The results showed that both Ant2 and Ant3 contained unfavorable features associated with poor solubility. Structural models were generated for Ant2 and Ant3 and solubility-based prediction tools were employed to determine the role of charge and hydrophobicity on protein production. Screening of different buffer/additives to improve stability showed that the addition of 1-15mM dithiothreitol (DTT) alone improved the stability of both Ant2 and Ant3. Changes to the construct design to include N-terminal solubility tags (Thioredoxin and N utilisation substance protein A) did not improve solubility. Optimisation of culture conditions such as varying the induction temperature and addition of heat-shock inducer benzyl alcohol to the growth media had no significant effect on the processing and protein production pattern for all four antigen molecules. Further, proteolytic cleavage of Ant2-3 was observed, potentially due to the presence of a large un-structured loop between the protein boundaries. Results: Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in BL21 CodonPlus (DE3) 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, different and 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, termed inclusion bodies.

Background: Bacterial expression systems remain a widely used host for recombinant protein production.

Absynth biologics limited