Comparison of methods for extraction of insect fusion antifreeze protein / 中国生物制品学杂志

ABSTRACT

@#Objective To compare the concentration and protein relative yield of insect fusion antifreeze protein EGFP-OcAFP4 extracted by low temperature ethanol method,ammonium sulfate salting-out method and isoelectric point method. Methods By screening different concentrations of ethanol(5%-65%)and ammonium sulfate(0. 2-3. 0 mol/L)and different the pH values(5. 6,5. 4,5. 2,5. 0,4. 8,4. 7,4. 6,4. 5,4. 4),EGFP-OcAFP4 was extracted from the supernatant of the induced expression solution of recombinant plasmid pET28a-EGFP-OcAFP4,and detected for the purity and protein relative yield.Results When prepared by ethanol method,EGFP-OCAFP4 had the highest purity of 86. 21% at the ethanol final concentration of 30%,and the relative yield was 6. 89%;when it was prepared by ammonium sulfate salting-out,the purity was up to 42. 33% with the protein relative yield of 30. 83% at the ammonium sulfate final concentration of 2. 0 mol/L;the purity of EGFP-OcAFP4 extracted by isoelectric point method with pH of 5. 0 was the highest 15. 46%,and the relative yield was 11. 97%. Conclusion Low temperature ethanol method,ammonium sulfate salting-out and isoelectric point method can all be used for the extraction of EGFP-OcAFP4,but low temperature ethanol method and ammonium sulfate salting-out method have higher extraction purity and protein relative yield,which are more suitable for the extraction of EGFP-OcAFP4