title: Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk.
abstract: Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to <20% of the world's population. We used transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock.
authors.affiliation: National Swine Resource and Research Center &Division of Animal Science, University of Missouri, Columbia, MO 65211, USA.|State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China, 100101.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Protein Purification and Characterization Laboratories, Department of Chemical and Biomolecular Engineering, 207 Othmer Hall, University of Nebraska, Lincoln 68588, USA.
authors.forename: Weijie
authors.initials: W
authors.lastname: Xu
authors.affiliation: National Swine Resource and Research Center &Division of Animal Science, University of Missouri, Columbia, MO 65211, USA.|Department of Animal Science, Iowa State University, Ames, IA 50011, USA.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Ross
authors.affiliation: National Swine Resource and Research Center &Division of Animal Science, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Walters
authors.affiliation: ProGenetics, LLC, Blacksburg, VA, 24060, USA.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Butler
authors.affiliation: National Swine Resource and Research Center &Division of Animal Science, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Jeff J
authors.initials: JJ
authors.lastname: Whyte
authors.affiliation: National Swine Resource and Research Center &Division of Animal Science, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Lindsey
authors.initials: L
authors.lastname: Kelso
authors.affiliation: Protein Purification and Characterization Laboratories, Department of Chemical and Biomolecular Engineering, 207 Othmer Hall, University of Nebraska, Lincoln 68588, USA.
authors.forename: Mostafa
authors.initials: M
authors.lastname: Fatemi
authors.affiliation: Protein Purification and Characterization Laboratories, Department of Chemical and Biomolecular Engineering, 207 Othmer Hall, University of Nebraska, Lincoln 68588, USA.
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Vanderslice
authors.affiliation: National Swine Resource and Research Center &Division of Animal Science, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Keith
authors.initials: K
authors.lastname: Giroux
authors.affiliation: National Swine Resource and Research Center &Division of Animal Science, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Lee D
authors.initials: LD
authors.lastname: Spate
authors.affiliation: National Swine Resource and Research Center &Division of Animal Science, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Samuel
authors.affiliation: National Swine Resource and Research Center &Division of Animal Science, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Cliff N
authors.initials: CN
authors.lastname: Murphy
authors.affiliation: National Swine Resource and Research Center &Division of Animal Science, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Wells
authors.affiliation: LFB USA, Inc.175 Crossing Blvd. Framingham, MA 01702, USA.
authors.forename: Nick C
authors.initials: NC
authors.lastname: Masiello
authors.affiliation: National Swine Resource and Research Center &Division of Animal Science, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Randall S
authors.initials: RS
authors.lastname: Prather
authors.affiliation: Protein Purification and Characterization Laboratories, Department of Chemical and Biomolecular Engineering, 207 Othmer Hall, University of Nebraska, Lincoln 68588, USA.
authors.forename: William H
authors.initials: WH
authors.lastname: Velander
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D019149:Bioreactors; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D005260:Female; D045683:Furin / Q000235:genetics* / Q000378:metabolism; D002836:Hemophilia B / Q000628:therapy*; D006801:Humans; D007774:Lactation / Q000378:metabolism; D008297:Male; D008321:Mammary Glands, Animal / Q000378:metabolism*; D008892:Milk / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D013552:Swine
chemical_list: D011994:Recombinant Proteins; D005164:Factor IX; D045683:Furin