Refold and Characterization of Recombinant Tissue Factor Pathway Inhibitor Expressed in Escherichia coli

Summary

Human tissue factor pathway inhibitor (TFPI) was expressed in E. coli as a non-glycosylated protein with an additional alanine attached to the aminoterminus of the wild type molecule. High-level expression was obtained with pMON6875, a plasmid containing a tac promoter, Gene 10 leader from bacteriophage T7, methionine-alanine-TFPI coding sequence, and the p22 transcriptional terminator. In this system, TFPI accounted for about 5-10% of the total cell protein. The inclusion bodies containing TFPI were sulfitolyzed, purified by anion-exchange chromatography, refolded through a disulfide interchange reaction, and further fractionated by Mono S cation exchange chromatography. The Mono S resin resolved a peak of highly active TFPI from relatively inactive and possibly misfolded molecules. The E. coli TFPI was shown to be about two-fold more active, on a molar basis, than full- length human SK hepatoma TFPI in a tissue factor-induced clotting assay in human plasma.

• References

• 1 Broze Jr GJ, Miletich JP. Isolation of the tissue factor inhibitor produced by HepG2 hepatoma. Proc Natl Acad Sci USA 1987; 84: 1886-90
• 2 Novotny WF, Girard TJ, Miletich JP, Broze Jr GJ. Purification and characterization of the lipoprotein-associated coagulation inhibitor from human plasma. J Biol Chem 1989; 264: 18832-7
• 3 Wun TC, Huang MD, Kretzmer KK, Palmier MO, Day KC, Bulock JW, Fok KF, Broze Jr GJ. Immunoaffinity purification of lipoprotein-associated coagulation inhibitors from HepG2 hepatoma, Chang liver, and SK hepatoma cells. J Biol Chem 1990; 265: 16096-101
• 4 Wun TC, Kretzmer KK, Girard TJ, Miletich JP, Broze Jr GJ. Cloning and characterization of a cDNA for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandom Kunitz-type inhibitory domains. J Biol Chem 1988; 263: 6001-4
• 5 Girard TJ, Warren LA, Novotny WF, Bejcek BE, Miletich JP, Broze Jr GJ. Identification of the 1.4 Kb and 4.0 Kb messages for the lipoprotein associated coagulation inhibitor and expression of the encoded protein. Thromb Res 1989; 55: 37-50
• 6 Girard TJ, Warren LA, Novotny WF, Likert KM, Brown SG, Miletich JP, Broze Jr GJ. Functional significance of the Kunitz type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 1989; 338: 518-20
• 7 Rapaport SI. Inhibition of factor Vlla/tissue factor-induced blood coagulation: with a particular emphsis upon a factor Xa-dependent inhibitory mechanism. Blood 1989; 73: 359-65
• 8 Broze Jr GJ, Girard TG, Novotny WF. Regulation of coagulation by a multivalent Kunitz-type inhibitor. Biochemistry 1990; 29: 7539-46
• 9 Day KC, Hoffman LC, Palmier MO, Kretzmer KK, Huang MD, Pyla EY, Spokas E, Broze Jr GJ, Warren TG, Wun TC. Recombinant lipoprotein-associated coagulation inhibitor inhibits tissue thromboplastin-induced intravascular coagulation in the rabbit. Blood 1990; 76: 1538-45
• 10 Pedersen AH, Nordfang O, Norris F, Wiberg FC, Christensen PM, Moeller KB, Meidahl-Pedersen J, Beck TC, Norris K, Hedner U, Kisiel W. Recombinant human extrinsic pathway inhibitor: production, isolation, and characterization of its inhibitory activity on tissue factor-initiated coagulations. J Biol Chem 1990; 265: 16786-93
• 11 Wun TC, Kretzmer KK, Paltmei MO, Day KC, Huaug MD, Welsch DJ, Lewis C, Wolfe RA, Zobel IF, Lange GW, Frazier RB, Bild GS, Peel MA, Shell RE, Horn NA, Junger KD, Foy BA, Gustafson ME, Leimgruber RM, Novotny WF, Broze Jr GJ, Pyla YE, Hippenmeyer PT, Warren TG. Comparison of recombinant tissue factor pathway inhibitors expressed in human SK hepatoma, mouse Cl27, baby hamster kidney, and Chinese hamster ovary cells. Thromb Haemostas 1992; 68: 54-9
• 12 Haskel EJ, Torr SR, Day KC, Palmier MO, Wun TC, Sobcl BE, Abcnd-sehein DR. Prevention of arterial reocclusion after thrombolysis with recombinant lipoprotein-associated coagulation inhibitor. Circulation 1991; 84: 821-7
• 13 Creasey AA, Chang ACK, Feigen L, Wun TC, Taylor Jr FB, Hinshaw LB. Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock. J Clin Invest 1993; 91: 2850-60
• 14 Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 1985; 32: 488-92
• 15 Olins PO, Rangwala SH. A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J Biol Chem 1989; 264: 16973-6
• 16 Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor laboratory; Cold Spring Harbor, N. Y.: 1982
• 17 Obukowicz MG, Gustafson ME, Junger KD, Leimgruber RM, Wittwer AJ, Wun TC, Warren TG, Bishop BF, Mathis KJ, McPherson DT, Siegel NR, Jennings MG, Brightwell BB, Diaz-Colier J, Bell LD, Craik CS, Tacon WC. Secretion of active Kringle-2-serine protease in Escherichia coli. . Biochemistry 1990; 29: 9737-45
• 18 Smith RL. Titration of activated bovine factor X. J Biol Chem 1973; 248: 2418-23
• 19 Covey TR, Bonner RF, Shushan BI, Henion JD. The determination of protein, oligonucleotide, and peptide molecular weights by ion spray mass spectrometry. Rapid Commun. Mass Spectrom. 1988; 2: 249-56
• 20 Bailey JL, Cole RD. Studies on the reaction of sulfite with proteins. J Biol Chem 1959; 231: 1733-9
• 21 Chan WC. A method for the complete S sulfonation of cysteine residues in proteins. Biochemistry 1968; 7: 4247-53
• 22 Furman TC, Epp J, Hsiung HM, Hoskins J, Long GL, Mendelsohn LG, Schoner B, Smith DP, Smith MC. Recombinant human insulin-like growth factor II expressed in Escherichia coli Rintechnnlngy. 1987; 5: 1017-51
• 23 Creighton TE, Goldenberg DP. Kinetic role of a meta-stable native-like two-disulphide species in the folding transition of bovine pancreatic trypsin inhibitor. J Mol Biol 1984; 179: 497-526