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1Department of Dermatology, Department of Obstetrics and Gynecology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois, 60611, USA.
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 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 transcriptional and translational controls of the lambdaP(L) promoter and lambdacII ribosome-binding site, respectively, with ATG initiation codon. TFPI-2 was produced as inclusion bodies and accounted for 25-30% of the total E. coli proteins. The inclusion bodies containing TFPI-2 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 (K(i)) of 5 nM that is similar with the TFPI-2 expressed in a 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, 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 nonglycosylated 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.
Insights
We successfully produced biologically active, nonglycosylated tissue factor pathway inhibitor-2 (TFPI-2) in E. coli. This recombinant TFPI-2 inhibits plasmin and matrix invasion, suggesting glycosylation is not essential for its function.
Area of Science:
- Biochemistry
- Molecular Biology
- Recombinant Protein Expression
Background:
- Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a serine protease inhibitor involved in regulating coagulation and matrix degradation.
- Understanding the functional domains and production methods of TFPI-2 is crucial for its therapeutic applications.
Purpose of the Study:
- To express and characterize recombinant, nonglycosylated TFPI-2 in Escherichia coli (E. coli).
- To assess the biological activity, including antiprotease and anti-invasion properties, of E. coli-expressed TFPI-2.
Main Methods:
- High-level expression of TFPI-2 in E. coli using the pRE1 vector and lambdaP(L) promoter.
- Solubilization, purification, and refolding of TFPI-2 inclusion bodies.
- Enzyme inhibition assays using plasmin.
- Cell-based assays to evaluate matrix degradation and invasion by fibrosarcoma and melanoma cells.
Main Results:
- Recombinant TFPI-2 was produced as a 25-kDa nonglycosylated protein in E. coli inclusion bodies.
- Refolded E. coli TFPI-2 exhibited potent plasmin inhibition (K(i) = 5 nM), comparable to mammalian-expressed TFPI-2.
- E. coli-derived TFPI-2 inhibited matrix degradation and cell invasion in vitro, demonstrating functional activity.
Conclusions:
- Glycosylation is not essential for the antiprotease, antitumor, and matrix-binding activities of TFPI-2.
- Biologically active, nonglycosylated TFPI-2 can be produced in high yields in E. coli.
- This recombinant TFPI-2 is suitable for in vivo studies to investigate its role in tumor invasion and metastasis.
