A clear band of purified protein
in the position corresponding to the overexpressed protein in the crude lysate was Vorinostat visualized on the gel (Figure 3B). This band cross-reacted with anti-Cam antiserum (Figure 3C). The recognition of recombinant Gca1 with heterologous antibody indicates significant similarity between Gca1 and Cam. Figure 3 Heterologous overexpression, purification and western blot analysis of recombinant Gca1 of A. brasilense (A) SDS-PAGE gel electrophoresis (15%) of uninduced (lane 2) and induced (lane 3) cell lysates of transformants harboring pSK7. The Gca1 protein overproduced in E. coli pSK7 is encircled. Low range molecular weight marker, Bangalore Genei (lane 1). (B). Purification of recombinant Gca1 of A. brasilense under denaturing conditions SDS-PAGE gel (15%) showing induced crude extract of transformant
harboring pSK7 (Lane 2); Ni-NTA purified His.Tag Gca1 (Lane 3); Low range molecular weight marker, Bangalore Genei (Lane 1). (C) Western blot analysis showing cross-reactivity of purified recombinant Gca1 with antisera raised against CAM. No CA activity could be detected in crude cell extracts of E. coli overexpressing recombinant Gca1 while under the same CA activity assay conditions, α-bovine CAII showed specific CA activity of about 1024 WAU/mg, respectively. These results indicate that the supernatant fractions containing soluble recombinant Gca1 lacked detectable CO2 hydration activity. Construction of gca1 knockout (Δgca1) mutant In order to gain an insight FLT3 inhibitor into the possible physiological role of Gca1 in A. brasilense, attempt was made to construct
a Δgca1 of A. brasilense Sp7 by inserting kanamycin resistance gene cassette into the coding region of gca1 but in spite of repeated attempts no gca1 mutant could be isolated. Since deletion of CA gene generally results in high Selleckchem Gefitinib CO2 requiring (HCR) phenotype , attempts were also made to isolate the desired mutants at 3% CO2 concentration (the highest CO2 concentration at which A. brasilense Sp7 is able to grow). The inability to obtain γ-CA knock-out mutant under aerobic atmosphere as well as under the atmosphere containing 3% CO2 probably reflects that this putative γ-CA might be essential for the survival and growth of A. brasilense in the atmosphere containing ambient to 3% levels of CO2. Since bicarbonate is a substrate for carboxylating enzymes central to many metabolic processes , attempts were also made to restore Δgca1 by check details supplementing the minimal medium with some metabolic intermediates (as mentioned in Methods). Unfortunately, none of these supplements rescued Δgca1 of A. brasilense suggesting that the putative Gca1 protein might have physiological implications other than hydration of CO2. Bioinformatic analysis of gca1 organization: Prediction of argC-gca1 operon in A. brasilense While analyzing the organization of gca1 chromosomal region of A.