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) (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.
Rao
C N
CN
Department of Dermatology, Department of Obstetrics and Gynecology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois, 60611, USA.
Reddy
P
P
Reeder
D J
DJ
Liu
Y
Y
Stack
S M
SM
Kisiel
W
W
Woodley
D T
DT
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D002465:Cell Movement / Q000187:drug effects; D018387:Codon, Initiator / Q000235:genetics; D003094:Collagen / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004338:Drug Combinations; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D005341:Fibrinolysin / Q000037:antagonists & inhibitors / Q000378:metabolism; D005354:Fibrosarcoma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism*; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007797:Laminin / Q000378:metabolism; D008545:Melanoma / Q000201:enzymology / Q000473:pathology; D051379:Mice; D009154:Mutation / Q000235:genetics; D009361:Neoplasm Invasiveness; D011257:Pregnancy Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D020673:Protein Renaturation; D011509:Proteoglycans / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D012995:Solubility; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D018387:Codon, Initiator; D004220:Disulfides; D004338:Drug Combinations; D006023:Glycoproteins; D007797:Laminin; D011257:Pregnancy Proteins; D011509:Proteoglycans; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C021240:tissue-factor-pathway inhibitor 2; C059485:matrigel; D006493:Heparin; D003094:Collagen; D005341:Fibrinolysin