isopropyl-thiogalactoside and Escherichia-coli-Infections

Toxoplasmosis, despite advances in science and technology, is a disease that requires attention since there is no vaccine capable of immunizing humans and animals against all isolated types of Toxoplasma gondii. Thus, the use of chimeric proteins, which can contain multiple antigens, is a very promising alternative for the process of obtaining a vaccine and diagnostic test for toxoplasmosis due to the great diversity of antigens presented by T. gondii. In this context, the present study evaluates batch culture strategies in the production of the multi-antigenic chimeric protein TgAGS/BsT from Toxoplasma gondii. Several exploratory cultures were initially carried out to observe the kinetic behavior of E. coli BL21 Star in five different medium compositions without the addition of IPTG (inducer). Cultures of E. coli B21 Star were carried out with 1.0 mM IPTG at different times of initiation of induction (0.5, 1, and 6 h) to evaluate the effects on cell growth, production of the protein of interest, culture pH, and acetic acid formation. The results showed that among the culture media evaluated, 2xTY and TB supplemented with glycerol had the best cell concentration values of 3.42 ± 0.05 g/L and 5.48 ± 0.05 g/L, respectively. In the assays induced by IPTG, a higher expression of TgAGS/BsT protein was observed, with induction beginning within 6 h of culture, with a maximum concentration of protein of interest of 1.82 ± 0.02 g/L for the 2xTY and 2.49 ± 0.03 g/L for the TB medium. In addition, later induction by IPTG provided greater stability of plasmid pET-TgAGS, remaining with values above 90% at the end of culture.

Topics: Animals; Antigens, Protozoan; Culture Media; Escherichia coli; Escherichia coli Infections; Glycerol; Humans; Isopropyl Thiogalactoside; Protozoan Proteins; Recombinant Fusion Proteins; Recombinant Proteins; Toxoplasma; Toxoplasmosis

The relationship between membrane damage and loss of viability following pressure treatment was examined in Escherichia coli strains C9490, H1071, and NCTC 8003. These strains showed high, medium, and low resistance to pressure, respectively, in stationary phase but similar resistance to pressure in exponential phase. Loss of membrane integrity was measured as loss of osmotic responsiveness or as increased uptake of the fluorescent dye propidium iodide. In exponential-phase cells, loss of viability was correlated with a permanent loss of membrane integrity in all strains, whereas in stationary-phase cells, a more complicated picture emerged in which cell membranes became leaky during pressure treatment but resealed to a greater or lesser extent following decompression. Strain H1071 displayed a very unusual pressure response in stationary phase in which survival decreased to a minimum at 300 MPa but then increased at 400 to 500 MPa before decreasing again. Membranes were unable to reseal after treatment at 300 MPa but could do so after treatment at higher pressures. Membrane damage in this strain was thus typical of exponential-phase cells under low-pressure conditions but of stationary-phase cells under higher-pressure conditions. Heat shock treatment of strain H1071 cells increased pressure resistance under low-pressure conditions and also allowed membrane damage to reseal. Growth in the presence of IPTG (isopropyl-beta-D-thiogalactopyranoside) increased resistance under high-pressure conditions. The mechanisms of inactivation may thus differ at high and low pressures. These studies support the view that membrane damage is an important event in the inactivation of bacteria by high pressure, but the nature of membrane damage and its relation to cell death may differ between species and phases of growth.

Topics: beta-Galactosidase; Cell Membrane; Colony Count, Microbial; Escherichia coli; Escherichia coli Infections; Escherichia coli O157; Hot Temperature; Humans; Hydrostatic Pressure; Isopropyl Thiogalactoside