Affordable Access

[Purification and identification of human recombinant IFN-beta expressed in yeast Pichia pastoris].

Authors
  • Yu, Qi-Wen
  • Li, Ning-Li
  • Nie, Hong
  • Ma, An-Lun
  • Xi, Bo
  • Gong, Yi
  • Zhang, Dong-Qing
Type
Published Article
Journal
Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica
Publication Date
Nov 01, 2003
Volume
35
Issue
11
Pages
1035–1039
Identifiers
PMID: 14614543
Source
Medline
License
Unknown

Abstract

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.

Report this publication

Statistics

Seen <100 times