Overexpression of MG207 in E coli Overexpression and purificatio

Overexpression of MG207 in E. coli Overexpression and purification of recombinant MG207 protein using pET16b were performed as detailed before [55, 56]. Briefly, E. coli strain BL21 (DE3) harboring the pMG207EX was induced with 0.5 mM IPTG at 37°C to overexpress the protein. The overexpressed protein was purified with Ni-NTA affinity column chromatography (Qiagen).

The E. coli extracts and purified protein were separated on 12% SDS-PAGE to assess the expression and purification. The purified recombinant protein was designated as His10MG207. All purification find more and desalting procedures were performed with buffers based on Tris–HCl pH8.0 and use of phosphate buffer was avoided. Enzyme assays To determine if the overexpressed and purified His10MG207 was functional, we performed phosphatase assay with p-nitrophenyl phosphate (pNPP) as substrate (Sigma-Aldrich, St. Louis, MO). The assay was conducted in 96 well plates and the assay mixture (120 μl) contained 1 mM pNPP in 20 mM Tris–HCl pH 8.0, 5 mM MgCl2 and His10MG207 protein. Control reactions had

no protein or heat inactivated His10MG207. Each reaction was done in triplicate wells. The reaction mixtures were incubated at 37°C for 1 h and the yellow color, developed due to the hydrolysis of pNPP, was read at 405 nm using a Spectramax plate reader (Molecular Devices, Sunnyvale, CA). To determine the specificity of His10MG207 towards buy Buparlisib serine or threonine residue, we used Alkaline/Acid Phosphatase assay kit (Millipore, Temecula, CA). This uses synthetic peptides for serine phosphate

(RRApSSVA) and threonine phosphate (KRpTIRR) as substrates for the enzyme assay. The reactions were done as described by the manufacturer in 96 well plates, except that the reaction mixture had MgCl2 instead of NiCl2. Amount of phosphate released was calculated using phosphate reference Baricitinib standards supplied with the kit. SDS-PAGE and immunoblot Premade SDS-PAGE gels (NuPAGE 12% Bis-Tris gel, Invitrogen, Carlsbad, CA) were used to separate proteins from E. coli and M. genitalium for coomassie staining of proteins and for Western blot. In these gels 50 μg of total protein was loaded per well. Protein concentration was determined by BCA method (Pierce). Western blots were probed with anti-MG207 rabbit antiserum (1:500 dilutions) to detect MG207 protein of M. genitalium strains. This rabbit antiserum was generated against purified His10MG207 protein using a commercial source (Alpha Diagnostic International Inc., San Antonio). Two-dimensional gel analysis of proteins Two-dimensional (2-D) gel analyses of total proteins of M. genitalium G37 and TIM207 strains were performed by Kendrick Lab Inc., (Madison, WI). Fifty μg of total proteins were separated by isoelectric focusing [IEF] in glass tubes with an inner diameter of 2.0 mm. The IEF gel contained 2% pH 4–6 ampholines (Servalytes, Serva, Heidelberg, Germany) and 2% pH 5–8 ampholines (GE Healthcare).

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