We found that induction of enzyme expression by IPTG at low temperature
(20°C) results in higher solubility than induction at 37°C. This last condition was critical for α-IPMS-14CR, as it is expressed to lower levels than α-IPMS-2CR. When expressed at 37°C, almost all of the α-IPMS-14CR selleck protein aggregates (i.e., is associated with an insoluble fraction, as assessed by SDS-PAGE (data not shown)). Figure 1 PCR amplification of leuA genes from M. tuberculosis strains. leuA genes were PCR amplified from H37Rv and Amnatchareon strain 731 with two and 14 copies of the tandem repeats, respectively. Lane M, 200 bp DNA markers. Figure 2 Analysis of His 6 -α-IPMS proteins on SDS-PAGE. A) Crude protein extracts of E. coli BL21 harboring p2C (α-IPMS-2CR) and p14C (α-IPMS-14CR). Cells were grown overnight at 20°C without (-) or with (+) Belnacasan ic50 0.5 mM IPTG. The cell pellets were sonicated, and the clear lysates were analyzed on 10% SDS-PAGE. Arrowheads indicate protein bands that were induced with IPTG. B) Purified His6-α-IPMS proteins from Ni-NTA agarose column. Lanes 1 and 2, elution fractions of His6-α-IPMS-14CR; Lane 3 and 4, elution fractions of His6-α-IPMS-2CR. MW, molecular weight markers. Purification of His6-tagged proteins under native conditions The purification of the His6-tagged proteins of α-IPMS-2CR
and α-IPMS-14CR under native conditions using a Ni-NTA column Selumetinib manufacturer yielded 90% and 80% pure protein, respectively. These proteins were further purified by gel filtration to approximately 99% purity. The yield of recombinant protein per gram of cell wet weight was 0.4–0.5 mg for α-IPMS-2CR and 0.1–0.2 mg for α-IPMS-14CR. The oligomeric state of each recombinant protein, as suggested by gel filtration analysis, was of a dimer (gel filtration profiles are presented in Additional file 1 and Additional file 2). Although purified α-IPMS-2CR was composed of both dimeric and tetrameric forms, the majority of the protein is in present as a dimer. In addition, the enzymatic activity of the dimeric form was three times higher than
that of the tetrameric protein (data not shown). The majority of purified α-IPMS-14CR was in dimeric form, with enzymatic activity six times higher than that of the minor fractions in monomeric form (data not shown). Enzymatic properties of His6-α-IPMS Both α-IPMS-2CR and α-IPMS-14CR enzymes worked well at a pH between Rucaparib research buy 7.5 and 8.5. At pH 9, α-IPMS-2CR lost much of its activity, while the activity of α-IPMS-14CR remained (Figure 3 panel A). The optimal temperature for both enzymes was approximately 37–42°C. At 50°C, the activity of α-IPMS-14CR remained at 75%, whereas the activity of α-IPMS-2CR dropped below 50% (Figure 3 panel B). Figure 3 Activities of His 6 -α-IPMS-2CR and His 6 -α-IPMS-14CR. Assays were performed as described in the Materials and Methods. Each point is the average of three assays and the vertical bars represent the standard deviations. A) Activities at various pH values at 37°C.