Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli

Marco, Ario de

doi:10.1186/1475-2859-8-26

Cited by 308 publications

(211 citation statements)

References 296 publications

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“…However, expression in E. coli has several drawbacks such as formation of inclusion bodies, low secretion efficiency, absence of splicing machinery and inability to perform post-translational modifications such as glycosylation explaining why it is not successfully being used for expression of fungal β-glucosidase enzymes [196,197,198]. E.coli is usually transformed with self-replicated plasmid that does not integrate to chromosomal DNA and keeps on replicating itself independently from cell divisions [199,200].…”

Section: Cloning and Expression Of Microbial β-Glucosidasesmentioning

confidence: 99%

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

Ahmed¹,

Aslam²,

Ashraf³

et al. 2017

JAEM

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Cellulose is the most abundant biomaterial in the biosphere and the major component of plant biomass.Cellulase is an enzymatic system required for conversion of renewable cellulose biomass into free sugar for subsequent use in different applications. Cellulase system mainly consists of three individual enzymes namely: endoglucanase, exoglucanase and β-glucosidases. β-Glucosidases are ubiquitous enzymes found in all living organisms with great biological significance. β-Glucosidases have also tremendous biotechnological applications such as biofuel production, beverage industry, food industry, cassava detoxification and oligosaccharides synthesis. Microbial β-glucosidases are preferred for industrial uses because of robust activity and novel properties exhibited by them. This review aims at describing the various biochemical methods used for screening and evaluating β-glucosidases activity from microbial sources. Subsequently, it generally highlights techniques used for purification of β-glucosidases. It then elaborates various biochemical and molecular properties of this valuable enzyme such as pH and temperature optima, glucose tolerance, substrate specificity, molecular weight, and multiplicity. Furthermore, it describes molecular cloning and expression of bacterial, fungal and metagenomic β-glucosidases. Finally, it highlights the potential biotechnological applications of β-glucosidases.

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Section: Cloning and Expression Of Microbial β-Glucosidasesmentioning

confidence: 99%

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

Ahmed¹,

Aslam²,

Ashraf³

et al. 2017

JAEM

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“…Furthermore, general limitations of bacterial expression over mammalian expression (e.g., protein solubility, disulfide bonds, post-translational modifications) need to be considered for individual target proteins. For instance, our system would need to be adapted to enable the selection of proteins that require disulfide bonds for proper folding in bacterial cells 37 . …”

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confidence: 99%

Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

2017

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Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. these include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. a typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (pM) library expressing variants of the gene of interest (GoI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. the system has been developed for the selection of new orthogonal transcription factors (tFs) but is capable of evolving any gene-or gene circuit function-that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage l cI tF against two synthetic bidirectional promoters. the evolved tF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. this protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ~7 weeks.© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. the chances of propagating nonfunctional library members because of multiple infections. A GOI with the desired characteristics upregulates gene VI expression on the AP, completing the phage life cycle. For example, a randomized TF library member that activates an artificial promoter upstream of gVI will increase its own phage production (Fig. 4a). In this way, a protein with novel desired properties can be selected after several rounds of reinfection. Applications of the methodThe method has been used to evolve a set of dual activator-repressor switches for orthogonal logic gates, based on bacteriophage λ cI variants, and multi-input promoter architectures, and these switches have been successfully applied in downstream synthetic gene circuits 19 . In general, the method is capable of evolving any gene-or gene circuit function-on the PM that can be linked to pVI production. This is analogous to previous uses of phageassisted continuous evolution (PACE) 22 (Fig. 4). With PACE, a wide range of medically and biotechnologically relevant biomolecules, including polymerases 22 , proteases 23 and genome-editing proteins 10 , as well as protein-protein interactions 24 , were linked to conditional M13 phage propagation. In principle, any application in which directed evolution approaches have been...

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“…The failure of NGF production as a soluble and functional protein in E. coli is due to the absence of post-translational modifications required for disulfide-bond formation. Disulfide-bond formation occurs in the periplasm of E. coli, which is a more oxidizing compartment due to the presence of Dsb proteins including Dsb A, B, C, D and G (18). However, the cytoplasm of E. coli can be genetically engineered to provide a suitable situation for successful expression of proteins containing disulfide bonds.…”

Section: Discussionmentioning

confidence: 99%

“…Disulfide bonds mainly form in the periplasm of E. coli, which is a more oxidizing environment due to the presence of Dsb proteins including DsbA, B, C, D and G (18). The cytoplasm of E. coli can be genetically engineered to provide a suitable situation for successful expression of proteins containing disulfide bonds.…”

mentioning

confidence: 99%