[Expression of human GDNF in methyltrophic yeast Pichia pastoris and silkworm larvae]

The cDNA encoding glial cell derived neurotrophic factor (GDNF) was cloned into the Pichia expression vector pPIC9K and then transformed into his4 mutant yeast GS115 by electroporation. Multicopy transformants were screened by various G418 concentrations and induced by methanol. The human GDNF gene was cloned into the baculovirus transfer vector pBacPAK8. The recombinant transfer vector pBacPAK-GDNF was coinfected with linear Bm-BacPAK6 DNA into BmN cells. The recombinant virus was screened and plaque-purified. The silkworm larvae were infected with the recombinant virus and collected 5 days later. SDS-PAGE and Western blot confirmed that GDNF was expressed in Pichia culture medium and silkworm larvae hemolymph. The GDNF protein expressed in Pichia and silkworm larvae could significantly promote the survival and neurite outgrowth of dopaminergic neurons.