An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived alpha-thrombin.
Human recombinant prethrombin-2 was produced in Escherichia coli. The expressed prethrombin-2 formed intracellular inclusion bodies from which the protein was refolded by a simple one-step dilution process in buffer consisting of 50 mM Tris-HCl, containing 20 mM CaCl(2), 500 mM NaCl, 1 mM EDTA, 600 mM arginine, 1 mM cysteine, 0.1 mM cystine, 10% (v/v) glycerol, and 0.2% (w/v) Brij-58 at pH 8.5. After refolding, prethrombin-2 was purified by hirudin-based COOH-terminal peptide affinity chromatography, and then activated with Echis carinatus snake venom prothrombin activator (ecarin). The activated protein, alpha-thrombin, was then tested for several activities including activity toward chromogenic substrate, release of fibrinopeptide A from fibrinogen, activation of protein C, and thrombin-activatable fibrinolysis inhibitor, reactivity with antithrombin, clotting activity, and platelet aggregation. The kinetic data showed no differences in activity between our recombinant alpha-thrombin and plasma-derived alpha-thrombin. The yield of refolded recombinant human prethrombin-2 was about 4-7% of the starting amount of solubilized protein. In addition, the final yield of purified refolded protein was 0.5-1%, and about 1 mg of recombinant prethrombin-2 could be isolated from 1 liter of E. coli cell culture.
Soejima
K
K
First Research Department, The Chemo-Sero-Therapeutic Research Institute, Kawabe, Kyokushi, Kikuchi, Kumamoto 869-1298, Japan. soejima@kaketsuken.or.jp
Mimura
N
N
Yonemura
H
H
Nakatake
H
H
Imamura
T
T
Nozaki
C
C
D000818:Animals; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D011516:Prothrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D011516:Prothrombin; C012007:prethrombins; D013917:Thrombin