Side effects of chaperone gene co-expression in recombinant protein production

Martínez-Alonso, Maira; García‐Fruitós, Elena; Ferrer-Miralles, Neus; Rinas, Ursula; Villaverde, Antonio

doi:10.1186/1475-2859-9-64

Cited by 89 publications

(58 citation statements)

References 77 publications

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“…Recently, it was reported that the promotion of proteolysis of target proteins was observed in DnaK and GroEL sets as folding assistant partner, resulting in the reduced yield or decreased expression level of foreign polypeptides [4,15]. In our archaea chaperonin system, this proteolysis on alginate lyase produced is likely less profound than GroEL/GroES and DnaK/DnaJ/GrpE sets.…”

Section: Comparison With Archaea Chaperonin and E Coli Molecular Chamentioning

confidence: 84%

Coexpression of Alginate Lyase with Hyperthermophilic Archaea Chaperonin in E. coli

Kim¹,

Kim²,

Nam³

2015

Journal of Life Science

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When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii IAM 14594 was expressed in E. coli, most of the gene product expressed was produced as aggregated insoluble particles known as inclusion bodies. In order to produce with an elevated level of a soluble and active form of alginate lyase in E. coli, the hyperthermophilic chaperonins (ApCpnA and ApCpnB) from archaeon Aeropyrum pernix K1 were employed as the coexpression partners. At 25℃ culture temperature, the level of alginate lyase activity was increased from 10.1 unit/g-soluble protein in aly single expression to 83.1 unit/g-soluble protein by coexpressing with ApCpnA and to 100.3 unit/g-soluble protein by coexpressing with ApCpnB. This results indicate that the coexpression of aly with ApCpnA and ApCpnB revealed a marked enhancement, about 8~10 fold, in the production of alginate lyase as a soluble and active form. Based on the results of various examinations on the expression variables, the optimal conditions for the maximal production of alginate lyase were determined as 1.0 mM IPTG for the inducer concentration, 25℃ for the culture temperature after IPTG induction, and ApCpnB for the coexpression partner. The coexpression set in the present report may be useful in the industrial production of functionally or medically important recombinant proteins in E. coli.

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Section: Comparison With Archaea Chaperonin and E Coli Molecular Chamentioning

confidence: 84%

Coexpression of Alginate Lyase with Hyperthermophilic Archaea Chaperonin in E. coli

Kim¹,

Kim²,

Nam³

2015

Journal of Life Science

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“…In fact, undesirable side effects derived from chaperone gene co-expression, such as growth inhibition, proteolysis, reduced yield, reduced solubility or reduced specific activity, have been reported (34).…”

Section: Discussionmentioning

confidence: 99%

Production of recombinant proteins from Plasmodium falciparum in Escherichia coli

et al. 2016

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Introduction:The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum. However, the proteins of P. falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies. Objective: To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8). Materials and methods:The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice. Results: The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli, compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies. Conclusion: The use of genetically modified strains of E. coli was essential to achieve high expression levels of the four evaluated P. falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P. falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.

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“…One approach to facilitating the productive folding of recombinant proteins is to overexpress molecular chaperones such as the DnaK/DnaJ and GroEL/GroES systems in the host Escherichia coli cells (8). Although a number of proteins have been produced successfully in these sophisticated bacterial strains, cellular growth impairment and unwanted proteolysis after refolding have been reported (9). In contrast to these multicomponent protein folding/disaggregation systems, other proteins exert the molecular chaperone function from within a much smaller ensemble or even without an obligatory cofactor (10).…”

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confidence: 99%

Protein Interaction Module–assisted Function X (PIMAX) Approach to Producing Challenging Proteins Including Hyperphosphorylated Tau and Active CDK5/p25 Kinase Complex

Sui

et al. 2015

Molecular & Cellular Proteomics

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Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction moduleassisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3␤ and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research. Molecular & Cellular Proteomics

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Side effects of chaperone gene co-expression in recombinant protein production

Cited by 89 publications

References 77 publications

Coexpression of Alginate Lyase with Hyperthermophilic Archaea Chaperonin in E. coli

Coexpression of Alginate Lyase with Hyperthermophilic Archaea Chaperonin in E. coli

Production of recombinant proteins from Plasmodium falciparum in Escherichia coli

Protein Interaction Module–assisted Function X (PIMAX) Approach to Producing Challenging Proteins Including Hyperphosphorylated Tau and Active CDK5/p25 Kinase Complex

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