Bacterial cultures were seeded from overnight cultures (1:100 dilution) in a total volume of 100ml of LB media and the optical density monitored at 600 nm (OD600) prior to induction. DNA constructs for Ant2 (A2), Ant3 (A3), Ant2-3 (A23) and Ant3-2 (A32) were transformed into BL21-CodonPlus (DE3) E.
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.Īnalysis of growth and antigen production in bacterial cultures. 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. 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. 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. Optimisation of culture conditions and changes to the construct design to include N-terminal solubility tags did not improve antigen solubility. Further, proteolytic cleavage of Ant2-3 was observed. Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in bacterial cells. 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. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase 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. Instantly grabbing visitor’s attention, partaking them, and building trust are the key factors of a strong video.Bacterial expression systems remain a widely used host for recombinant protein production.Video is a web reflection of your business and guests choose your business supported expertise from looking your video.
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