Synthesis of Human Neurotrohin-3 Gene and Its Expression in Pichia pastoris

A 357bp DNA fragment encoding Human neurotrohin-3 (hNT-3) was generated by five rounds of PCR from a total of ten 36～59nt oligoes after modification of the original sequence to the optional codon usage of Pichia pastoris.The gene(called NT-3b gene)was cloned into pPIC9K to construct a recombinant vector pPIC9K-NT-3b. This recombinant vector and another recombinant vector pPIC9K-NT-3 containing the hNT-3 gene were transformed into P.pastoris strain GS115 respectively by electroporation. The transformants were induced by methanol and the expression product were analyzed by SDS-PAGE, Western blot and ELISA. The results showed that soluble NT-3 were secreted into culturesupernatant .The NT-3b gene is better than the hNT-3 gene for expression in the GS115 yeast strain(x 2=4.334,P0.05) and the expression level was higher between 72～96h after induction, reaching about 31mg/L.