Zn(2+)-selective purification of recombinant proteins from the milk of transgenic animals.
The milk of transgenic livestock is becoming a viable, large-scale source of post-translationally complex, recombinant therapeutic proteins. Recombinant vitamin K-dependent proteins such as human protein C (rhPC) and Factor IX can be produced in milk. However, rate limitations in post-translational modification such as intrachain proteolytic cleavage and gamma-carboxylation occur in the mammary gland. Thus, most desirable recombinant products often exist as sub-populations in milk because the mammary gland tends to secrete incompletely processed polypeptides. In general, a nonaffinity purification strategy by which to purify mature recombinant proteins from milk is desirable. Zn2+ is used to selectively modify ion-exchange adsorption behavior of endogenous and recombinant milk proteins through conformational changes which cause aggregation and or precipitation. Zn(2+)-selective precipitation of milk and recombinant proteins results in the purification of active rhPC at high yield from the milk of transgenic pigs using expanded bed chromatography. This method selects for rhPC which is both heterodimeric and properly gamma-carboxylated. Due to the homology of milk proteins among different species, this same Zn(2+)-selective precipitation strategy is useful for developing purification methods for other recombinant proteins from the milk of transgenic livestock.
Degener
A
A
Department of Chemical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, USA.
Belew
M
M
Velander
W H
WH
D000818:Animals; D030801:Animals, Genetically Modified; D002118:Calcium / Q000737:chemistry; D006801:Humans; D008027:Light; D008274:Magnesium / Q000737:chemistry; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000737:chemistry; D011486:Protein C / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012542:Scattering, Radiation; D013552:Swine; D015032:Zinc / Q000737:chemistry*
D008894:Milk Proteins; D011486:Protein C; D011994:Recombinant Proteins; D008274:Magnesium; D015032:Zinc; D002118:Calcium