Tissue inhibitor of metalloproteinase-2 (TIMP-2) from red seabream (Pagrus major): Molecular cloning and biochemical characterization of highly expressed recombinant protein
Tissue inhibitor of metalloproteinase-2 (TIMP-2) from red seabream (Pagrus major): Molecular cloning and biochemical characterization of highly expressed recombinant protein
复制标题
红鲷（真鲷）金属蛋白酶 2 (TIMP-2) 组织抑制剂：高表达重组蛋白的分子克隆和生化表征
DOI：
10.1016/j.fsi.2019.11.006
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发表时间：
2019-12-01
影响因子：
4.7
通讯作者：
Wu，Guo Ping
中科院分区：
文献类型：
--
作者：
Zhong，Chan;Cao，Min Jie;Wu，Guo Ping
金属蛋白酶组织抑制剂-2（TIMP-2）最初是一种内源性的基质金属蛋白酶抑制剂，用于应答免疫刺激引起的胶原溶解。本研究克隆了真鲷（Pagrus major）肌肉TIMP-2a基因的cDNA。该基因全长585 bp，编码194个氨基酸，包含所有已知的功能结构域，与其他硬骨鱼类TIMP-2a同源性较高，属于TIMP-2a家族。利用新构建的表达载体pPIC 9 K-rTIMP-2a高效表达了可溶性rTIMP-2a，并对MMP-2和MMP-9具有高抑制活性。重组TIMP-2a的分子量为23 kDa，等电点为6.50。此外，由12个保守的半胱氨酸残基形成的6个二硫键被鉴定为结构稳定的功能基序。此外，rTIMP-2a对内源性MMPs诱导的肌肉I型胶原蛋白的水解和降解具有较强的抑制作用。研究结果揭示了rTIMP-2a的特性和抑制功能，推测其可能在防御机制中调节明胶降解MMPs的代谢。
The tissue inhibitor of metalloproteinase-2 (TIMP-2) is originally characterized as an endogenous inhibitor of matrix metalloproteinases (MMPs) to response collagenolysis associated with immune challenge. In this study, the cDNA encoding TIMP-2a gene from red seabream (Pagrus major) muscle was cloned. It was 585 bp encoding a putative protein of 194 amino acids, which comprised all recognized functional domains and showed the high identity to TIMP-2as from other teleost fishes, revealing it belongs to TIMP-2a family. Soluble rTIMP-2a was efficiently expressed using a new constructed pPIC9K-rTIMP-2a vector with high inhibitory activity against to MMP-2 and MMP-9. The recombinant TIMP-2a tagged with 6 histidine residues showed the molecular mass of 23 kDa and isoelectric point of 6.50. Furthermore, the 6 disulfide bonds formed by 12 conserved cysteine residues were identified as functional motifs for its structural stability. In addition, rTIMP-2a possessed the high inhibitory activity against gelatinolytic hydrolysis and degradation of type I collagen which induced by endogenous MMPs in muscle. The results revealed the properties and inhibitory function of rTIMP-2a, which may be a pivotal role in regulation gelatinolytic MMPs metabolization during defense mechanism.