Prokaryotic expression, purification, and reconstitution of biological activities (antiprotease, antitumor, and heparin-binding) for tissue factor pathway inhibitor-2

We report the expression of tissue factor pathway inhibitor-2 (TFPI-2) (als o known as PP-5, placental protein-5; MSPI, matrix-associated serine protea se inhibitor) in E. coli as a 25-kDa nonglycosylated 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 transcription al and translational controls of the lambda P-L promoter and lambda c(II) r ibosome-binding site, respectively, with ATG initiation codon. TFPI-2 was p roduced as inclusion bodies and accounted for 25-30% of the total E. coli p roteins. The inclusion bodies containing TFPI-2 were solubilized with urea, sufitolyzed, purified, and refolded through a disulfide interchange reacti on. The refolded E. coli TFPI-2 inhibited plasmin with an inhibition consta nt (K-i) of 5 nM that is similar with the TFPI-2 expressed in a mammalian s ystem. 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 t he membranes of HT-1080 fibrosarcoma cells. In addition, refolded E. coli T FPI-2 inhibited radiolabeled matrix degradation and Matrigel matrix invasio n by HT-1080 fibrosarcoma cells and B16-F10 melanoma cells. Together, our r esults suggest that glycosylation is not essential for antiprotease, antitu mor, and matrix-binding activities of TFPI-2, Based on these collective dat a, we conclude that a biologically active nonglycosylated TFPI-2 can be pro duced in E. coli and that the protein can be produced in high-enough quanti ties to conduct in vivo studies for determination of the role of this inhib itor in tumor invasion and metastasis. (C) 2000 Academic Press.