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

Authors

Jianguo Zhao, University of Missouri, Columbia
Weijie Xu, University of Nebraska-LincolnFollow
Jason W. Ross, University of Missouri, Columbia
Eric M. Walters, University of Missouri, Columbia
Stephen P. Butler, ProGenetics, LLC
Jeff J. Whyte, University of Missouri, Columbia
Lindsey Kelso, University of Missouri, Columbia
Mostafa Fatemi, University of Nebraska-Lincoln
Nicholas C. Vanderslice, University of Nebraska-LincolnFollow
Keith Giroux, University of Missouri, Columbia
Lee D. Spate, University of Missouri, Columbia
Melissa S. Samuel, University of Missouri, Columbia
Cliff N. Murphy, University of Missouri, Columbia
Kevin D. Wells, University of Missouri, Columbia
Nick C. Masiello, LFB USA, Inc.
Randall S. Prather, University of Missouri, Columbia
William H. Velander, University of Nebraska-LincolnFollow

Date of this Version

2015

Document Type

Article

Citation

Scientific Reports, 5:14176

Comments

This work is licensed under a Creative Commons Attribution 4.0 International License.

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 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.