Expression And Functional Characterization Of Antifreeze Protein BkAFP972 From Blaps Kashgarensis

Antifreeze proteins (AFPs), known as thermal hysteresis proteins have the ability to inhibit ice nucleators and ice recrystallization. In recent years, researches on antifreeze proteins are mainly concentrated on the cryoprotection of AFP to organisms. However, the purification of enough amounts of natural antifreeze proteins is difficulty, which leads to the restriction of the application of AFP. To obtain mass production of antifreeze proteins, the employment of bioengineering technique might be essential.The antifreeze protein gene Bkafp972 from beetle Blaps kashgarensis was inserted into the Pichia pastorris expression vector pPIC9K, which containing AOX 1 promotor andαsecreting signal peptide. The recombinant expression plasmid pPIC9K- Bkafp972 was constructed and transformed into E. coli Top10 F'. The recombinant plasmid was transformed in to Pichia pastorris GS115. The transformants with multiple copies of insert were screened by DNA sequence analysis. The selected transformants were fermented and induced by 0.5% methanol in flasks. The protein expression was detected by TricineSDS-PAGE,Western blotting and HPLC. The biological activity of the recombinant protein was confirmed via low temperature bacteria protection experiment, and the morphology of ice crystals in the AFP solution.The results of all above showed that Bkafp972 was inserted into pPIC9K. Western blotting indicated that the recombinan protein was expressed and the product had antigenicity. The product could prevent the bacteria from freezing damage, as well as decorated the ice crystal shape when freezing occurred.In addition, Bkafp972 were prokaryotic expressed, and its antiserum was prepared. A novel strategy, vein blood transmission, for delivering AFP to protect mouse liver at -20°C was test, Microscopic cross-section showed that this method could protect the organ effectively against the low temperature freeze injury.