Tissue Inhibitor of Metalloprotease-2: bioprocess development, physicochemical, biochemical and biological characterization of highly expressed recombinant protein

Published

December 1, 2017

Author(s)

Ananda Chowdhury, Robert G. Brinson, Beiyang Wei, William G. Stetler-Stevenson

Abstract

Tissue Inhibitor of Metalloprotease -2 (TIMP-2) is a secreted, 21 kDa, multifunctional protein. It was first described as an endogenous inhibitor of matrix metalloproteases (MMPs) that prevents protease-mediated breakdown of the extracellular matrix often observed in chronic diseases. TIMP-2, independent of its MMP inhibitory activity, also diminished growth factor- mediated cell proliferation in vitro, as well as neoangiogenesis and tumor growth in vivo. TIMP-2 is an excellent candidate for further pre-clinical development as a biologic therapy for cancer. Here we present a straightforward bioprocessing methodology that yields >35 mg/L TIMP- 2-6XHis-tagged protein from suspension HEK 293F cells. These enhanced TIMP-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 TIMP-2-6XHis was free of mouse antigen and endotoxin contamination. Using Circular Dichroism and 2D nuclear magnetic resonance spectroscopies, TIMP-2-6XHis was determined to be monodisperse, properly folded, and refractory to pH changes, all important critical quality attributes for therapeutic development. Purified TIMP-2-6XHis inhibited MMP-2 enzymatic activity in a dose dependent fashion with an IC50 of 1.4 nmol / L. TIMP-2-6XHis pre-treatment of A549 lung cancer and JygM(A) triple-negative breast cancer cells in low nanomolar amounts inhibited the EGF-induced proliferation of these cells to basal (unstimulated) levels. The present study therefore not only offers a robust bioprocess methodology for TIMP-2 production but also characterizes critical physicochemical and biological attributes useful for monitoring quality control of the production process

Citation

Biochemistry

Volume

56

Issue

49

Pub Type

Journals

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DOI Link

Keywords

TIMP-2, ECM, MMP, MMP-2, anti-proliferative, bio-therapeutic

Citation

Chowdhury, A. , Brinson, R. , Wei, B. and Stetler-Stevenson, W. (2017), Tissue Inhibitor of Metalloprotease-2: bioprocess development, physicochemical, biochemical and biological characterization of highly expressed recombinant protein, Biochemistry, [online], https://doi.org/10.1021/acs.biochem.7b00700 (Accessed October 10, 2025)