Yang Mingying, Tanaka Chikako, Yamauchi Kazuo, Ohgo Kosuke, Kurokawa Masato, Asakura Tetsuo
Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
J Biomed Mater Res A. 2008 Feb;84(2):353-63. doi: 10.1002/jbm.a.31348.
Two silklike proteins, [TGRGDSPAGG(GAGAGS)3AS]5 (FS5) and [TGRGDSPA-(GVPGV)2GG(GAGAGS)3AS]8 (FES8) were designed to demonstrate the superior performance as biomaterials of silklike proteins. The former protein consists of the crystalline domain sequence, (GAGAGS)n from Bombyx mori silk fibroin and cell-adhesive sequence TGRGDSPA coming from fibronectin-containing RGD triplet. The additional sequence (GVPGV)n from elastin was included in the latter protein. The considerably higher cell-adhesion activities of these proteins for NHDF and VERO cells were observed by comparing with those of silklike materials without RGD sequences and also the crystalline fraction of B. mori silk fibroin. This tendency was independent of the treatments, 4.5M LiClO4 or formic acid (FA), on silklike proteins. Their activities are also higher than those of commercial Fibronectin F for NHDF cell. Their structural characterization was studied using 13C solid-state NMR. Although the overlapped peaks in usual 13C CP/MAS NMR spectra make the detailed structural analysis difficult, the methyl resonance regions observed using dipolar dephasing NMR were very useful for the analysis. The presence of both random coil and beta-sheet structures was observed in these proteins clearly. The content of beta-sheet structure in both proteins increases after FA treatment when compared with the lyophilized samples. The production of electrospun nanofibers from their hexafluoroacetone solution was also tried. The silklike protein FES8 could prepare nonwoven silk fibers although FS5 could not.
设计了两种丝样蛋白，即[TGRGDSPAGG(GAGAGS)3AS]5（FS5）和[TGRGDSPA-(GVPGV)2GG(GAGAGS)3AS]8（FES8），以证明丝样蛋白作为生物材料的卓越性能。前一种蛋白由家蚕丝素蛋白的结晶结构域序列(GAGAGS)n和来自含纤连蛋白的RGD三联体的细胞粘附序列TGRGDSPA组成。后一种蛋白中包含了来自弹性蛋白的额外序列(GVPGV)n。通过与不含RGD序列的丝样材料以及家蚕丝素蛋白的结晶部分进行比较，观察到这些蛋白对人正常皮肤成纤维细胞（NHDF）和非洲绿猴肾细胞（VERO）具有显著更高的细胞粘附活性。这种趋势与丝样蛋白的处理方式无关，处理方式为4.5M高氯酸锂（LiClO4）或甲酸（FA）。它们对NHDF细胞的活性也高于商业纤连蛋白F。使用13C固态核磁共振（NMR）对其结构特征进行了研究。尽管常规13C交叉极化/魔角旋转（CP/MAS）NMR谱中的重叠峰使详细的结构分析变得困难，但使用偶极去相NMR观察到的甲基共振区域对分析非常有用。在这些蛋白中清楚地观察到了无规卷曲和β-折叠结构的存在。与冻干样品相比，FA处理后两种蛋白中β-折叠结构的含量均增加。还尝试了从它们的六氟丙酮溶液中制备电纺纳米纤维。丝样蛋白FES8可以制备无纺丝纤维，而FS5则不能。
