Production of bioactive recombinant ovine cysteine-rich secretory protein 1 in Escherichia coli.

Ovine cysteine-rich secretory protein 1 (CRISP-1) is an acidic glycoprotein of epididymal origin under CRISP, antigen 5, pathogenesis-related protein 1 (CAP) super-family. The aim of the present study was the optimization of bacterial production and partial characterization of putative mature ovine CRISP-1 protein. The cDNA corresponding to T23 - C242 peptide fragment of ovine CRISP-1 protein was cloned into THE pET32b(+) expression vector using E. coli DH5α. Protein expression was carried out in E. coli BL21(DE3) by inducition with 1 mM IPTG at 37°C for 4 h. The recombinant protein was expressed as inclusion bodies and purified by Ni-NTA affinity chromatography using a pH gradient. Further purification of the protein was carried out by gel extraction following zinc sulfate negative staining. SDS-PAGE analysis of the purified recombinant CRISP-1 protein revealed a 43.8 kDa band. Bioactivity of the purified CRISP-1 protein was examined on sperm motility and capacitation. The recombinant ovine CRISP-1 protein at 5 µg/ml caused significant inhibition of sperm motility, and the activity was lost following heating the protein at 100°C for 5 min. The protein also demonstrated decapacitation activity, and at a concentration of 2 µg/ml, it caused a significant (P < 0.05) reduction in sperm capacitation. In conclusion, the thioredoxin-tagged ovine CRISP-1 protein was successfully produced in E. coli and purified in the soluble form by a combination of Ni-NTA affinity chromatography, gel purification, and dialysis. The recombinant protein exhibited both motility-inhibiting and decapacitating activities. Further study is needed to elucidate the mechanism of action and evaluate it's possible use in semen preservation.Abbreviations: CRISP-1: Cysteine-rich secretory protein-1; PCR: polymerase chain reaction; IPTG: isopropyl-ß-D-thiogalactopyranoside; LB: Luria Bertani; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; EDTA: ethylene diamine tetraacetic acid; Ni-NTA: Nickel nitrilotriacetic acid.

Environment friendly biomass gasifier cookstove for community cooking.

Traditional community cookstoves have a low level of efficiency due to their poor heat transfer efficiency and incomplete combustion. The low efficiency results in a high consumption of fuel wood, thereby creating a need of more fuel wood. This paper deals with the development of a biomass cookstove suitable for community cooking. The stove exhibits approximately 36.38% thermal efficiency and has a thermal power rating of 5 kW. The maximum flame temperature recorded was 712°C. The data indicate that the developed cookstove can save approximately 7155 kg of CO2 per annum.

Mathematical modeling of cadmium(II) solvent extraction from neutral and acidic chloride media using Cyanex 923 extractant as a metal carrier.

This paper describes experimental work and the mathematical modeling of solvent extraction of cadmium(II) from neutral and acidic aqueous chloride media with a Cyanex 923 extractant in Exxol D-100. Solvent extraction experiments were carried out to analyze the influence of variations in the composition of the aqueous and organic phases on the efficiency of cadmium(II) extraction. In neutral and acidic chloride conditions, the extraction of cadmium(II) by the organophosphorous extractant Cyanex 923 (L) is based on the solvation mechanism of neutral H(n)CdCl((2+n)) species and the formation of H(n)CdCl((2+n))L(q) complexes in the organic phase, where n=0, 1, 2 and q=1, 2. The mathematical model of cadmium(II) extraction was derived from the mass balances and chemical equilibria involved in the separation system. The model was computed with the Matlab software. The equilibrium parameters for metal extraction, i.e. the stability constants of the aqueous Cd-Cl complexes, the formation constants of the acidic Cd-Cl species and the metal equilibrium extraction constants, were proposed. The optimized constants were appropriate, as there was good agreement when the model was fitted to the experimental data for each of the experiments.