Expression, refolding, and characterization of human soluble BAFF synthesized in Escherichia coli.
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. Here, a recombinant form of the extracellular domain of the BAFF (hsBAFF) was expressed in Escherichia coli BL21(DE3) under the control of a T7 promoter. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 8 M urea. A rapid and simple on-column refolding procedure was developed. It was applied and then the refolded hsBAFF was purified by anion-exchange. The purified final product was >98% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 17.5 kDa, which equalled the theoretically expected mass. The N-terminal sequencing of refolding hsBAFF showed the sequence corresponded to the designed protein. The correct refolding of the recombinant protein was verified in the recovery of its secondary and tertiary structures as assessed by circular dichroism and fluorescence emission spectra. The renatured protein displayed its immunoreactivity with the antibodies to BAFF protein by Western blotting. The final purified material was biologically active in a validated induced human B lymphocyte proliferation bioassay. The expression and in vitro refolding of hsBAFF resulted in production of an active molecule in a yield of 15 mg/L flask cultivation.
Cao
Peng
P
Jiang su Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210097, Jiangsu, PR China. pcao79@yahoo.com
Mei
Jing Jing
JJ
Diao
Zhen Yu
ZY
Zhang
Shuang quan
Sq
D000595:Amino Acid Sequence; D053264:B-Cell Activating Factor; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D002479:Inclusion Bodies / Q000737:chemistry; D008213:Lymphocyte Activation / Q000187:drug effects; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012995:Solubility; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology
D053264:B-Cell Activating Factor; D018076:DNA, Complementary; D008565:Membrane Proteins; D011994:Recombinant Proteins; C505719:TNFSF13B protein, human; D014409:Tumor Necrosis Factor-alpha