Secretion expression of BPI23-haFGF fusion gene in Pichia pastoris X-33 and identification of its biological activity
Ma, Y.; Zhu, J.; Jin, X.; Peng, H.; Wang, Y.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 26(2): 379-384
2009
ISSN/ISBN: 1001-5515 PMID: 19499807 Document Number: 633256
The BPI23-haFGF fusion gene was subcloned to the yeast expression vector pPICZaA and the recombinant plasmid pPICZaA-BPI23-haFGF was constructed. After linearization by sac I, the construct was introduced into X-33 yeast cells. The efficient engineering strain was obtained by the resistance and phenotype selection and identified by specific PCR. SDS-PAGE and Western blot analysis indicated that a 43 KD protein band coincident with the anticipated fusion protein size expressed in the culture supernatant of the transformed yeast cells, which accounted for above 50% of the total proteins of the culture supernatant. About 90% purity of recombinant BPI23-haFGF fusion protein was obtained by affinity chromatography. The in vitro bioactivity testing showed that the purified fusion protein killed E. coli and promoted proliferation of NIH3T3 cells, suggesting that the recombinant BPI23-haFGF fusion protein possessed both of BPI and FGF functions.