Expression of human nerve growth factor gene in E. coli
He, X.; Chen, J.; Wang, R.; You, L.; Liu, J.; Chen, J.; Lin, J.
Hua Xi Yi Ke da Xue Xue Bao 33(1): 94-97
2002
ISSN/ISBN: 0257-7712 PMID: 12599440 Document Number: 546476
To investigate the structure and function of human nerve growth factor (beta-NGF) and the gene encoding beta-NGF. A pair of specific primers (29 mer) for the sequence encoding human beta-NGF was designed and synthesized. A 380 bp fragment was amplified from human blood genomic DNA by polymerase chain reaction, and cloned into pGEM-T Easy vector. The identified insert fragment from the recombinant pGEM-T-NGF was directionally ligated with linearized pGEX-5T with the compatible termini. E. coli JM 109 was transformed with the expression recombinant p5TNGF and induced by IPTG. The cloned DNA fragment was identified as the full-length sequence encoding human beta-NGF by restriction analysis and DNA sequencing. SDS-PAGE and Western blot revealed the cloned NGF gene expressed as a fusion protein (40.5 x 10(3) u) in the cells transformed by p5TNGF. The soluble fusion protein was determined to be 503.2 mg/L, accounting for 6.8% of the total soluble protein (7.4 g/L) of bacterial cells. This fusion protein was found to have antigenic activities of NGF. The clone containing the full-length sequence encoding human beta-NGF is obtained and successfully expressed in E. coli to be of use for studying the biological functions of human beta-NGF gene.