Recombinant Bacterial Expression of the Lysozyme from the Tobacco-Hornworm Manduca sexta with Activity at Low Temperatures

Abstract

A gene coding for lysozyme from the insect Manduca sexta (Ms-lyz) was expressed in Escherichia coli. The protein was produced as an insoluble cytoplasmic inclusion body which was denatured in 8 M: guanidine-HCl, renatured and purified by affinity and ion-exchange chromatography. The N-terminal sequence and the activity of the recombinant protein against Micrococcus luteus confirmed that correct expression had occurred. When Ms-lyz activity was compared to hen egg white lysozyme, the insect lysozyme was active at lower temperatures. These results demonstrate the feasibility of producing a disulfide-bonded lysozyme enzyme in bacteria and suggest that the insect Ms-lyz is an interesting system for further development of an antibacterial functional at low temperatures.

Over-expression and characterization of recombinant prefoldin from hyperthermophilic archaeum Pyrococcus furiosus in E. coli

“…Lysozyme activity was determined according to Garcia-Orozco et al (2005) by following the decrease in absorbance at 450 nm of a Micrococcus lysodeikticus suspension in 0.1 M potassium phosphate, pH 6.2.…”

White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity against Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae

“…The N-terminal sequence of the recombinant protein was determined by the Edman degradation method at the University of California at Davis, and it was found to be identical to the cDNA deduced amino acid sequence. The shrimp lysozyme was refolded in vitro and purified by affinity to a Micrococcus luteuseagarose chromatographic matrix [11]. The purified lysozyme was extensively dialyzed against lysozyme buffer (300 mM NaCl, 50 mM Tris, pH 8.0), then it was sterilized by filtration through a 0.2-mm membrane.…”

Over-expression and characterization of the recombinant small heat shock protein from Pyrococcus furiosus

“…The aggregation of lysozyme upon heat denaturation was determined by measuring apparent absorption due to light scattering at 360 nm. Lysozyme activity was determined according to Garcia-Orozco et al (2005) by following the decrease in absorbance at 450 nm of a Micrococcus lysodeikticus suspension in 0.1 M potassium phosphate, pH 6.2.…”