Prokaryotic Expression, Purification, and Reconstitution of Biological Activities (Antiprotease, Antitumor and Heparin-Binding) for Tissue Factor Pathway Inhibitor- 2

Published

October 5, 2000

Author(s)

C N. Rao, Prasad T. Reddy, D J. Reeder, Yonglin Liu, S M. Stack, W Kisiel, D T. Woodley

Abstract

We report the expression of tissue factor pathway inhibitor-2 (TFPI-2) (also known as PP-5, placental protein-5; MSPI, matrix-associated serine protease inhibitor) in E. coli as a 25 kDa non-glycosylated protein with a glycine substituted for aspartic acid at the amino terminus. High level expression of TFPI-2 was obtained with pRE1 expression vector under the transcriptional and translational controls of the lPL promoter and lcII ribosome-binding site, respectively, with ATG initiation codon. TFPI-2 was produced as inclusion bodies and accounted for 25 % to 30 % of the total E. coli proteins. The inclusion bodies containing TFPI-2 protein were solubilized with urea, sulfitolyzed, purified, and refolded through a disulfide interchange reaction. The refolded E. coli TFPI-2 inhibited plasmin with an inhibition constant (Ki) of 5 nM that is similar with the TFPI-2 expressed in a mammalian system using baby hamster kidney cells. Similar to TFPI-2 from the mammalian system, the refolded E.coli TFPI-2 bound heparin and also inhibited plasmin, regardless of whether the enzyme was in the fluid-phase or was bound to the membranes of HT-1080 fibrosarcoma cells. In addition, similar to TFPI-2 from the mammalian system, refolded E.coli TFPI-2 inhibited radiolabeled matrix degradation and Matrigel matrix invasion by HT-1080 fibrosarcoma cells and B16-F10 melanoma cells. Together, our results suggest that glycosylation is not essential for antiprotease, antitumor and matrix-binding activities of TFPI-2. Based on these collective data, we conclude that a biologically active non-glycosylated TFPI-2 can be produced in E.coli and that the protein can be produced in high-enough quantities to conduct in vivo studies for determination of the role of this inhibitor in tumor invasion and metastasis.

Citation

Biochemical and Biophysical Research Communications

Volume

276

Issue

3

Pub Type

Journals

Keywords

antiprotease activity, antitumor activity, glycosylation, matrix-binding activity, prokaryotic expression, protein refolding, serine protease inhibitors, TFPI-2

Citation

Rao, C. , Reddy, P. , Reeder, D. , Liu, Y. , Stack, S. , Kisiel, W. and Woodley, D. (2000), Prokaryotic Expression, Purification, and Reconstitution of Biological Activities (Antiprotease, Antitumor and Heparin-Binding) for Tissue Factor Pathway Inhibitor- 2, Biochemical and Biophysical Research Communications (Accessed April 19, 2024)