Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens

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.

Predictive Approaches to Guide the Expression of Recombinant Vaccine Targets in Escherichia Coli: A Case Study Presentation Utilising Absynth Biologics Ltd Proprietary C.Difficile Vaccine Antigens

Background: 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, termed inclusion bodies. As a consequence, different and 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. 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. Further, proteolytic cleavage of Ant2-3 was observed, potentially due to the presence of a large un-structured loop between the protein boundaries. 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. Changes to the construct design to include N-terminal solubility tags (Thioredoxin and N utilisation substance protein A) did not improve solubility. 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. 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. The results showed that both Ant2 and Ant3 contained unfavorable features associated with poor solubility. A large non-polar region was detected on the surface of Ant2 structures, whereas, positively charged regions were observed for Ant3.Conclusions: Commonly used strategies to enhance recombinant protein production in bacterial systems did not act to increase production of model ‘difficult-to-express’ antigens, Ant2 and Ant3 and their fusion proteins. Sequence and structural analysis of antigens identified unfavorable features that potentially result in the increased tendency of these antigens to aggregate and/or lead to improper processing. 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 of single and potentially multi-domain (or fusion) antigens in bacterial expression systems.

A Review on Molecular Functional and Structural Characterization of Human Cysteine Cathepsins in Bacterial Expression Systems

Cysteine cathepsins, a class of proteinaceous enzymes, regulate a wide variety of metabolic processes in human including protein breakdown and turnover and immune functions. Eleven cysteine cathepsins have been identified so far and a wide array of studies related to identifying their specific functions, regulation and distribution patterns in tissues have been conducted. However, in recent past, the association of cysteine cathepsins in occurrence and progression of cancers have been identified and this has caused unrest in scientists triggering them to investigate the physiology, biochemical pathways and interactions of these cathepsins in cancer metastasis and therefore has become a noteworthy topic of intensive research. This review focusses and collects together the published work on molecular functional and structural characterization studies that have been done so far on in vitro expression of genes encoding for cysteine cathepsins in the Escherichia coli bacterial expression system. Accordingly, it was found out that all cathepsins except for cathepsins K, C, H, X and W have been expressed this way and the majority of them were found to be expressed in E. coli BL21(DE3) pLysS expression host via pET3 expression vector. In addition, it was also noted that in most of the expression studies, the substrate that was used to validate the enzymatic activity of the recombinant enzyme that was produced was a cysteine residue along with a benzyloxy-carbonyl salt. Through this review, the authors suggest that there is a very high need that all cysteine cathepsins need to be characterized both structurally and functionally on a molecular platform to better understand their interactions including the biochemical pathways. It is also momentous that the mass production of the recombinant forms of these enzymes are facilitated via expression in such bacterial expression systems and in turn, would also provide a strong platform for the development and progression of studies related to human physiology including oncological studies such as cancer metastasis. Moreover, as per biochemical features of the enzymes that could be identified, the production of efficient inhibitors or inducers as per the necessity to improve health and promote wellbeing among the mankind could be facilitated.

An open library of human kinase domain constructs for automated bacterial expression

Kinases play a critical role in many cellular signaling pathways and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Since the FDA approval of imatinib in 2001, therapeutics targeting kinases now account for roughly 50% of current cancer drug discovery efforts. The ability to explore human kinase biochemistry, biophysics, and structural biology in the laboratory is essential to making rapid progress in understanding kinase regulation, designing selective inhibitors, and studying the emergence of drug resistance. While insect and mammalian expression systems are frequently used for the expression of human kinases, bacterial expression systems are superior in terms of simplicity and cost-effectiveness but have historically struggled with human kinase expression. Following the discovery that phosphatase coexpression could produce high yields of Src and Abl kinase domains in bacterial expression systems, we have generated a library of 52 His-tagged human kinase domain constructs that express above 2 µg/mL culture in a simple automated bacterial expression system utilizing phosphatase coexpression (YopH for Tyr kinases, Lambda for Ser/Thr kinases). Here, we report a structural bioinformatics approach to identify kinase domain constructs previously expressed in bacteria likely to express well in a simple high-throughput protocol, experiments demonstrating our simple construct selection strategy selects constructs with good expression yields in a test of 84 potential kinase domain boundaries for Abl, and yields from a high-throughput expression screen of 96 human kinase constructs. Using a fluorescence-based thermostability assay and a fluorescent ATP-competitive inhibitor, we show that the highest-expressing kinases are folded and have well-formed ATP binding sites. We also demonstrate how the resulting expressing constructs can be used for the biophysical and biochemical study of clinical mutations by engineering a panel of 48 Src mutations and 46 Abl mutations via single-primer mutagenesis and screening the resulting library for expression yields. The wild-type kinase construct library is available publicly via Addgene, and should prove to be of high utility for experiments focused on drug discovery and the emergence of drug resistance.