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A putative beta-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-beta-D-1-thiogalactopyranoside, the enzyme expressed at approximately 40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A(600) 12 in auto-inducing M9NG medium, beta-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the beta-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45 degrees C and 8.0, respectively, under the assay conditions. K(m) and k(cat) against p-nitrophenyl-beta-D-glucopyranoside were 4 mM and 0.75 sec(-1), respectively. To our knowledge, this is the first report of high-level expression and characterization of a beta-glucosidase from B. halodurans.

Cloning, high-level expression, and characterization of the somatotropin (ST) gene of an indigenous Nili-Ravi breed of water buffalo Bubalus bubalis (BbST) are described. Coding, non-coding, and promoter regions of BbST were amplified and sequenced. Sequence analysis revealed several silent and two interesting point mutations on comparison with STs of other vertebrate species. One interesting variation in the BbST sequence was the replacement of a conserved glutamine residue by arginine. A plasmid was also constructed for the production of BbST in Escherichia coli BL21 (RIPL) CodonPlus, under the control of IPTG-inducible T7-lac promoter. High-level expression could be obtained by synthesizing a codon-optimized ST gene and expressing it in the form of inclusion bodies. The inclusion bodies represented over 20% of the E. coli cellular proteins. The biologically active conformation of purified BbST was confirmed by its efficient growth promoting activity in Nb2 cell proliferation assay. The expression system and purification strategy employed promise to be a useful approach to produce BbST for further use in structure-function studies and livestock industry.