Purification and Characterization of a 44-kDa Recombinant Collagen I α 1 Fragment from Corn Grain

This paper demonstrates that a fibrous, repetitive amino acid sequence collagen-related protein, a 44-kDa fragment of human collagen I α 1 (CIα1), was expressed in corn grain molecularly equivalent to that produced in recombinant yeast. The recombinant CIα1 was extracted and purified from early generation plants having low levels of recombinant protein accumulation. It was selectively extracted at low pH and purified by ion exchange and gel filtration chromatography, resulting in a 44-kDa CIα1 with >70% purity and 60% recovery. The N-terminal sequence, amino acid composition, and immunoreactivity closely matched those of an analogous 44-kDa CIα1 fragment produced by the yeast Pichia. The corn-derived 44-kDa CIα1 had an intact protein mass of 44088 Da, which is within 0.2% of the mass calculated from the expected sequence. Tandem mass spectrometry confirmed the primary sequence with 78% coverage. The amino acid composition analysis indicated a low level of prolyl hydroxylation. Glycoprotein staining revealed no glycosylation.