title: Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Interdepartmental Plant Biology Major, Iowa State University, Ames, IA 50011-1010, USA.
authors.forename: Xing
authors.initials: X
authors.lastname: Xu
authors.forename: Qinglei
authors.initials: Q
authors.lastname: Gan
authors.forename: Richard C
authors.initials: RC
authors.lastname: Clough
authors.forename: Kameshwari M
authors.initials: KM
authors.lastname: Pappu
authors.forename: John A
authors.initials: JA
authors.lastname: Howard
authors.forename: Julio A
authors.initials: JA
authors.lastname: Baez
authors.forename: Kan
authors.initials: K
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002853:Chromatography, Liquid; D024042:Collagen Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000090602:Collagen Type I, alpha 1 Chain; D006801:Humans; D006900:Hydroxylation; D006909:Hydroxyproline / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry; D003313:Zea mays / Q000235:genetics* / Q000378:metabolism
chemical_list: C000716127:COL1A1 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D011994:Recombinant Proteins; D011393:Procollagen-Proline Dioxygenase; D006909:Hydroxyproline