Optimization and One-Step Purification of Recombinant V Antigen Production from Yersinia pestis.

The purpose of this study was to develop an efficient and inexpensive method for the useful production of recombinant protein V antigen, an important virulence factor for Yersinia pestis. To this end, the synthetic gene encoding the V antigen was subcloned into the downstream of the intein (INT) and chitin-binding domain (CBD) from the pTXB1 vector using specific primers. In the following, the produced new plasmid, pTX-V, was transformed into E. coli ER2566 strain, and the expression accuracy was confirmed using electrophoresis and Western blotting. In addition, the effects of medium, inducer, and temperature on the enhancement of protein production were studied using the Taguchi method. Finally, the V antigen was purified by a chitin affinity column using INT and CBD tag. The expression was induced by 0.05 mM IPTG at 25 °C under optimal conditions including TB medium. It was observed that the expression of the V-INT-CBD fusion protein was successfully increased to more than 40% of the total protein. The purity of V antigen was as high as 90%. This result indicates that V antigen can be produced at low cost and subjected to one-step purification using a self-cleaving INT tag.

A new method for simultaneous gene deletion and down-regulation in Brucella melitensis Rev.1.

In this study, our aim was to integrate an antisense expression cassette in bacterial chromosome for providing a long-term expression down-regulation in a bid to develop a new approach for simultaneous deletion and down-regulation of target genes in bacterial system. Therefore, we were used this approach for simultaneous deletion of the perosamine synthetase (per) gene and down-regulation of the virB1 expression in Brucella melitensis Rev.1. The per gene, which is one of the LPS O-chain coding genes, was replaced by homologous recombination with an antisense virB1 expression cassette together with kanamycin resistance cassette (kan(R)). Deletion of the per gene was characterized by PCR analysis and DNA sequencing. The expression of antisense virB1 cassette was confirmed by RT-PCR. Down-regulation of the virB1 mRNA expression was quantified by real-time RT-PCR using virB1 specific primers relative to the groEL reference gene. The survival rate of mutant strain was evaluated by CFU count in the BALB/c mice. The virB1 mRNA expression was down-regulated on average 10-fold in mutant strain as compared to parental strain. The loss of per gene function and decrease of the virB1 mRNA expression resulted in reduced entry and survival of the mutant Rev.1 strain in BALB/c mice splenocytes. We propose that this method can be used for simultaneous regulation of multiple genes expression.

Stabilisation of recombinant aequorin by polyols: activity, thermostability and limited proteolysis.

The photoprotein aequorin is a calcium-dependent bioluminescent enzyme which is most widely used in biotechnology processes, but this protein is susceptible to aggregation and proteolysis degradation. Various additives such as polyols are known to enhance the stability of proteins and protect them in native folded and functional state. In this work, for study of aequorin stability, the histidine-tagged apoaequorin was expressed in Escherichia coli and purified by nickel chelate affinity chromatography. Kinetics of light emission of purified aequorin upon addition of Ca(2+) showed a linear dependency on aequorin concentration. Furthermore, the effect of some stabilisers, such as glycerol, glucose, lactose, terehalose, sucrose and sorbitol on thermostability of recombinant aequorin was measured. Results indicate that the recombinant aequorin is very stable in phosphate buffer including 30 mM sorbitol, since after heat shock of 30 min at different temperatures, a slight decrease in activity was observed. However, flexibility and exposure of tryptophan residues of aequorin to the solvent, in the presence and absence of stabilisers, with respect to fluorescence quenching by acrylamide, indicated identical characterisation. In addition, according to limited proteolysis of aequorin demonstrating that this enzyme is sensitive to proteases as in the presence of 2 ng/ml of protease, aequorin was completely digested. In conclusion, sorbitol increases stability of aequorin with high photoactivity and not effect for flexibility and limited proteolysis of this photoprotein.

Design and characterization of an aequorin-based bacterial biosensor for detection of toluene and related compounds.

An aequorin-based Escherichia coli strain JM109 biosensor was constructed and characterized for its potential to detect toluene and related compounds in aqueous solutions. The biosensor was constructed based on a PGL2 plasmid carrying the lower pathway promoter (Pu) of the xyl operon of Pseudomonas putida mt-2, which was incorporated with transcriptional activator xylR and fused to aequorin cDNA named pGL2-aequorin. Binding of xylR protein to a subset of toluene-like compounds activates transcription at the Pu promoter, thus expression of aequorin is controlled by xylR and Pu. In this work we have compared the effect of Shine-Dalgarno (SD) and T2 rrnß terminator sequence in the expression of aequorin. According to the sensitivity of aequorin and increase in the signal-to-noise ratio, this reporter enzyme has reasonable sensitivity compared with other reporter systems. The results indicate higher expression of aequorin in the presence of SD and T2 rrnß. The activity of aequorin in recombinant whole-cell biosensor was linear from 1 to 500 µm of toluene. The bioluminescence response was specific for toluene-like molecules, so this biosensor cells would be able to detect toluene derivative contamination in environmental samples, accurately.