RESUMEN
The ß-sandwich domain 1 (SD1) of islandisin is a stable thermophilic protein with surface loops that can be redesigned for specific target binding, architecturally comparable to the variable domain of immunoglobulin (IgG). SD1's propensity to aggregate due to incorrect folding and subsequent accumulation in Escherichia coli inclusion bodies limits its use in biotechnological applications. We rationally designed SD1 for improved variants that were expressed in soluble forms in E. coli while maintaining the intrinsic thermal stability of the protein (melting temperature (Tm) = 73). We used FoldX's ΔΔG predictions to find beneficial mutations and aggregation-prone regions (APRs) using Tango. The S26K substitution within protein core residues did not affect protein stability. Among the soluble mutants studied, the S26K/Q91P combination significantly improved the expression and solubility of SD1. We also examined the effects of the surface residue, pH, and concentration on the solubility of SD1. We showed that the surface polarity of proteins had little or no effect on solubility, whereas surface charges played a substantial role. The storage stability of several SD1 variants was impaired at pH values near their isoelectric point, and pH levels resulting in highly charged groups. We observed that mutations that create an uneven distribution of charged groups on the SD1 surface could enhance protein solubility by eliminating favorable protein-protein surface charge interactions. Our findings suggest that SD1 is mutationally tolerant to new functionalities, thus providing a novel perspective for the application of rational design to improve the solubility of targeted proteins.
Asunto(s)
Escherichia coli , Dominios Proteicos , Estabilidad Proteica , Solubilidad , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Ingeniería de Proteínas/métodos , Pliegue de Proteína , Mutación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Infectious bronchitis (IB) and Newcastle disease (ND) are two important diseases of poultry and have remained a threat to the development of the poultry industry in many parts of the world. The immunology of avian has been well studied and numerous vaccines have been developed against the two viruses. Most of these vaccines are either inactivated vaccines or live attenuated vaccines. Inactivated vaccines induce weak cellular immune responses and require priming with live or other types of vaccines. Advanced technology has been used to produce several types of vaccines that can initiate prime immune responses. However, as a result of rapid genetic variations, the control of these two viral infections through vaccination has remained a challenge. Using various strategies such as combination of live attenuated and inactivated vaccines, development of IB/ND vaccines, use of DNA vaccines and transgenic plant vaccines, the problem is being surmounted. It is hoped that with increasing understanding of the immunological mechanisms in birds that are used in fighting these viruses, a more successful control of the diseases will be achieved. This will go a long way in contributing to global food security and the economic development of many developing countries, given the role of poultry in the attainment of these goals.
RESUMEN
Herpes simplex virus (HSV) infections are among the most common viral infections and usually last for a lifetime. The virus can potentially be controlled with vaccines since humans are the only known host. However, despite the development and trial of many vaccines, this has not yet been possible. This is normally attributed to the high latency potential of the virus. Numerous immune cells, particularly the natural killer cells and interferon gamma and pathways that are used by the body to fight HSV infections have been identified. On the other hand, the virus has developed different mechanisms, including using different microRNAs to inhibit apoptosis and autophagy to avoid clearance and aid latency induction. Both traditional and new methods of vaccine development, including the use of live attenuated vaccines, replication incompetent vaccines, subunit vaccines and recombinant DNA vaccines are now being employed to develop an effective vaccine against the virus. We conclude that this review has contributed to a better understanding of the interplay between the immune system and the virus, which is necessary for the development of an effective vaccine against HSV.