Expression, purification, and characterization of recombinant human soluble BAFF secreted from the yeast Pichia pastoris.
The B lymphocyte stimulator (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family which is important in B lymphocyte maturation and survival. Herein, the cDNA coding for the extracellular domain of the BAFF (hsBAFF) has been cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hsBAFF, a 20.2 kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using DEAE-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 102 mg of the protein was obtained in high purity from 1 L of the supernatant and its identity to hsBAFF was confirmed by NH(2)-terminal amino acid sequence analysis Bioactivity of the recombinant hsBAFF was confirmed by the ability of the protein to stimulate human B lymphocyte proliferation in vitro. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hsBAFF for both research and industrial purpose.
Diao
Zhenyu
Z
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, 210097, Jiangsu, PR China.
Ye
Tingmei
T
Cao
Peng
P
Zhang
Jing
J
Mei
Jingjing
J
Lin
Zhihua
Z
Zhang
Shuangquan
S
D053264:B-Cell Activating Factor / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D001402:B-Lymphocytes / Q000187:drug effects; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D002241:Carbohydrates / Q000032:analysis; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007218:Industrial Microbiology / Q000379:methods*; D000432:Methanol / Q000494:pharmacology; D010843:Pichia / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D020539:Sequence Analysis, Protein
D053264:B-Cell Activating Factor; D002241:Carbohydrates; D011994:Recombinant Proteins; D000432:Methanol