Expression, Purification, And Characterization Of Recombinant Human Neurturin Derived From Escherichia Coli And The Yeast Pichia Pastoris

Neurotrophic factors are a kinds of peptides, which can promote neuron survival, growth, and differentiation. There are important actions in developping, differentiating, damaging and repairing of nervous system. NTN is a recently discovered neurotrophic factor that can act specifically on the dopaminergic neurons of the midbrain and motor neurons. This has important clinical significance for the treatment of nervous system diseases characterized by dopaminergic metabolic disorders, such as Parkinson's disease. The naturally occurring NTN content in the human body is very low and difficult to isolate and purify. In view of this problem, it is quite important for producing recombinant human NTN by the methods of gene engineering. In the present study, we expressed, purified and characterized of recombinant hNTN in prokaryote and eukaryote cell system.Firstly, The 5' modified hNTN gene was cloned to plasmid pThioHisA to construct the thioredoxin(Trx) and hNTN fusion expression vetor with hydroxylamine site between the two fused genes.High levels of expression of hNTN fusion protein was obtained in E. coli .The expressed fusion protein existed in inclusion body. About 90% of the purity of rhNTN was obtained after cleavaging with hydroxylamine in denaturation, purifying with SP-Sepharose fast flow and renaturing.Secondly, a gene for mature human NTN using preferred codons by the yeast Pichia pastoris was synthesized. This synthesized gene was fused with a factor signal peptide gene of Saccharomyces cerevisiae and the fused gene was cloned into yeast expression vector pPIC9k. Then recombinant plasmid pPIC9k-α-hNTN was transformed into the Pichia pastoris cell GS115. A strain of Pichia pastoris capable of secreting hNTN into the medium at a level up to 100mg/L was constructed. The recombinant protein was purified to about 95% using CM-Sepharose ion exchange and Superdex-75 size-exclusion chromatographies.Lastly, the biological activities of recombinant hNTN from E.coli and Pichia pastoris were characterized. Recombinant hNTN from E.coli and Pichia pastoris stimulated growth of nerve fibers from the dorsal root ganglia of the chick embryos and promoted survival of mesencephalon dopaminergic neurons of rat embryos in vitro. Furthermore, the hNTN promoted survival of facial neurons in rat facial paralysis model and prevent PD...