Bacterial expression systems for recombinant protein production: E. coli and beyond

Abstract 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.

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Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

10.21203/rs.2.19057/v2 ◽

2020 ◽

Author(s):

Artur Schuller ◽

Monika Cserjan-Puschmann ◽

Christopher Tauer ◽

Johanna Jarmer ◽

Martin Wagenknecht ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Expression System ◽

Cellular Level ◽

Lac Repressor ◽

Expression Systems ◽

E Coli ◽

Basal Expression

Abstract Background The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy of. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators ( lacO ) into the constitutive T5 and A1 promoter sequences. Results We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

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Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

10.21203/rs.2.19057/v1 ◽

2019 ◽

Author(s):

Artur Schuller ◽

Monika Cserjan-Puschmann ◽

Christopher Tauer ◽

Johanna Jarmer ◽

Martin Wagenknecht ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Expression System ◽

Lac Repressor ◽

Expression Systems ◽

E Coli ◽

Basal Expression ◽

T7 Expression System

Abstract Background The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy of. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators ( lacO ) into the constitutive T5 and A1 promoter sequences.Results We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level.Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

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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

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11405-9 ◽

2021 ◽

Author(s):

Hirra Hussain ◽

Edward A McKenzie ◽

Andrew M Robinson ◽

Neill A Gingles ◽

Fiona Marston ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Clostridium Difficile ◽

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Expression Systems ◽

Predictive Approaches ◽

Bacterial Expression Systems

AbstractBacterial 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.

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Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1

Microbial Cell Factories ◽

10.1186/s12934-021-01661-9 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Jan Weber ◽

Zhaopeng Li ◽

Ursula Rinas

Keyword(s):

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Expression System ◽

Limiting Factors ◽

Expression Vectors ◽

Metabolic Enzyme ◽

E Coli ◽

Metabolic Burden ◽

Fructose 1,6 Bisphosphate

Abstract Background Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. Results Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. Conclusions Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated “metabolic burden”. Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.

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