Chowdhury Anandã, Brinson Robert, Wei Beiyang, Stetler-Stevenson William G
Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute , Bethesda, Maryland 20892, United States.
Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology and University of Maryland , 9600 Gudelsky Drive, Rockville, Maryland 20850, United States.
Biochemistry. 2017 Dec 12;56(49):6423-6433. doi: 10.1021/acs.biochem.7b00700. Epub 2017 Nov 28.
Tissue inhibitor of metalloprotease-2 (TIMP-2) is a secreted 21 kDa multifunctional protein first described as an endogenous inhibitor of matrix metalloproteinases (MMPs) that prevents breakdown of the extracellular matrix often observed in chronic diseases. TIMP-2 diminishes the level of growth factor-mediated cell proliferation in vitro, as well as neoangiogenesis and tumor growth in vivo independent of its MMP inhibitory activity. These physiological properties make TIMP-2 an excellent candidate for further preclinical development as a biologic therapy of cancer. Here we present a straightforward bioprocessing methodology that yields >35 mg/L recombinant human TIMP-2 6XHis-tagged protein (rhTIMP-2) from suspension cultures of HEK-293-F cells. Enhanced rhTIMP-2-6XHis yields were achieved by optimization of both TIMP-2 cDNA codon sequence and cell culture conditions. Using a two-step chromatographic process, we achieved >95% purity with minimal processing losses. Purified rhTIMP-2-6XHis was free of mouse antigen contamination. Circular dichroism spectroscopy indicated a well-folded rhTIMP-2-6XHis that is highly stable and refractory to pH changes. Two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance of full length rhTIMP-2-6XHis also indicated a monodisperse, well-folded protein preparation. Purified rhTIMP-2-6XHis inhibited MMP-2 enzymatic activity in a dose-dependent fashion with an IC of ∼1.4 nM. Pretreatment of A549 lung cancer and JygMC(A) triple-negative breast cancer cells with rhTIMP-2-6XHis in low-nanomolar amounts inhibited EGF-induced proliferation to basal (unstimulated) levels. This study therefore not only offers a robust bioprocess methodology for rhTIMP-2 production but also characterizes critical physicochemical and biological attributes that are useful for monitoring quality control of the production process.
金属蛋白酶组织抑制剂-2（TIMP-2）是一种分泌型的21 kDa多功能蛋白，最初被描述为基质金属蛋白酶（MMPs）的内源性抑制剂，可防止在慢性疾病中经常观察到的细胞外基质分解。TIMP-2在体外可降低生长因子介导的细胞增殖水平，在体内可抑制新生血管形成和肿瘤生长，且与其MMP抑制活性无关。这些生理特性使TIMP-2成为作为癌症生物治疗进一步临床前开发的优秀候选物。在此，我们提出了一种直接的生物加工方法，可从HEK-293-F细胞的悬浮培养物中产生>35 mg/L的重组人TIMP-2 6XHis标签蛋白（rhTIMP-2）。通过优化TIMP-2 cDNA密码子序列和细胞培养条件，提高了rhTIMP-2-6XHis的产量。使用两步色谱法，我们以最小的加工损失实现了>95%的纯度。纯化的rhTIMP-2-6XHis不含小鼠抗原污染。圆二色光谱表明rhTIMP-2-6XHis折叠良好，高度稳定且对pH变化具有抗性。全长rhTIMP-2-6XHis的二维异核单量子相干核磁共振也表明是一种单分散、折叠良好的蛋白质制剂。纯化的rhTIMP-2-6XHis以剂量依赖性方式抑制MMP-2酶活性，IC约为1.4 nM。用低纳摩尔量的rhTIMP-2-6XHis预处理A549肺癌细胞和JygMC(A)三阴性乳腺癌细胞可将表皮生长因子（EGF）诱导的增殖抑制至基础（未刺激）水平。因此，本研究不仅为rhTIMP-2的生产提供了一种强大的生物加工方法，还表征了对监测生产过程质量控制有用的关键物理化学和生物学特性。
