Cloning, expression and characterization of the recombinant Yersinia pseudotuberculosis L-asparaginase.
We have cloned ansB (YPTB1411) gene from Yersinia pseudotuberculosis Q66CJ2 and constructed stable inducible expression system that overproduce L-asparaginase from Y. pseudotuberculosis (YpA) in Escherichiacoli BL21 (DE3) cells. For purification of YpA we used Q-Sepharose and DEAE-Toyopearl column chromatography. We examined kinetics of the enzyme reaction, catalytic activity as a function of pH, temperature and ionic strength, thermostability and other enzyme properties. Biochemical properties of YpA are similar with those of E. coli type II L-asparaginase. K(m) for L-asparagine is 17 ± 0.9 μM and pI 5.4 ± 0.3. Enzyme demonstrates maximum activity at pH 8.0 and 60 °C. YpA L-glutaminase activity is relatively low and more than 15 times less than specific activity towards L-asn. We evaluated also the antiproliferative effect of YpA in vitro and in vivo with E. colil-asparaginase (EcA) as the reference substance at similar conditions.
Institute of Biomedical Chemistry, RAMS, Moscow, Russia.
M V
MV
Pokrovskaya
S S
SS
Aleksandrova
V S
VS
Pokrovsky
N M
NM
Omeljanjuk
A A
AA
Borisova
N Yu
NY
Anisimova
N N
NN
Sokolov
D000595:Amino Acid Sequence; D000818:Animals; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use*; D001216:Asparagine / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D006801:Humans; D008223:Lymphoma / Q000188:drug therapy* / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D015011:Yersinia pseudotuberculosis / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D001216:Asparagine; D001215:Asparaginase