High Prokaryotic Expression and Purification of Human sΔBAFF

Objective To clone the cDNA encoding the mutant(sΔBAFF) of human B cell activating factor to the TNF family(BAFF), with the amino acid residues at sites 142-160 of extracellular domain(sBAFF142-160) deleted, then express the gene in prokaryotic cells and purify the expressed product.Methods The nucleic acid sequence encoding amino acids 142-160 of sBAFF was deleted by one-step reverse PCR using the constructed recombinant plasmid pUC19/sBAFF(containing the cDNA encoding sBAFF_ 134-285 ) as a template. The amplified cDNA was identified by sequencing and cloned into prokaryotic expression vector pQE-80L for expression under induction of IPTG. The expressed product was analyzed by SDS-PAGE and Western blot, then purified by Ni 2+ -NTA chromatography.Results A cDNA at length of 401 bp was amplified by one-step reverse PCR, and its sequence was consistent with that encoding human sΔBAFF amino acids reported in GeneBank. Human sΔBAFF with a relative molecular weight of 18 000 was highly expressed in a form of inclusion body in E. coli and reached a purity of 95.5% after purification by Ni 2+ -NTA chromatography.Conclusion The successful expression of human sΔBAFF laid a foundation of further study on its function.