Neurotrophic Factor Gene Expression And Protein Purification

Neurotrophic factors are believed to play an essential role in the proliferation , differentiation and survival of neurons in the nervous system. The ability of neurotrophic factors to promote the proliferation, differentiation of peripheral and central neurons during development and survival after neuronal damage, has stimulated an interest in these molecules as potential therapeutic agents for the treatment of nerve injuries and neurodegenerative disease. There are two types of neurotrophic factors: One is the Nerve Growth Factor family, structurally and functionally related polypeptides, which include NGF,BDNF etc.. Another is the type unrelated to the NGF, e.g. GDNF,CNTF etc. Recent studies suggest that 11-6 signal may also play an important role in nerve regeneration after trauma in vivo.Since there are only limited amounts of neurotrophic factors in vivo, overproduction of these polypeptides will greatly promote the process of basic research, and pave the way to clinical trial.This work focus on the genes of GDNF, BDNF and IL-6 .First , the cloning of the genes and the construction of the expression vectors. The genes of GDNF,BDNF and 11-6 mature peptides were cloned by PCR separately. The sequences of GDNF and BDNF are identical with the references. Although ten sites of 11-6 gene are different from the sequence registered in GenBank, there is no change in the amino-acid sequence. This may indicates the important roles of the conservative amino acid sequence in maintaining the normal function of proteins.The GDNF,BDNF and 11-6 gene are then cloned into the E.coli high expression vector pET30a or pET16b. The pETR vector with E.coli thioredoxin gene (trxA) was constructed by ourselves, by using it GDNF gene are fused to trxA.Second , acquirement of high level expressive engineered strains. By optimizing the culture conditions, high level expression of recombinant BDNF,GDNF and 11-6 in E.coli were achieved. The ratio of expressed recombinant proteins to total cell protein was 52.6%, 16.3%,and 16%. The amount of Thio-GDNF fusion protein were 50.4%, increased (?)arkedly compared with the level of nature GDNF protein(16%).This confirmed the superiority of pETR in high-level expression of exogenous gene.In the process of the induction of engineered bacterial cells, two phase growth phenomena were observed, which had great significance in guiding high-level expression of eukaryotic gene in E.coli.