Expression, refolding, and activation of the catalytic domain of human blood coagulation factor XII.
Human blood coagulation factor XII (FXII; 80 kDa) contains a C-terminal serine protease zymogen domain, which becomes activated upon contacting a negative surface. Activated FXII (alphaFXIIa) brings about reciprocal activation of FXII and kallikrein that by further hydrolysis produces the free catalytic domain (betaFXIIa; 28 kDa). Increased levels of alphaFXIIa are associated with coronary heart disease, sepsis, and diabetes. Biophysical investigation of the structural basis of activation, substrate specificity, and regulation of FXII requires an efficient bacterial system for producing the wild-type and mutant recombinant proteins. Here, the cDNA of the zymogen domain of FXII (betaFXII) was cloned into the pET-28a(+) vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) and overexpressed. The multi-disulfide, recombinant protein, His(6)-betaFXII (rbetaFXII), expressed as an inclusion body, was purified by means of a Ni(2+)-charged resin. The matrix-bound rbetaFXII was subjected to refolding with the glutathione redox system and activated by the in vivo activator, kallikrein. The active form, rbetaFXIIa, obtained in milligram quantities, exhibited similar structural and comparable functional properties relative to human betaFXIIa, as indicated by circular dichroism spectroscopy and kinetics of substrate hydrolysis. Thermodynamics of enzyme:inhibitor complex formation, including the expected 1:1 stoichiometry, was determined for rbetaFXIIa by isothermal calorimetric titration with a specific recombinant protein inhibitor, Cucurbita maxima trypsin inhibitor-V (rCMTI-V; 7kDa).
Shan
Jixiu
J
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Baguinon
Marilyn
M
Zheng
Li
L
Krishnamoorthi
Ramaswamy
R
D001777:Blood Coagulation; D002151:Calorimetry; D020134:Catalytic Domain; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005174:Factor XII / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D005174:Factor XII