title: Purification and characterization of a 44-kDa recombinant collagen I alpha 1 fragment from corn grain.
abstract: This paper demonstrates that a fibrous, repetitive amino acid sequence collagen-related protein, a 44-kDa fragment of human collagen I alpha 1 (CIalpha1), was expressed in corn grain molecularly equivalent to that produced in recombinant yeast. The recombinant CIalpha1 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 CIalpha1 with >70% purity and 60% recovery. The N-terminal sequence, amino acid composition, and immunoreactivity closely matched those of an analogous 44-kDa CIalpha1 fragment produced by the yeast Pichia . The corn-derived 44-kDa CIalpha1 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.
authors.affiliation: Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, USA.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhang
authors.forename: Julio
authors.initials: J
authors.lastname: Baez
authors.forename: Charles E
authors.initials: CE
authors.lastname: Glatz
mesh_terms: D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D024042:Collagen Type I / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000090602:Collagen Type I, alpha 1 Chain; D006031:Glycosylation; D006801:Humans; D008970:Molecular Weight; D010766:Phosphorylation; D030821:Plants, Genetically Modified / Q000737:chemistry*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012639:Seeds / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D003313:Zea mays / Q000737:chemistry* / Q000235:genetics
chemical_list: D000596:Amino Acids; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D011994:Recombinant Proteins