title: Process integration for recovery of recombinant collagen type I α1 from corn seed.
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.
authors.affiliation: Koch Industries - Flint Hills Resources, 510 S.16th Street, suite 104, Ames, IA, 50010.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Setina
authors.affiliation: Dow Chemical Co., 7600 Metro Blvd., Edina, MN, 55439.
authors.forename: Jason P
authors.initials: JP
authors.lastname: Haase
authors.affiliation: Dept. of Chemical and Biological Engineering, Iowa State University, 2114 Sweeney Hall, Ames, IA, 50011.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Glatz
mesh_terms: D024042:Collagen Type I / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000090602:Collagen Type I, alpha 1 Chain; D010434:Pepsin A / Q000737:chemistry; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry; D003313:Zea mays / Q000201:enzymology
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D011994:Recombinant Proteins; D010434:Pepsin A
keywords: Pichia; collagen; corn; purification; transgenic