Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk

Jianguo Zhao, Weijie Xu, Jason W. Ross, Eric M. Walters, Stephen P. Butler, Jeff J. Whyte, Lindsey Kelso, Mostafa Fatemi, Nicholas C. Vanderslice, Keith Giroux, Lee D. Spate, Melissa S. Samuel, Cliff N. Murphy, Kevin D. Wells, Nick C. Masiello, Randall S. Prather, William H. Velander

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 3-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 3-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 3-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock.

Original languageEnglish (US)
Article number14176
JournalScientific reports
Volume5
DOIs
StatePublished - Sep 21 2015

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Hemophilia B
Protein Engineering
Factor IX
Human Mammary Glands
Milk
Bioreactors
Therapeutics
Furin
trans-Golgi Network
Livestock
Lactation
Recombinant Proteins
Swine
Cell Count
Enzymes
Population

ASJC Scopus subject areas

  • General

Cite this

Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk. / Zhao, Jianguo; Xu, Weijie; Ross, Jason W.; Walters, Eric M.; Butler, Stephen P.; Whyte, Jeff J.; Kelso, Lindsey; Fatemi, Mostafa; Vanderslice, Nicholas C.; Giroux, Keith; Spate, Lee D.; Samuel, Melissa S.; Murphy, Cliff N.; Wells, Kevin D.; Masiello, Nick C.; Prather, Randall S.; Velander, William H.

In: Scientific reports, Vol. 5, 14176, 21.09.2015.

Research output: Contribution to journalArticle

Zhao, J, Xu, W, Ross, JW, Walters, EM, Butler, SP, Whyte, JJ, Kelso, L, Fatemi, M, Vanderslice, NC, Giroux, K, Spate, LD, Samuel, MS, Murphy, CN, Wells, KD, Masiello, NC, Prather, RS & Velander, WH 2015, 'Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk', Scientific reports, vol. 5, 14176. https://doi.org/10.1038/srep14176
Zhao, Jianguo ; Xu, Weijie ; Ross, Jason W. ; Walters, Eric M. ; Butler, Stephen P. ; Whyte, Jeff J. ; Kelso, Lindsey ; Fatemi, Mostafa ; Vanderslice, Nicholas C. ; Giroux, Keith ; Spate, Lee D. ; Samuel, Melissa S. ; Murphy, Cliff N. ; Wells, Kevin D. ; Masiello, Nick C. ; Prather, Randall S. ; Velander, William H. / Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk. In: Scientific reports. 2015 ; Vol. 5.
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