Expression Of Human Neurotrophin-4 Gene In Escherichia Coli And Purification Of The Product

Object: Neurotrophins have been demonstrated to play important roles in the development and functioning of the nervous system, they are specific proteins which can maintain and promote the survival, develop and differentiation of neuron. Neurotrophin-4 is the fourth neurotrophin which gene was first been cloned from Xenopus in 1990. But human neurotrophin-4 (hNT-4) has only a very little content in all human tissues which makes a very difficulty to the research of hNT-4' s functions. So it' s very necessary to obtain a certain amount of hNT-4 through some other way. Our research object is to obtain the human matured neurotrophin-4 with high purity by the technique of genomic engineering.Method: With the chromosomal DNA of human blood lymphocytes as template, the gene of the human matured neurotrophin-4 was amplified by polymerase chain reaction (PCR), and the gene was recombined into phage vector pET32a which was a efficient prokaryotic vector of protein expression. The recombined phage was called pET-NT4, and transformed into E.coli BL21(DE3) for expression, the recombined bacteria was induced to express the target protein. After detected by SDS-PAGE, the expressed protein would be purified through Ni²⁺ affinity chromatography.Result: The human matured neurotrophin-4 gene was amplified successfully from the chromosomal DNA. The gene was integrated into phage vector pET32a and formed a recombination plasmid (pET-NT4). After transformed into E.coli BL21(DE3) and induced by IPTG, the recombined bacteria expressed a special protein , the molecule weight of the special protein is about 35KD which is according with what wehave anticipated before, and the protein mainly existed in the formation of inclusion bodies. After purified with Ni"* affinity chromatography, the hmNT-4 fusion protein(hmNT-4-TrxA) was obtained to a 94% purity.Conclusion: The human matured neurotrophin-4 gene was amplified, the hmNT-4 fusion protein(hmNT-4-TrxA) was expressed in the pET-32a prokaryotic system, and its purified protein was obtained.