Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin.
Bovine prethrombin-2 has been produced in Escherichia coli using a T7 expression system. The expressed prethrombin-2 formed intracellular inclusion bodies which were solubilized by reversible sulfonation of the cysteines in the presence of 7 M guanidine hydrochloride. Sulfonated prethrombin-2 was refolded in the presence of 4 M guanidine hydrochloride, using oxidized and reduced glutathione as the redox couple. The folded protein was purified by heparin affinity chromatography and activated to thrombin with Echis carinatus snake venom. The resulting thrombin was also purified by heparin affinity chromatography. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline by recombinant thrombin (kcat = 123 +/- 10 s-1 and Km = 2.91 +/- 0.3 microM). These values are in good agreement with those determined for wild-type thrombin (kcat = 97 +/- 8 s-1 and Km = 2.71 +- 0.25 microM). From the thrombin-mediated release of fibrinopeptide A from fibrinogen, kcat/Km was found to be the same for recombinant (17.3 +/- 1.2 microM-1 s-1) and wild-type (16.7 +/- 2.0 microM-1 s-1) thrombin. These results, taken together with circular dichroism spectra and the elution position of prethrombin-2 from a heparin affinity resin, indicate that prethrombin-2 was folded into a conformation similar to that of the wild-type protein. In addition, since E. coli produces deglycosylated enzymes, these findings suggest that the carbohydrate on the B chain of wild-type thrombin does not affect the amidolytic and fibrinolytic activities of thrombin. Finally, this expression system can be used to prepare mutants of prethrombin-2 for future structure-function studies involving thrombin and its substrates; some preliminary results of this type are presented here.
DiBella
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Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301.
Maurer
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Scheraga
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D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D017510:Protein Folding; D011516:Prothrombin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D014757:Viper Venoms / Q000494:pharmacology
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D014757:Viper Venoms; D011516:Prothrombin; C012007:prethrombins; D013917:Thrombin