Direct production of functional matrix metalloproteinase-14 without refolding or activation and its application for in vitro inhibition assays: Periplasmic Production of MMP
Direct production of functional matrix metalloproteinase-14 without refolding or activation and its application for in vitro inhibition assays: Periplasmic Production of MMP
复制标题
无需重折叠或激活的功能性基质金属蛋白酶-14的直接生产及其在体外抑制测定中的应用：MMP的周质生产
DOI：
10.1002/bit.25840
复制
发表时间：
2016
影响因子：
3.8
通讯作者：
Ge, Xin
中科院分区：
文献类型：
--
作者：
Nam, Dong Hyun;Ge, Xin
人基质金属蛋白酶 (MMP)-14 是一种膜结合锌内肽酶，是最重要的癌症靶标之一，因为它在肿瘤生长和侵袭中发挥着核心作用。癌症研究和化学或生物 MMP-14 抑制剂的开发需要大量活性 MMP-14。目前通过重折叠和激活生产 MMP-14 的方法是劳动密集型、耗时的，并且通常与回收率低、批次间差异和产品异质有关。在这里，我们报道了在大肠杆菌周质空间中直接产生 MMP-14 催化结构域。 0.5 mg/L 的功能性 MMP-14 的产生无需繁琐的重折叠或有问题的激活过程。通过简单的周质处理制备的 MMP-14 可轻松用于评估化学和抗体抑制剂的效力。此外，MMP-14 和抗体 Fab 片段在周质中的共表达通过避免 MMP-14 或 Fab 的纯化来促进抑制性抗体筛选。我们预计这种 MMP-14 表达策略可以加速开发具有生物学意义的针对 MMP 的治疗药物。生物技术。生物工程。 2016；113：717–723。 © 2015 Wiley 期刊公司。
Human matrix metalloproteinase (MMP)‐14, a membrane‐bound zinc endopeptidase, is one of the most important cancer targets because it plays central roles in tumor growth and invasion. Large amounts of active MMP‐14 are required for cancer research and the development of chemical or biological MMP‐14 inhibitors. Current methods of MMP‐14 production through refolding and activation are labor‐intensive, time‐consuming, and often associated with low recovery rates, lot‐to‐lot variation and heterogeneous products. Here, we report direct production of the catalytic domain of MMP‐14 in the periplasmic space ofEscherichia coli. 0.5 mg/L of functional MMP‐14 was produced without tedious refolding or problematic activation process. MMP‐14 prepared by simple periplasmic treatment can be readily utilized to evaluate the potencies of chemical and antibody‐based inhibitors. Furthermore, co‐expression of both MMP‐14 and antibody Fab fragments in the periplasm facilitated inhibitory antibody screening by avoiding purification of MMP‐14 or Fabs. We expect this MMP‐14 expression strategy can expedite the development of therapeutic drugs targeting MMPs with biological significance. Biotechnol. Bioeng. 2016;113: 717–723. © 2015 Wiley Periodicals, Inc.