title: Purification and characterization of a recombinant version of human alpha-fetoprotein expressed in the milk of transgenic goats.
abstract: Alpha-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 beta-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.
authors.affiliation: Merrimack Pharmaceuticals, Inc., 101 Binney St., Cambridge, MA 02142, USA.
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Parker
authors.forename: Eszter
authors.initials: E
authors.lastname: Birck-Wilson
authors.forename: Greg
authors.initials: G
authors.lastname: Allard
authors.forename: Nick
authors.initials: N
authors.lastname: Masiello
authors.forename: Maria
authors.initials: M
authors.lastname: Day
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Murphy
authors.forename: Violette
authors.initials: V
authors.lastname: Paragas
authors.forename: Sandra
authors.initials: S
authors.lastname: Silver
authors.forename: Mark D
authors.initials: MD
authors.lastname: Moody
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D005260:Female; D005312:Fetal Blood / Q000737:chemistry; D006041:Goats / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008892:Milk / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000493:pharmacokinetics; D019076:Transgenes; D020298:U937 Cells; D000509:alpha-Fetoproteins / Q000302:isolation & purification* / Q000378:metabolism / Q000493:pharmacokinetics
chemical_list: D011993:Recombinant Fusion Proteins; D000509:alpha-Fetoproteins