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- W2392877273 abstract "Objective To express recombinant human heparin binding neurite promoting factor (hrHBNF) with yeast expression system, and to observe its in vitro activity of stimulating neurites outgrowth. Methods The cDNA encoding mature human heparin binding neurite-promoting factor (HBNF) was amplified from total RNA isolated from an aborted human fetus brain (18 weeks) by RT PCR method. After amplification, the HBNF cDNA gene was cloned with pPIC9K, an expression vector for yeast system. The recombinant expression vector bearing HBNF cDNA gene was obtained by screening. The verified recombinant vector was then used to transform Pichia strain GS115 by electroporation. His+ transformants were selected to grow on plates of histidine deficient medium. His+ recombinants with multiple inserts were screened in vivo by their resistance to G418. PCR analysis was used to confirm the integration of the HBNF cDNA gene into the Pichia genome. Secreted expression of hrHBNF protein in culture medium was obtained when the positive clone containing HBNF cDNA gene was induced by methanol. The hrHBNF product purified by gel chromatograph was added to the cultured rat pheochromocytoma (PC12) cells to help observe its activity of stimulating neurites outgrowth. Results In the recombinant expression vector, the insert was exactly the sequence encoding human HBNF according to Genebank data. The open reading frame of this cDNA gene was cloned in frame with the factor α signal sequence in pPIC9K. SDS PAGE showed that the molecular weight of the induced expression product was about 18 000, consistent with human HBNF reported in literatures. The protein product did promote neurites outgrowth in cultured rat pheochromocytoma (PC12) cells. Conclusions The hrHBNF can be steadily expressed through Pichia, and product of hrHBNF has the biological activity to stimulate neurites outgrowth." @default.
- W2392877273 created "2016-06-24" @default.
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- W2392877273 date "2001-01-01" @default.
- W2392877273 modified "2023-09-24" @default.
- W2392877273 title "Recombinant human heparin-binding neurite-promoting factor expressed with yeast and its neurite-promoting activity" @default.
- W2392877273 hasPublicationYear "2001" @default.
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