RESUMEN
Bacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production. In this case study, we present the strategies used to increase the recombinant production and solubility of 'difficult-to-express' bacterial antigens, termed Ant2 and Ant3, from Absynth Biologics Ltd.'s Clostridium difficile vaccine programme. Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in bacterial cells. Further, proteolytic cleavage of Ant2-3 was observed. Optimisation of culture conditions and changes to the construct design to include N-terminal solubility tags did not improve antigen solubility. However, screening of different buffer/additives showed that the addition of 1-15 mM dithiothreitol alone decreased the formation of insoluble aggregates and improved the stability of both Ant2 and Ant3. Structural models were generated for Ant2 and Ant3, and solubility-based prediction tools were employed to determine the role of hydrophobicity and charge on protein production. The results showed that a large non-polar region (containing hydrophobic amino acids) was detected on the surface of Ant2 structures, whereas positively charged regions (containing lysine and arginine amino acids) were observed for Ant3, both of which were associated with poor protein solubility. We present a guide of strategies and predictive approaches that aim to guide the construct design, prior to expression studies, to define and engineer sequences/structures that could lead to increased expression and stability of single and potentially multi-domain (or fusion) antigens in bacterial expression systems.
Asunto(s)
Productos Biológicos , Clostridioides difficile , Escherichia coli/genética , Proteínas Recombinantes de Fusión , Proteínas Recombinantes/genética , Solubilidad , Vacunas Sintéticas/genéticaRESUMEN
MALDI-ToF MS can be used to identify microorganisms directly from blood cultures. This study compared two methods of sample preparation. Similar levels of genus- (91% vs 90%) and species-level identifications (79% vs 74%) were obtained with differential centrifugation and SDS methods. The SDS method is faster and requires minimal handling.
Asunto(s)
Bacterias/aislamiento & purificación , Cultivo de Sangre/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Bacteriemia/diagnóstico , Bacteriemia/microbiología , Cultivo de Sangre/economía , Humanos , Dodecil Sulfato de Sodio , Manejo de Especímenes/métodos , Factores de TiempoRESUMEN
AIM: Antibiotic resistance mediated by extended-spectrum ß-lactamases (ESBL) and AmpC ß-lactamases is widespread and increasingly common, often rendering empiric antibiotic therapy ineffective. In septicemia, delays in initiating effective antibiotic therapy are associated with worse clinical outcomes. With current phenotypic antimicrobial susceptibility testing methods, there is often a delay of 18-24 h before the susceptibility of an isolate is known. RESULTS: Using an HPLC assay, breakdown of the third-generation cephalosporin cefotaxime by ESBL- and AmpC- ß-lactamase-producing organisms could be detected within 90 min with 86.4% sensitivity and 100% specificity; sensitivity for ESBL detection was 100%. CONCLUSION: This assay could be readily established in any clinical laboratory with an HPLC to rapidly detect ESBL-producing Enterobacteriaceae.