Process integration for recovery of recombinant collagen type I α1 from corn seed

Christopher M. Setina, Iowa State University
Dow Chemical Co.
Charles E. Glatz, Iowa State University

This is the pre-peer reviewed version of the following article: Setina, Christopher M., Jason P. Haase, and Charles E. Glatz. "Process integration for recovery of recombinant collagen type I α1 from corn seed." Biotechnology progress 32, no. 1 (2016): 98-107, which has been published in final form at DOI: 10.1002/btpr.2191. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. Posted with permission.

Abstract

Because of safety concerns and product consistency issues with the use of animal-derived collagen, several recombinant protein expression hosts have been considered for recombinant collagen corn seed. Full length, triple-helical, recombinant collagen (rCIα1) is expressed as a fusion with a foldon domain, which must later be removed. Here we have examined integration of purification and foldon removal by comparing advantages of removal before or after purification, using salt precipitation as the main purification step. Because expression levels in available maize lines are low, Pichia-produced recombinant collagens, both with and without foldon, were added to corn seed germ at the extraction step.

Salt precipitation of an acidic corn seed extract yielded 100% of the collagen without foldon at >70% purity without the pepsin pretreatment. With pepsin pretreatment, yield was 94.0% with purity of 76.5%. Analysis of the protein molecular weight distribution of the pre- and post-treatment extracts showed that the corn proteins are largely resistant to pepsin proteolysis, explaining why little benefit was obtained by pepsin treatment. In the absence of pepsin treatment, the recovery of rCIα1 with foldon was still above 90% but the purity was only 44%. This still represented at about 13-fold purification with a 2.7-fold volume reduction which would reduce the pepsin requirement for post-recovery foldon cleavage. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:98–107, 2016