Bacteriophage Lambda Expression Systems for Enhanced Recombinant Protein Production

1992 ◽  
pp. 367-372
Author(s):  
N. Padukone ◽  
S. W. Peretti ◽  
D. F. Ollis
Keyword(s):  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Bacteriophage Lambda ◽  
Expression Systems ◽  
Lambda Expression

scholarly journals Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Javier Garrigós-Martínez ◽  
Kiira Vuoristo ◽  
Miguel Angel Nieto-Taype ◽  
Juha Tähtiharju ◽  
Jaana Uusitalo ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Cell Factory ◽  
The Novel ◽  
Expression Systems ◽  
Fed Batch ◽  
Model Protein

Abstract Background Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (PAOX1), and the constitutive GAP promoter (PGAP). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (PPDF, PUPP) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter PGAP, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. Results Both the PPDF and PUPP-based expression systems outperformed similar PGAP-based expression in chemostat cultivations, reaching ninefold higher specific production rates (qp). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both PPDF and PUPP expression systems were also successfully implemented in 15 L fed-batch cultivations where qp and product to biomass yield (YP/X*) values were similar than those obtained in chemostat cultivations. Conclusions As an outcome of the macrokinetic characterization presented, the novel PPDF and PUPP were observed to offer much higher efficiency for CalB production than the widely used PGAP-based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris-based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest.

scholarly journals 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

2021 ◽  
Author(s):  
HIRRA HUSSAIN ◽  
Edward A McKenzie ◽  
Andrew M Robinson ◽  
Neill A Gingles ◽  
Fiona Marston ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Fusion Proteins ◽  
Recombinant Protein Production ◽  
Inclusion Bodies ◽  
Protein Production ◽  
Expression Systems ◽  
Predictive Approaches ◽  
Bacterial Expression Systems

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.

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

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.

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

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.

scholarly journals Recombinant Protein Production of Earthworm Lumbrokinase for Potential Antithrombotic Application

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Kevin Yueju Wang ◽  
Lauren Tull ◽  
Edwin Cooper ◽  
Nan Wang ◽  
Dehu Liu
Keyword(s):  
Recombinant Protein ◽  
Oral Administration ◽  
Transgenic Plant ◽  
Recombinant Protein Production ◽  
Blood Circulation ◽  
Protein Production ◽  
Far East ◽  
Genetic Technology ◽  
Expression Systems ◽  
Fibrinolytic Agents

Earthworms have been used as a traditional medicine in China, Japan, and other Far East countries for thousands of years. Oral administration of dry earthworm powder is considered as a potent and effective supplement for supporting healthy blood circulation. Lumbrokinases are a group of enzymes that were isolated and purified from different species of earthworms. These enzymes are recognized as fibrinolytic agents that can be used to treat various conditions associated with thrombosis. Many lumbrokinase (LK) genes have been cloned and characterized. Advances in genetic technology have provided the ability to produce recombinant LK and have made it feasible to purify a single lumbrokinase enzyme for potential antithrombotic application. In this review, we focus on expression systems that can be used for lumbrokinase production. In particular, the advantages of using a transgenic plant system to produce edible lumbrokinase are described.

scholarly journals 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

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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