Expression PEBP Gene From Saussurea Involucr Ate Kar.er Kir In E.coli And Pichia Pastoris

PEBP gene is new Antifreeze gene that was separated by our laboratory in Saussurea involucrate Kar.er kir. The length of PEBP is 510bp,Homology search with PEBP were performed against GenBank using BLAST, the identity with PEBP of Arabidopsis thaliana were 81%.ln this research, Using Teasy-PEBP plasmid as a template ,the PEBP gene was amplified by PCR .PEBP gene was inserted into pET30a(+) andpPIC9K, then expressed in E. coli BL21 and pichia pastoris GS115, respectively. The expression products were purified by affinity chromatography, and the optimal expression condition was obtained by preliminary research. The main results were as follows:A pair of specific primers PEBP sense and PEBP anti-sense were designed based on the multi clone site (MCS) of pET30a(+), introduced restriction site EcoRâ… and Ncoâ… respectively. The PEBP gene was amplified from Teasy-PEBP plasmid by PCR and inserted into the pET30a(+) vector. The recombinant vector was transferred into Ecoli BL21(DE3)and induced the expression of protein by low concentration of IPTG and low temperature overnight. Fusion protein was purified by Ni-NTA affinity chromatography. SDS-PAGE and Western blot analysis showed that the fusion protein relative molecular weight was 28KD that was consistent with the theoretical value, and its product was 26.8% in total protein.Expression primers PEBP sense and PEBP anti-sense were designed based on the MCS)of pPIC9K, introduced restriction site EcoRâ… and Notâ… respectively. The PEBP gene was amplified from Teasy-PEBP plasmid by PCR and inserted into the Pichia pastoris expression vector. The recombinant plasmid named as pPIC9K-PEBP was linearized by Salâ… ,and men transformed into Pichia pastoris GS115. Mut^s (Methanol utilization slow) transformants were screened by MD and MM plates, confirmed by PCR. Supernatant of the culture was analyzed by SDS-PAGE, a size of specific strip was found, indicating that the PEBP gene was expressed and its relative molecular weight is 20KD, and target protein was 90% in total protein. The PEBP has been definiteanti-freeze character by the biological activity detection of the supernatant.The optimal expression parameters of GS115 were 0.5% ( V/V ) CH₃OH concentration, PH 6.0 culture medium. 30℃culture temperature, 216 hours inducing time.The prokaryotic and Eukaryotic expression system of PEBP is successfully constructed. It lays the foundation for the further study on the Antifreeze characters of the PEBP...