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

Download Paper

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 April 24, 2024)