Prospects and limitations of the rational engineering of fibrillar collagens
Prospects and limitations of the rational engineering of fibrillar collagens
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DOI：
10.1110/ps.0385103
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发表时间：
2003-09-01
期刊：
影响因子：
8
通讯作者：
Fertala, A
中科院分区：
文献类型：
--
作者：
Majsterek, I;McAdams, E;Fertala, A
重组胶原蛋白对于许多生物医学应用来说是有吸引力的蛋白质。迄今为止，非修饰重组胶原蛋白的规模化生产取得了重大进展；然而，根据定制规格设计新型胶原蛋白样蛋白的问题尚未得到解决。在此，我们研究了合理工程具有特定特征的类胶原蛋白的可能性。我们通过基因工程改造了两种编码多 D4 胶原蛋白的 DNA 结构，这些胶原蛋白被定义为类胶原蛋白，主要由对应于生物活性区域的胶原 Pi D4 周期串联组成。我们还尝试通过突变 D4 期中存在的基质金属蛋白酶 I 裂解位点来减少新型胶原蛋白的酶促降解。我们证明，重组胶原α链主要由D4期组成，但缺乏天然胶原中发现的大多数其他D期，折叠成典型的胶原三螺旋，并且新型前胶原被前胶原N-蛋白酶和前胶原C-蛋白酶正确加工。未突变的多-D4胶原蛋白具有41℃的正常熔点和与对照相似的碳水化合物含量。相比之下，突变型多D4胶原蛋白的36℃热稳定性明显较低，碳水化合物含量明显较高。两种胶原蛋白均被基质金属蛋白酶 1 在多个位点裂解，但突变型多 D4 胶原蛋白的水解率较低。这些结果为胶原蛋白的合理工程和识别改变胶原氨基酸序列的任何不良后果提供了基础。
Recombinant collagens are attractive proteins for a number of biomedical applications. To date, significant progress was made in the large-scale production of nonmodified recombinant collagens; however, engineering, of novel collagen-like proteins according to customized specifications has not been addressed. Herein we investigated the possibility of rational engineering of collagen-like proteins with specifically assigned characteristics. We have genetically engineered two DNA constructs encoding multi-D4 collagens defined as collagen-like proteins, consisting primarily of a tandem of the collagen Pi D4 periods that correspond to the biologically active region. We have also attempted to decrease enzymatic degradation of novel collagen by mutating a matrix metalloproteinase I cleavage site present in the D4 period. We demonstrated that the recombinant collagen alpha-chains consisting predominantly of the D4 period but lacking most of the other D periods found in native collagen fold into a typical collagen triple helix, and the novel procollagens are correctly processed by procollagen N-proteinase and procollagen C-proteinase. The non-mutated multi-D4 collagen had a normal melting point of 41degreesC and a similar carbohydrate content as that of control. In contrast, the mutant multi-D4 collagen had a markedly lower thermostability of 36degreesC and a significantly higher carbohydrate content. Both collagens were cleaved at multiple sites by matrix metalloproteinase 1, but the rate of hydrolysis of the mutant multi-D4 collagen was lower. These results provide a basis for the rational engineering of collagenous proteins and identifying any undesirable consequences of altering the collagenous amino acid sequences.