昆虫融合抗冻蛋白提取方法的比较

Comparison of methods for extraction of insect fusion antifreeze protein

目的对用低温乙醇法、硫酸铵盐析法及等电点法提取的昆虫融合抗冻蛋白EGFP-OcAFP4浓度和蛋白相对得率进行比较。方法选取不同终浓度的乙醇(5%~65%)、硫酸铵(0.2~3.0 mol/L)及pH(5.6、5.4、5.2、5.0、4.8、4.7、4.6、4.5、4.4)提取重组质粒pET28a-EGFP-OcAFP4诱导表达液破菌上清中的EGFP-OcAFP4,并检测其纯度及蛋白相对得率。结果乙醇终浓度为30%时提取EGFP-OcAFP4的纯度最高,达86.21%,蛋白相对得率为6.89%;硫酸铵终浓度为2.0 mol/L时提取EGFP-OcAFP4的纯度最高,达42.33%,蛋白相对得率为30.83%;pH为5.0时提取EGFP-OcAFP4的纯度最高,达15.46%,蛋白相对得率为11.97%。结论低温乙醇法、硫酸铵盐析法和等电点法均可用于EGFP-OcAFP4的提取,但低温乙醇法和硫酸铵盐析法提取蛋白的纯度及相对蛋白得率更高,更适用于EGFPOcAFP4的提取。

Objective Tocomparetheconcentration and protein relativeyield ofinsectfusion antifreezeprotein 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.