Production, purification and characterization of recombinant human interferon gamma.
An essentially three-step chromatographic purification procedure, i.e., ion-exchange, immobilized metal ion affinity and size-exclusion chromatography, is described for the purification to homogeneity of recombinant human interferon-gamma (rhIFN-gamma) from the inclusion bodies produced in genetically transformed Escherichia coli cells. Batchwise adsorption of the cloudy solution of renatured rhIFN-gamma obviated the need for high-speed centrifugation to clarify the suspension. This step effectively removed about 70% of extraneous protein impurities. The established purification process is reproducible and leads to a total recovery of 32%. Pilot-scale processing of E. coli cells grown in a 30-l fermentor gave about 70 mg of a homogeneous preparation of rhIFN-gamma. The specific biological activity of purified rhIFN-gamma is ca. 3.4 x 10(7) I.U./mg protein, which is comparable to that of its natural counterpart. It is basic protein (pI greater than pH 9) with a monomer relative molecular mass of 15,000. It behaves, however, as a dimer on size-exclusion chromatography. Its partial NH2-terminal sequence is identical with that established for the rhIFN-gamma. However, its amino acid composition and its relative molecular mass (15,067 as determined by electrospray mass spectrometry) indicate that the purified protein is a truncated form lacking fifteen amino acid residues from its carboxyl-terminal side. This modification does not seem to have any adverse effect on its biological potency. The levels of DNA, bacterial endotoxins and Ni(II) ions in the final product were determined.
Zhang
Z
Z
Institute of Virology, Chinese Academy of Preventive Medicine, Beijing.
Tong
K T
KT
Belew
M
M
Pettersson
T
T
Janson
J C
JC
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry; D011786:Quality Control; D011994:Recombinant Proteins; D013056:Spectrophotometry, Ultraviolet; D014461:Ultracentrifugation
D011994:Recombinant Proteins; D009532:Nickel; D007371:Interferon-gamma