Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase

• Published in: BMC biotechnology Vol. 11; no. 1; p. 69
• Main Authors: Xu, Xing, Gan, Qinglei, Clough, Richard C, Pappu, Kameshwari M, Howard, John A, Baez, Julio A, Wang, Kan
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 24.06.2011; BioMed Central; BMC
• Subjects: Amino Acid Sequence; Blotting, Western; Chromatography, Liquid; Collagen; Collagen Type I - chemistry; Collagen Type I - genetics; Collagen Type I - metabolism; Corn; Gene expression; Genetic aspects; Humans; Hydroxylation; Hydroxyproline - chemistry; Hydroxyproline - metabolism; Molecular Sequence Data; Physiological aspects; Pichia; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Procollagen-Proline Dioxygenase - chemistry; Procollagen-Proline Dioxygenase - genetics; Procollagen-Proline Dioxygenase - metabolism; Proline; Protein Engineering; Protein Stability; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Seeds - genetics; Seeds - metabolism; Sequence Alignment; Tandem Mass Spectrometry; Zea mays - genetics; Zea mays - metabolism; United States
• ISSN: 1472-6750; 1472-6750
• DOI: 10.1186/1472-6750-11-69

Collagens require the hydroxylation of proline (Pro) residues in their triple-helical domain repeating sequence Xaa-Pro-Gly to function properly as a main structural component of the extracellular matrix in animals at physiologically relevant conditions. The regioselective proline hydroxylation is catalyzed by a specific prolyl 4-hydroxylase (P4H) as a posttranslational processing step. A recombinant human collagen type I α-1 (rCIα1) with high percentage of hydroxylated prolines (Hyp) was produced in transgenic maize seeds when co-expressed with both the α- and β- subunits of a recombinant human P4H (rP4H). Germ-specific expression of rCIα1 using maize globulin-1 gene promoter resulted in an average yield of 12 mg/kg seed for the full-length rCIα1 in seeds without co-expression of rP4H and 4 mg/kg seed for the rCIα1 (rCIα1-OH) in seeds with co-expression of rP4H. High-resolution mass spectrometry (HRMS) analysis revealed that nearly half of the collagenous repeating triplets in rCIα1 isolated from rP4H co-expressing maize line had the Pro residues changed to Hyp residues. The HRMS analysis determined the Hyp content of maize-derived rCIα1-OH as 18.11%, which is comparable to the Hyp level of yeast-derived rCIα1-OH (17.47%) and the native human CIa1 (14.59%), respectively. The increased Hyp percentage was correlated with a markedly enhanced thermal stability of maize-derived rCIα1-OH when compared to the non-hydroxylated rCIα1. This work shows that maize has potential to produce adequately modified exogenous proteins with mammalian-like post-translational modifications that may be require for their use as pharmaceutical and industrial products.