Aqueous two-phase partitioning of milk proteins. Application to human protein C secreted in pig milk.
Milk of transgenic pigs secreting recombinant human Protein C (rHPC) was used as a model system to determine the utility of aqueous two-phase extraction systems (ATPS) for the initial step in the purification of proteins from milk. The major challenges in purification of recombinant proteins from milk are removal of casein micelles (that foul processing equipment) and elimination of the host milk proteins from the final product. When milk was partitioned in ATPS composed of polyethylene glycol (PEG) and ammonium sulfate (AS), the phases were clarified and most of the caseins precipitated at the interphase. The partition coefficients of the major milk proteins and rHPC were dependent upon the molecular weight of the PEG used in the ATPS. Higher-partition coefficients of the major whey proteins, beta-lactoglobulin, and alpha-lactalbumin were observed in ATPS made up of lower molecular-weight PEG (1000 or 1450) as compared to systems using higher molecular-weight PEG. Lowering the pH of the ATPS from 7.5 to 6.0 resulted in increased precipitation of the caseins and decreased their concentration in both phases. rHPC had a partition coefficient of 0.04 in a system composed of AS and PEG 1450. The rHPC in pig milk was shown to be highly heterogenous by two-dimensional gel electrophoresis. The heterogeneity was owing to inefficient proteolytic processing of the single chain to the heterodimeric form and differences in glycosylation and other post-translational processing. Differential partitioning of the multiple forms of purified rHPC in the ATPS was not observed. rHPC after processing in ATPS was recovered in a clear phase free of most major milk proteins. ATPS are useful as the initial processing step in the purification of recombinant proteins from milk because clarification and enrichment in combined in a single step.
Cole
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KD
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. Cole@mailserver.nist.gov
Lee
T K
TK
Lubon
H
H
D000645:Ammonium Sulfate; D000818:Animals; D030801:Animals, Genetically Modified; D002364:Caseins / Q000302:isolation & purification; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008823:Micelles; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D011486:Protein C / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D013045:Species Specificity; D013552:Swine
D002364:Caseins; D008823:Micelles; D008894:Milk Proteins; D011486:Protein C; D011092:Polyethylene Glycols; D000645:Ammonium Sulfate