Optimizing a production strategy for a nonspecific nuclease from Yersinia enterocolitica subsp. palearctica in genetically engineered Escherichia coli

ABSTRACT A nuclease from Yersinia enterocolitica subsp. palearctica (Nucyep) is a newly found thermostable nonspecific nuclease. The heat-resisting ability of this nuclease would be extremely useful in biological research or pharmaceutical production. However, the application of this nuclease is limited because of its poor yield. This research aimed to improve Nucyep productivity by producing a novel genetically engineered Escherichia coli and optimizing the production procedures. After 4 h of induction by lactose, the new genetically engineered E. coli can express a substantial amount of Nucyep in the form of inclusion bodies. The yield was approximately 0.3 g of inclusion bodies in 1 g of bacterial pellets. The inclusion bodies were extracted by sonication and solubilized in an 8 M urea buffer. Protein renaturation was successfully achieved by dilution method. Pure enzyme was obtained after subjecting the protein solution to anion exchange. The Nucyep showed its nonspecific and heat resistant properties as previously reported (Boissinot et al. 2016). Through a quantification method, its activity was determined to be 1.3 × 10 6 Kunitz units (K.U.)/mg. These results can serve as a reference for increasing Nucyep production.

Optimization of the fusion protein rhIL7-BAPmut renaturation process from the Escherichia coli inclusion bodies and its practical application

Aim. The aim of our work was to optimize the renaturation method of the rhIL7-BAPmut fusion protein based on recombinant human interleukin-7 (rhIL7) and bacterial alkaline phosphatase with enhanced catalytic properties (BAPmut) for its obtaining in functionally active form. Methods. The cells of E. coli strain BL21(DE3) were transformed with pET24-IL7-BAPmut plasmid vector. Protein synthesis was induced by autoinduction protocol. Immobilized-metal affinity chromatography (IMAС) and slow dilution methods were applied for rhIL7-BAPmut fusion protein renaturation from bacterial inclusion bodies in vitro. Results. Combination of IMAС method and slow dilution at the presence of arginine, GSH/ GSSG and Mg2+ ions provided obtaining of rhIL7-BAPmut in pure and active form. Bifunctional activity of rhIL7-BAPmut after refolding is confirmed immunochemically by binding with specific antibodies. Conclusions. It was shown that application of rhIL7-BAPmut allows to reduce the time of the screening of immune combinatory libraries of variable genes of IgG and does not require specific primary and secondary antibodies. The rhIL7-BAPmut fusion protein also can be used for qualitative and quantitative analysis of IL-7 receptors.Keywords: IL-7, BAPmut, inclusion bodies, renaturation.