification process also leads to a significant decrease in DNA and endotoxin levels in the final product. Of the three gel media used, Phenyl Sepharose 6 FF (high sub) was most effective in reducing the DNA content (by a factor of ca. 2000) while Superdex 75 prep grade was more effective for removing endotoxins (reduction factor ca. 15). The recovery of purified rhGM-CSF was 35% by enzyme-linked immunosorbent assay and 70% by a biological assay method. The overall purification factor obtained was about 4.6, which is in the range of those reported for recombinant proteins produced in E. coli as inclusion bodies. The purified rhGM-CSF is an acidic protein (pI = 5.4) and has a specific activity of ca. 3.3 x 10(7) units/mg, which is in excellent agreement with that reported for its natural counterpart. Its monomer molecular mass of 14,605, as determined by electrospray mass spectrometry, corresponds exactly to the mass calculated from its cDNA sequence. Its amino acid composition and partial NH2-terminal sequence (up to seventeen residues) are also identical with those reported for this protein. These and other results confirm the identity of the purified rhGM-CSF with its natural counterpart. However, the results also showed that it is apparently heterogeneous from its NH2-terminal side as it is composed of three polypeptides having Met, Ala and Pro as the NH2-terminal residues in which the intact Met analogue accounts for 60% for the mixture. This heterogeneity does not seem to have any biological significance since the specific activity of the purified rhGM-CSF is identical with that of its natural counterpart.
Pharmacia Bioprocess Technology, Uppsala, Sweden.
M
M
Belew
Y
Y
Zhou
S
S
Wang
L E
LE
Nyström
J C
JC
Janson
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011786:Quality Control; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor