[Purification and behavior of recombinant human gamma-interferon on reversed-phase high performance liquid chromatography].
A high performance liquid chromatographic system is described for the purification to homogeneity of recombinant human gamma-interferon (gamma-IFN) from the inclusion bodies produced in genetically transformed Escherichia Coli cells. Crude products of gamma-IFN obtained from typical preparative liquid chromatographic separation on Sephacryl S200 column was applied to analytical HPLC with RP-C18 column and eluted with convex gradient of 0-82% acetonitrile in 30 min followed by linear gradient of 82%-100% in 30 min. A constant level of 0.1% trifluoroacetic acid was maintained throughout the gradient process. The retention of gamma-IFN was investigated as a function of pH, temperature as well as concentration of salt in the mobile phase. For denatured and renatured gamma-IFN the chromatographic profiles were analyzed under various conditions. The retention time of renatured gamma-IFN was compared with that of denatured gamma-IFN and a significant difference in the retention time of profiles was observed. Both biological activities were reduced to different degree followed with incorrect disulfide bond gamma-IFN or oligomer gamma-IFN. The purified gamma-IFN has a specific activity about 6 x 10(6) units/mL. The eluats were assayed for identification of gamma-IFN using Enzyme-Linked Immunosorbent Assays (ELISA). Its molecular weight was 17.5 kD, as determined by SDS-PAGE. The results support that renatured sample with tR = 24.8 min and denatured one with tR = 30.1 min were identical protein with different conformation. The chromatographic behavior is explained by the change in net charge and polarity of gamma-IFN when the mobile phase condition and the conformation vary. The oligomer or incorrect disulfide bonds were formed during gamma-IFN renaturation and also the biological activity was found to be native conformation dependent.
Kang
J
J
General Hospital of Shenyang Command of PLA, Shenyang, 110015.
Liu
Y
Y
Meng
L
L
Sun
Y
Y
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D007371:Interferon-gamma