[Preparation and identification of recombinant human interferon-gamma].
The renaturation with simultaneous purification of recombinant human interferon-gamma (rhIFN-gamma) expressed as inclusion bodies in Escherichia coli (E. coli) was accomplished by the stationary phase of hydrophobic interaction chromatography (STHIC) with the end group of poly(ethylene glycol) (PEG)(PEG200) packed in a chromatographic column and a chromatographic pie by nonlinear gradient, separately. In order to provide more selections for the chromatographic separation of rhIFN-gamma from different sources, the chromatographic behavior of rhIFN-gamma in reversed-phase liquid chromatography, ion-exchange chromatography and immobilized-nickel affinity chromatography were also studied. The fraction of the renatured and purified rhIFN-gamma from HIC was desalted by the size exclusion chromatography, subsequently freeze-dried to powder. With matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), monomeric and dimeric rhIFN-gamma were found in the powder due to the freeze-dried process and their relative molecular masses were 17 184.0 and 34 204.4, respectively. With the bioactivity assay by cytopathic effect inhibition (CPEI), the specific bioactivity of rhIFN-gamma was 9.5 x 10(8) IU/mg, which was higher than that of the required criteria in the phar macopoeia of China, because the presence of dimeric rhIFN-gamma which has much higher specific bioactivity than its monomer in the powder. The obtained mass recovery, purity, specific bioactivity of the purified monomeric rhIFN-y were 93.7%, > 95%, and 4.3 x 10(7) IU/mg, respectively. The results showed that the renaturation with simultaneous purification of rhIFN-gamma by PEG200-STHIC is a kind of efficient method.
Wu
Dan
D
Institute of Modern Separation Science, Key Laboratory of Modern Separation Science in Shaanxi Province, Northwest University, Xi'an 710069, China. wd.imss@163.com
Gao
Dong
D
Bai
Quan
Q
Geng
Xindu
X
D002845:Chromatography / Q000379:methods*; D015925:Cryopreservation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007371:Interferon-gamma / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D007371:Interferon-gamma