Purification and characterization of a transgenic corn grain-derived recombinant collagen type I alpha 1.
Corn offers advantages as a transgenic host for producing recombinant proteins required at large volumes (1,000's of tons per year) and low cost (less than US$50/kg) by generating them as co-products of biorefining. We describe the purification and characterization of a corn grain-derived mammalian structural protein having such market characteristics: a full length recombinant collagen type I alpha 1 (rCI alpha 1) chain. Material properties of interest are gelation behavior, which would depend on as yet unverified ability of corn to carry out post-translational prolyl hydroxylation and formation of triple helical conformation. The starting material was grain where the expression of rCI alpha 1 had been directed by an embryo-specific promoter. Purification consisted of extraction at low pH followed by membrane and chromatographic steps to isolate rCI alpha 1 for characterization. The amino acid composition and immunoreactivity of CI alpha 1 was similar to that of an analogous native human CI alpha 1 and to rCI alpha 1 produced by the yeast Pichia pastoris. Tandem mass spectrometry confirmed the primary sequence of the corn-derived rCI alpha 1 with 46% coverage. Fragments of the rCI alpha 1 chains were also observed, possibly caused by endogenous plant proteases. The corn-derived rCI alpha 1 had a low level of prolyl hydroxylation (approximately 1% versus 11%) relative to animal-derived CI alpha 1 and folded into its characteristic triple-helical structure as indicated by its resistance to pepsin digestion below its melting temperature of 26(o)C. The 29 amino acid foldon fused to the C-terminus to initiate triple helix formation was not cleaved from the rCI alpha 1 chains, but could be removed by pepsin treatment.
Zhang
Cheng
C
Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, USA.
Baez
Julio
J
Pappu
Kameshwari M
KM
Glatz
Charles E
CE
D000595:Amino Acid Sequence; D015153:Blotting, Western; D024042:Collagen Type I / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000090602:Collagen Type I, alpha 1 Chain; D004591:Electrophoresis, Polyacrylamide Gel; D005374:Filtration; D006801:Humans; D006900:Hydroxylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D030821:Plants, Genetically Modified / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry; D003313:Zea mays / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C000716127:COL1A1 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D011994:Recombinant Proteins