Purification and characterization of a recombinant version of human α-fetoprotein expressed in the milk of transgenic goats

https://doi.org/10.1016/j.pep.2004.07.007Get rights and content

Abstract

α-Fetoprotein (AFP) is a 68 kDa glycoprotein expressed at high levels by the fetal liver and yolk with transcription repressed to very low levels after birth. Transfer of fetal AFP through the placenta into the circulation of the mother is correlated with remission of rheumatoid arthritis, multiple sclerosis, and other autoimmune disorders. AFP is therefore under development as a biopharmaceutical for the treatment of autoimmune diseases. The clinical evaluation of AFP requires the production of hundreds of grams of highly purified and biologically active protein. We have produced goats that express a form of the human AFP transgene under the control of the β-casein promoter. In this form of rhAFP, the single N-linked glycosylation site was removed by mutagenesis (N233Q). Here, we describe a purification protocol for this recombinant human (rh)AFP from the milk of these transgenic goats. A three-column procedure was developed to produce gram quantities of highly purified rhAFP. Near- and far-UV circular dichroism spectra of human umbilical cord blood AFP and rhAFP were essentially identical, suggesting that the structure is not affected by removal of the glycosylation site. Furthermore, the cell binding and pharmacokinetics of purified rhAFP were similar to human AFP isolated from cord blood. Our results demonstrate that an active form of rhAFP can be produced on industrial scale by expression in transgenic goat milk.

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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