doi: 10.15171/apb.2019.050.īordoli L, Kiefer F, Arnold K, Benkert P, Battey J, Schwede T. Two simple methods for optimizing the production of “difficult-to-express” GnRH-DFF40 chimeric protein. doi: 10.1093/bioinformatics/bti770.īarazesh M, Mostafavipour Z, Kavousipour S, Mohammadi S, Mokarram P. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Data from the addition of reducing agents dithiothreitol (DTT) and β-mercaptoethanol (BME) is plotted for both Ant2 and Ant3 ( a– b)Īrnold K, Bordoli L, Kopp J, Schwede T. The data was analysed and plotted for each condition using Protein Dashboard™. Absorbance readings (at 280 nm) were taken of the plate with reagent alone (blank) and after the collection of soluble protein following incubation under stressed conditions. Following incubation the soluble protein was collected by centrifugation. Fifteen nicrolitres of protein were mixed with 150 μl of reagent per well and incubated at 37☌ for 24 h (stressed conditions). The protein concentration was determined via the Bio-Rad DC protein assay kit (Table SI), and the top three concentrated samples (*) were pooled for the OptiSol™ protein solubility screen. Proteins were eluted with imidazole in 5 × 1 ml stages (E1-E5). Proteins were re-solubilised in urea buffer from the insoluble fraction and purified via the His-tag. Ant2 ( a) and Ant3 ( b) were expressed in the BL21-CodonPlus (DE3) E. Protein solubilisation screen of the single antigens (Ant2 and Ant3). Data is representative of at least four biological replicates The band position for each antigen is indicated with an arrow. Bovine serum albumin (BSA) was used as a protein standard. The cell pellet was lysed via sonication and a sample of the crude lysate (total fraction) was analysed by SDS-PAGE ( b). Bacterial cultures for each antigen were harvested post-IPTG induction (20 h at 18☌). Error bars shown are the mean value ± SEM of three biological replicates (n=3). The table summarises the estimated doubling time (min) of each culture analysed using the exponential growth equation in GraphPad Prism. This figure shows the OD600 plotted against the time (min) for bacterial cultures transformed with all four target antigens ( a). 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. Antigens Bacterial expression Predictive tools Recombinant protein production Sequences and structural analysis ‘Difficult-to-express’.Īnalysis of growth and antigen production in bacterial cultures. 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.Ībsynth Biologics Ltd. 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. Bacterial expression systems remain a widely used host for recombinant protein production.
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