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 Escherichia coli 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. Km 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. coli l-asparaginase (EcA) as the reference substance at similar conditions.

Recombinant E. coli strain producing Y. pseudotuberculosis Q66CJ2 (YpA) L-asparaginase II was created. Gene ansB homologue encoding Y. pseudotuberculosis IP 32953 L-asparaginase precursor was synthesized. The gene was cloned in pBad24 expression vector and expressed in E. coli BL21 (DE3) strain. Optimal conditions for the producer strain culturing were selected. An effective method for isolation and purification of the enzyme by two-staged column chromatography was developed.

The cytotoxic activity of L-asparaginases from Yersinia pseudotuberculosis and from Erwinia carotovora were investigated in vitro using human T-lymphoblastic leukemia (Jurkat and Molt-4) and also solid tumor cell lines MCF-7 (human breast adenocarcinoma), LnCap (human prostate carcinoma), NGUK1 (rat Gasser node neurinoma). E.coli L-asparaginase produced by Medak (Germany) was used as a reference preparation. The data obtained indicate that Y. pseudotuberculosis L-asparaginase significantly inhibits growth of leukemic and solid tumor cells. Its antitumor activity is comparable to that of the reference preparation of L-asparaginase (Medak). These results suggest that the recombinant L-asparaginase can be used for the development of new preparations for the therapy of different types of tumors.

Human cytochrome P450 2B6 gene from plasmid pUC9, carrying cytochrome CYP2B6 cDNA was cloned into eucariotic expression vector pcDNA 3.1 (+). Cytochrome P450 2136 gene in recombinant plasmid pcDNA 3.1 (+) /CYP 2B6 was expressed in E.coli cells. The expression of catalytically active recombinant protein was 60-100 nm per liter of the culture medium.