RÉSUMÉ
@#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
RÉSUMÉ
OBJECTIVE: To establish a method for detecting the impurity components in human serum albumin by electrospray ionization mass spectrometry (ESI-Q-TOF MS) and investigate the impurity components in 28 batches of post-marketing albumin products from 27 companies. METHODS: According to Chinese Pharmacopoeia′s General Principles 4121, gel filtration column (Hiprep16/60 SepharylS-200HR) was used to separate the protein components. The dimer and multimer components were collected, then desalted, concentrated and digested by trypsin. The peptide was eluted by BEH C18(2.1 mm×100 mm,130) column with mobile phase consisting of water (0.1% formic acid)-acetonitrile in gradient elution mode. Chromatography-mass spectrometry was used to determine the secondary sequences of the peptides and the searching software (PLGS3.0) was used to identify the impurity protein components in albumin. RESULTS: A total of 23 impurity proteins were detected, among which apolipoprotein A-II, haptoglobin, and hempoexin, α-1B-glycoprotein and α-2-HS-glycoprotein were the impurity proteins common to all enterprise products; α-albumin (VE binding protein), N-acetyl-L-alanine amidase, haptoglobin-related proteins, hemoglobin (β subunit, α subunit), transthyretin and other proteins were found in the products of most companies. The number of heterologous proteins in each enterprise was between 8-17, and the numbers of impurity proteins in 27 companies were quite different. CONCLUSION: This method can be used for the investigation of unknown components in human serum albumin, which provides a guidance for the optimization of human albumin production process.