文献类型：期刊文献

英文题名：Use of aqueous two-phase systems in the purification of human interferon-alpha 1 from recombinant Escherichia coli

作者：Guan, Y; Lilley, TH; Treffry, TE; Zhou, CL; Wilkinson, PB

机构：[1]UNIV SHEFFIELD, DEPT MOL BIOL & BIOTECHNOL, SHEFFIELD S10 2TN, S YORKSHIRE, ENGLAND;[2]UNIV SHEFFIELD, DEPT CHEM, SHEFFIELD S10 2TN, S YORKSHIRE, ENGLAND;[3]UNIV SHEFFIELD, KREBS INST BIOMOLEC RES, SHEFFIELD S10 2TN, S YORKSHIRE, ENGLAND;[4]E CHINA UNIV SCI & TECHNOL, DEPT BIOCHEM ENGN, SHANGHAI 200237, PEOPLES R CHINA

年份：1996

卷号：19

期号：6

起止页码：446

外文期刊名：ENZYME AND MICROBIAL TECHNOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:A1996VL25700006)】；

语种：英文

外文关键词：interferon; interferon-alpha; recombinant protein; aqueous two-phase systems; partitioning; Escherichia coli; polyethylene glycol; polyethylene glycol derivative

摘要：Intracellular human recombinant interferon alpha 1 (rhIFN-alpha 1) has been purified using two consecutive aqueous two-phase extractions: selective extraction followed by back extraction. In the selective extraction step using polyethylene glycol (PEG)-potassium phosphate aqueous two-phase system, optimization of the systems was achieved by varying the concentrations of PEG-phosphate ester, phosphate, added inorganic salts, pH, and biomass load. The optimum condition was found to be 22% (w/w) synthesized PEG-phosphate ester, 16% (w/w) potassium phosphate and 3% (w/w) NaCl at pH 6.9 with 10% (wet w/w) biomass load. The corresponding top phase yield of rhIFN-alpha 1 was 99.6% with a purification factor of 25. It was observed that most of the modified PEG is partitioned to the phosphate-enriched bottom phase, so the mechanism for enhancement to the extraction is likely to be an electrostatic expulsion effect. In the back extraction step where the partially purified IFN is transferred from the PEG-rich top phase to a phosphate buffer, the concentrations of PEG-phosphate ester and phosphate, pH, and other parameters have been adjusted. The pH was found to be the most sensitive. A back extraction system using 20% (w/w) PEG-phosphate ester and 10% (w/w) potassium phosphate system at pH 6.0 gives the yield of rhIFN-alpha 1 in the bottom phase (approximately 76%). This newly established back extraction procedure avoids the use of a cumbersome gel chromatography while the yield achieved is fairly high.