Zhang Cheng, Baez Julio, Glatz Charles E
Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, USA.
J Agric Food Chem. 2009 Feb 11;57(3):880-7. doi: 10.1021/jf8026205.
This paper demonstrates that a fibrous, repetitive amino acid sequence collagen-related protein, a 44-kDa fragment of human collagen I alpha 1 (CIalpha1), was expressed in corn grain molecularly equivalent to that produced in recombinant yeast. The recombinant CIalpha1 was extracted and purified from early generation plants having low levels of recombinant protein accumulation. It was selectively extracted at low pH and purified by ion exchange and gel filtration chromatography, resulting in a 44-kDa CIalpha1 with >70% purity and 60% recovery. The N-terminal sequence, amino acid composition, and immunoreactivity closely matched those of an analogous 44-kDa CIalpha1 fragment produced by the yeast Pichia . The corn-derived 44-kDa CIalpha1 had an intact protein mass of 44088 Da, which is within 0.2% of the mass calculated from the expected sequence. Tandem mass spectrometry confirmed the primary sequence with 78% coverage. The amino acid composition analysis indicated a low level of prolyl hydroxylation. Glycoprotein staining revealed no glycosylation.
本文证明，一种纤维状、重复氨基酸序列的胶原蛋白相关蛋白，即人胶原蛋白Iα1（CIα1）的44 kDa片段，在玉米籽粒中的表达在分子水平上等同于在重组酵母中产生的表达。从重组蛋白积累水平较低的早期世代植物中提取并纯化了重组CIα1。它在低pH值下被选择性提取，并通过离子交换和凝胶过滤色谱法进行纯化，得到了纯度>70%、回收率60%的44 kDa CIα1。其N端序列、氨基酸组成和免疫反应性与酵母毕赤酵母产生的类似44 kDa CIα1片段的序列、组成和免疫反应性密切匹配。玉米来源的44 kDa CIα1的完整蛋白质质量为44088 Da，与根据预期序列计算出的质量相差在0.2%以内。串联质谱法证实了78%的序列覆盖率。氨基酸组成分析表明脯氨酰羟化水平较低。糖蛋白染色显示没有糖基化。
