[Expression, purification and identification of recombinant human lymphotoxin alpha deletant (rhLT-alphaDeltaN27)]

Beijing Tri-prime Genetic Engineering Co. Ltd., Beijing 102600, China. yongquanl@yahoo.com

AIM: To construct prokaryotic expression vector of recombinant human lymphotoxin alpha deletant (rhLT-alphaDeltaN27) and express the protein in E.coli. METHODS: The rhLT-alphaDeltaN27 gene was amplified by RT-PCR using total RNA extracted from Jurkat cells,cloned into prokaryotic expression vector pET-23b, and transformed into E.coli BL21(DE3). The recombinant protein was expressed after IPTG induction and purified by DEAE Sepharose FF and Phenyl-Sepharose FF. RESULTS: The recombinant protein was expressed as inclusion bodies with the yield of more than 30% of total bacterial protein.After purification, the purity of rhLT-alphaDeltaN27 was 99%, and the biological activity was more than 8x10(7) U/mg. Other characteristics of rhLT-alphaDeltaN27, such as relative molecular mass(M(r)), pI and N-terminal amino acid sequence, all corresponded to theoretical prediction. CONCLUSION: The expression vector of rhLT-alphaDeltaN27 gene was constructed, and the recombinant protein was expressed in E.coli successfully.A method of for purifying rhLT-alphaDeltaN27 was established.

PMID: 15182635 [PubMed - indexed for MEDLINE]