Optimization and efficient purification of recombinant Omp28 protein of Brucella melitensis using Triton X-100 and β-mercaptoethanol

The high level expression of recombinant proteins in Escherichia coli often leads to the formation of inclusion bodies that contain most of the expressed protein held together by non-covalent forces. The inclusion bodies are usually solubilized using strong denaturing agents like urea and guanidium hydrochloride. In this study recombinant Omp28 (rOmp28) protein of Brucella melitensis was expressed in two different vector systems and further efficient purification of the protein was done by modification in buffers to improve the yield and purity. Different concentrations of Triton X-100 and β-mercaptoethanol were optimized for the solubilization of inclusion bodies. The lysis buffer with 8M urea alone was not sufficient to solubilize the inclusion bodies. It was found that the use of 1% Triton X-100 and 20mM β-mercaptoethanol in lysis and wash buffers used at different purification steps under denaturing conditions increased the yield of purified rOmp28 protein. The final yield of purified protein obtained with modified purification protocol under denaturing conditions was 151 and 90mg/l of the culture or 11.8 and 9.37mg/g of wet weight of cells in pQE30UA and pET28a(+) vector respectively. Thus modified purification protocol yielded more than threefold increase of protein in pQE30UA as compared with purification by conventional methods.     

A rapid and simple method of evaluating the dimeric tendency of fluorescent proteins in living cells using a truncated protein of importin α as fusion tag

Enhanced green fluorescent protein (EGFP) and its yellow variant (Venus) are weakly dimeric under physiological conditions. We designed a simple method to evaluate the dimeric tendency of fluorescent proteins in living mammalian cells. A novel single mutation, A206L, interfering with the hydrophobic interactions of the dimer interface in Venus, contributed to its monomerization, and was as effective as the A206K mutation in this assay.     

Production of β-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of β-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of β-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular β-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 °C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 °C. β-Fructofuranosidase activity was slightly activated by Cu²⁺, Mn²⁺, Mg²⁺, and Na⁺ at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K_d values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively.     

Assay for the (R)- and (S)-enantiomers of salsolinols in biological samples and foods with ion-pair high-performance liquid chromatography using β-cyclodextrin as a chiral mobile phase additive

A chromatographic procedure was devised for the quantitative determination of the enantiomers of salsolinol and N-methylsalsolinol, which are biologically important alkaloids. The enantiomers of salsolinol and N-methylsalsolinol were completely separated using β-cyclodextrin in a reversed-phase ion-pair system. The HPLC method was sensitive enough to detect the isoquinolines at a concentration less than 0.1 pmol per injection. The presence of (R)- and (S)-salsolinol was confirmed in fermented foods and beverages, while N-methylsalsolinol was not detected. On the other hand, the (R)-enantiomers of both salsolinol and N-methylsalsolinol were found to predominate in the human brain.