Purification and characterization of a recombinant version of human α-fetoprotein expressed in the milk of transgenic goats
Section snippets
Materials
Milk containing rhAFP was obtained from transgenic goats which contain the genomic DNA for human α-fetoprotein N251Q under the control of the goat β-casein promoter. To generate these transgenic goats, genomic DNA encoding human AFP was isolated and engineered to remove the single glycosylation site by changing the asparagine codon to a glutamine codon at amino acid 251 (position 233 of the mature secreted form of rhAFP which has been processed to remove the 18 amino acid secretion signal). The
Purification of recombinant hAFP expressed in the milk of transgenic goats
The concentration of rhAFP in the milk of transgenic goats, as determined by ELISA, was in the range of 0.6–1.1 g/L. Recombinant proteins are expressed in transgenic goat milk as whey proteins [20], and substantial initial purification away from fats, caseins, and other components is typically achieved by several precipitation and centrifugation steps or by tangential flow filtration—this initial purification is typically referred to as clarification. The clarification of milk containing rhAFP
Conclusions
In summary, a recombinant version of human AFP, engineered to remove its glycosylation site, was expressed in transgenic goat milk and purified at gram scale by a three-column procedure. This expression and purification system produced highly purified protein that, apart from the absence of glycosylation, was identical to human AFP as judged by mass spectroscopy, circular dichroism, pharmacokinetics, and cell-binding activity. Recombinant human AFP produced by this method will be a useful
Acknowledgments
We would like to acknowledge Dr. Joe Nawrocki for his help with the tandem mass spectroscopy analysis of MM-093, Dr. John Anders for his help with the absorptivity constant determination, and Drs. Ulrik Nielsen, Michael Cardone, Anna Voronova, and Elise Tran for their helpful suggestions for the manuscript.
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