Interdepartmental Plant Biology Major, Iowa State University, Ames, IA 50011-1010, USA.
BMC Biotechnol. 2011 Jun 24;11:69. 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.
胶原蛋白的三螺旋结构域重复序列 Xaa-Pro-Gly 中的脯氨酸（Pro）残基需要羟化，才能在生理相关条件下作为动物细胞外基质的主要结构成分正常发挥功能。这种区域选择性脯氨酸羟化是由特定的脯氨酰 4-羟化酶（P4H）作为翻译后加工步骤来催化的。
当与重组人 P4H（rP4H）的α和β亚基共表达时，在转基因玉米种子中生产出具有高百分比羟脯氨酸（Hyp）的重组人 I 型胶原蛋白 α-1（rCIα1）。使用玉米球蛋白-1 基因启动子在生殖细胞中特异性表达 rCIα1，导致在没有共表达 rP4H 的情况下，全长 rCIα1 在种子中的平均产量为 12mg/kg，而在共表达 rP4H 的情况下，rCIα1（rCIα1-OH）的产量为 4mg/kg。高分辨率质谱（HRMS）分析表明，从共表达 rP4H 的玉米系中分离出的 rCIα1 中，近一半的胶原蛋白重复三肽的 Pro 残基被改变为 Hyp 残基。HRMS 分析确定玉米来源的 rCIα1-OH 的 Hyp 含量为 18.11%，与酵母来源的 rCIα1-OH（17.47%）和天然人 CIa1（14.59%）的 Hyp 水平相当。与非羟化的 rCIα1 相比，增加的 Hyp 百分比与玉米来源的 rCIα1-OH 的热稳定性显著增强有关。
这项工作表明，玉米具有生产具有哺乳动物样翻译后修饰的外源性蛋白质的潜力，这些修饰可能是其作为药物和工业产品使用所必需的。
