Expression, purification and characterization of Solanum tuberosum recombinant cytosolic pyruvate kinase.

摘要: Abstract The cDNA encoding for a Solanum tuberosum cytosolic pyruvate kinase 1 (PKc1) highly expressed in tuber tissue was cloned the bacterial expression vector pProEX HTc. construct carried hexahistidine tag N-terminal position to facilitate purification of recombinant protein. Production high levels soluble PKc1 Escherichia coli only possible when using co-expression strategy with chaperones GroES-GroEL. Purification protein by Ni2 + chelation chromatography yielded single an apparent molecular mass 58 kDa and specific activity 34 units mg−1 enzyme had optimum pH between 6 7. It relatively heat stable as it retained 80% its after 2 min at 75 °C. Hyperbolic saturation kinetics were observed ADP UDP whereas sigmoidal during analysis phosphoenolpyruvate binding. Among effectors tested, aspartate glutamate no effect on activity, α-ketoglutarate citrate most potent inhibitors. When tested kinetics, these latter compounds increased S0.5. These findings suggest that S. is subject strong control respiratory metabolism exerted via other tricarboxylic acid cycle intermediates.