Peng Yong Y, Stoichevska Violet, Schacht Kristin, Werkmeister Jerome A, Ramshaw John A M
CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, 3169, Australia.
J Biomed Mater Res A. 2014 Jul;102(7):2189-96. doi: 10.1002/jbm.a.34898. Epub 2013 Aug 10.
Bacterially derived triple-helical, collagen-like proteins are attractive as potential biomedical materials. The collagen-like domain of the Scl2 protein from S. pyogenes lacks any specific binding sites for mammalian cells yet possesses the inherent structural integrity of the collagen triple-helix of animal collagens. It can, therefore, be considered as a structurally-stable "blank slate" into which various defined, biological sequences, derived from animal collagens, can be added by substitutions or insertions, to enable production of novel designed materials to fit specific functional requirements. In the present study, we have used site directed mutagenesis to substitute two functional sequences, one for heparin binding and the other for integrin binding, into different locations in the triple-helical structure. This provided three new constructs, two containing the single substitutions and one containing both substitutions. The stability of these constructs was marginally reduced when compared to the unmodified sequence. When compared to the unmodified bacterial collagen, both the modified collagens that contain the heparin binding site showed marked binding of fluorescently labeled heparin. Similarly, the modified collagens from both constructs containing the integrin binding site showed significant adhesion of L929 cells that are known to possess the appropriate integrin receptor. C2C12 cells that lack any appropriate integrins did not bind. These data show that bacterial collagen-like sequences can be modified to act like natural extracellular matrix collagens by inserting one or more unique biological domains with defined function.
细菌衍生的三螺旋胶原样蛋白作为潜在的生物医学材料具有吸引力。化脓性链球菌Scl2蛋白的胶原样结构域缺乏与哺乳动物细胞的任何特异性结合位点，但具有动物胶原蛋白胶原三螺旋的固有结构完整性。因此，它可以被视为一个结构稳定的“空白画布”，通过替换或插入来添加源自动物胶原蛋白的各种特定生物序列，从而能够生产出符合特定功能要求的新型设计材料。在本研究中，我们使用定点诱变将两个功能序列，一个用于肝素结合，另一个用于整合素结合，替换到三螺旋结构的不同位置。这产生了三种新构建体，两种含有单取代，一种含有双取代。与未修饰的序列相比，这些构建体的稳定性略有降低。与未修饰的细菌胶原蛋白相比，含有肝素结合位点的两种修饰胶原蛋白均显示出荧光标记肝素的明显结合。同样，来自两种含有整合素结合位点的构建体的修饰胶原蛋白均显示出已知具有适当整合素受体的L929细胞的显著粘附。缺乏任何适当整合素的C2C12细胞不结合。这些数据表明，通过插入一个或多个具有特定功能的独特生物结构域，细菌胶原样序列可以被修饰成类似于天然细胞外基质胶原蛋白的作用。
