On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows.
Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni2+ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%.
Institute of Fundamental Sciences, Massey University, New Zealand.
Medhat A
MA
Al-Ghobashy
Martin A K
MA
Williams
Brigid
B
Brophy
Götz
G
Laible
David R K
DR
Harding
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005260:Female; D006801:Humans; D008823:Micelles; D008892:Milk / Q000737:chemistry*; D004676:Myelin Basic Protein / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D002364:Caseins; D008823:Micelles; D004676:Myelin Basic Protein; D011993:Recombinant Fusion Proteins