Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, USA.
Biotechnol Prog. 2009 Nov-Dec;25(6):1660-8. doi: 10.1002/btpr.257.
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.
玉米作为生产大量（每年数千吨）和低成本（低于 50 美元/公斤）重组蛋白的转基因宿主具有优势，这些蛋白可以作为生物炼制的副产物产生。我们描述了一种从玉米籽粒中提取的哺乳动物结构蛋白的纯化和特性，该蛋白具有以下市场特征：全长重组胶原蛋白 I 型α1（rCIα1）链。感兴趣的材料特性是胶凝行为，这将取决于玉米是否具有尚未验证的进行翻译后脯氨酰羟化和形成三螺旋构象的能力。起始材料是籽粒，其中 rCIα1 的表达由胚胎特异性启动子指导。纯化包括在低 pH 下提取，然后通过膜和色谱步骤分离 rCIα1 进行特性分析。CIα1 的氨基酸组成和免疫反应性与类似的天然人 CIα1 和酵母毕赤酵母生产的 rCIα1 相似。串联质谱法证实了玉米来源的 rCIα1 的一级序列，覆盖率为 46%。还观察到 rCIα1 链的片段，可能是由内源性植物蛋白酶引起的。与动物来源的 CIα1 相比，玉米来源的 rCIα1 的脯氨酰羟化程度较低（约 1%对 11%），并且在低于其 26°C 的熔点时对胃蛋白酶消化具有抗性，表明其折叠成其特征性的三螺旋结构。融合到 C 末端以启动三螺旋形成的 29 个氨基酸折叠结构未从 rCIα1 链中切割，但可以通过胃蛋白酶处理去除。
