title: Efficient separation of homologous alpha-lactalbumin from transgenic bovine milk using optimized hydrophobic interaction chromatography.
abstract: Transgenic bovine milk could be a rich source of recombinant human proteins. However, the co-presence of bovine and human homologous proteins can be a challenge for product purification. In this study, the average surface hydrophobicity and electric potential of human alpha-lactalbumin (HLA) and bovine alpha-lactalbumin (BLA) were analyzed and compared through the exposure area calculation of different amino acids. Based on the analysis, calcium independent hydrophobic interaction chromatography was selected for separation of recombinant human alpha-lactalbumin (rHLA) from BLA in transgenic bovine milk. The operating conditions for the best separation of two proteins were predicted by fluorescence data. Three commercially available HIC resins (Butyl Sepharose 4 FF, Octyl Sepharose 4 FF, Phenyl Sepharose 6 FF) were compared. The transgenic milk was skimmed and treated by pH adjustment to remove a large quantity of casein protein. The supernatant was loaded on the hydrophobic interaction chromatographic matrix. The correct elution fraction was further treated with gel filtration chromatography. The overall recovery of rHLA was up to 67.1% with the purity greater than 95%. Circular dichroism spectroscopy (CD) and mass spectrogram (MS) confirmed the native state and glycosylated form of the purified rHLA.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 1, Bei-er-tiao, Zhong-guan-cun, Beijing 100190, China; Graduate School of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Luo
authors.forename: Jingxiu
authors.initials: J
authors.lastname: Bi
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Lijing
authors.initials: L
authors.lastname: Sun
authors.forename: Yongdong
authors.initials: Y
authors.lastname: Liu
authors.forename: Guifeng
authors.initials: G
authors.lastname: Zhang
authors.forename: Guanghui
authors.initials: G
authors.lastname: Ma
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle; D002850:Chromatography, Gel; D002853:Chromatography, Liquid / Q000379:methods*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D030841:Food, Genetically Modified; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007768:Lactalbumin / Q000737:chemistry / Q000302:isolation & purification*; D008892:Milk / Q000737:chemistry*; D008958:Models, Molecular; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D011994:Recombinant Proteins; D007768:Lactalbumin