One-step purification and kinetic properties of the recombinant L-asparaginase from Erwinia carotovora.
ECAR-LANS, the recombinant L-asparaginase from Erwinia carotovora, is a prospective therapeutic enzyme for leukaemia treatment. An efficient and economical scheme was developed for the purification, cloning and expression in Eschericha coli of ECAR-LANS. More than 90% purity, complemented with 72% active enzyme recovery, was achieved with a single chromatographic purification step. The activity of purified L-asparaginase was 630 i.u./mg. The ECAR-LANS K (m) value was 98x10(-6) M for the main physiological substrate L-Asn and 3400x10(-6) M for L-Gln. ECAR-LANS was found to have low relative glutaminase activity (1.2%) at physiological concentrations of L-Asn and L-Gln in blood. Kinetic studies of ECAR-LANS showed that the recombinant asparaginase combined the main advantages of Erw. chrysanthemi and E. coli L-asparaginases II, currently used in the treatment of acute lymphoblastic leukaemia.
Krasotkina
Julya
J
Laboratory of Medical Biotechnology, Institute for Biomedical Chemistry, Russian Academy of Medical Sciences, 559-B, 10 Pogodinskay St, Moscow 119121, Russia. julya_k@bmh.msk.su
Borisova
Anna A
AA
Gervaziev
Yuri V
YV
Sokolov
Nikolay N
NN
D000595:Amino Acid Sequence; D001215:Asparaginase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D016973:Pectobacterium carotovorum / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D001215:Asparaginase