[Purification and identification of human recombinant IFN-beta expressed in yeast Pichia pastoris].
To find an effective and quick way of purifying and identifying recombinant human IFN-beta (rhIFN-beta) expressed in yeast Pichia pastoris, Blue Sepharose 6 fast flow (Blue S6FF) and immunological affinity chromatography (IAC) were compared in this report. rhIFN-beta was produced in 15 liter bioreactor and purified using the two methods mentioned above. The protein concentrations of rhIFN-beta and residual mouse IgG in purified rhIFN-beta were determined with ELISA. The molecular weight and specificity were demonstrated by PAGE and Western blot. The density of the specific precipitation bands was determined by gel scanning. The relative bioactivities were determined by cyto pathogenic effect inhibition (CPEI). The results showed that 2.65 and 3.03 mg of rhIFN-beta were obtained, respectively, by purifying with Blue S6FF or IAC from 2 liter of fermentation supernatant. The molecular weight was 22 kD. The concentrations of the special precipitation of rhIFN-beta were 95.1% and 96.2% respectively. The relative bioactivity of rhIFN-beta purified by Blue S6FF and IAC were 1.63x10(7) IU/mg and 1.43x10(7) IU/mg, respectively. The residual mouse IgG in purified rhIFN-beta by IAC was less than 50 microg/L. The results indicated that rhIFN-beta could be purified effectively and quickly from fermentation supernatant of yeast Pichia pastoris by IAC. The rhIFN-beta products purified by Blue S6FF and IAC had almost the same purity and bioactivity. The data accumulated from the experiment are useful to the preparation of rhIFN-beta on a larger scale.
Yu
Qi-Wen
QW
Shanghai Institute of Immunology, Shanghai Second Medical University, Shanghai 200025, China.
Li
Ning-Li
NL
Nie
Hong
H
Ma
An-Lun
AL
Xi
Bo
B
Gong
Yi
Y
Zhang
Dong-Qing
DQ
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016899:Interferon-beta / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives
D011994:Recombinant Proteins; D016899:Interferon-beta; D012685:Sepharose