Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity

Zhang, Zixu; Nong, Fang-Tong; Wang, Yu‐Zhou; Yan, Chun-Xiao; Gu, Yang; Song, Ping; Sun, Xiao‐Man

doi:10.1186/s12934-022-01917-y

Cited by 31 publications

(14 citation statements)

References 151 publications

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“…In this work, an increase in the IPTG concentration did not have an improved effect on enzyme expression, suggesting that 0.1 mM IPTG is a suitable concentration to induce expression. A possible explanation for this fact may include toxicity, saturation of the lac operon, or cell stress response promoted by high concentrations of IPTG, as previously reported (Dvorak et al 2015 ; Gomes et al 2020 ; Zhang et al 2022 ).…”

Section: Discussionmentioning

confidence: 67%

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library

Sousa,

Santos-Pereira,

Gomes

et al. 2024

Appl Microbiol Biotechnol

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Xylanases are key biocatalysts in the degradation of the β‐1,4‐glycosidic linkages in the xylan backbone of hemicellulose. These enzymes are potentially applied in a wide range of bioprocessing industries under harsh conditions. Metagenomics has emerged as powerful tools for the bioprospection and discovery of interesting bioactive molecules from extreme ecosystems with unique features, such as high temperatures. In this study, an innovative combination of function-driven screening of a compost metagenomic library and automatic extraction of halo areas with in-house MATLAB functions resulted in the identification of a promising clone with xylanase activity (LP4). The LP4 clone proved to be an effective xylanase producer under submerged fermentation conditions. Sequence and phylogenetic analyses revealed that the xylanase, Xyl4, corresponded to an endo-1,4-β-xylanase belonging to glycosyl hydrolase family 10 (GH10). When xyl4 was expressed in Escherichia coli BL21(DE3), the enzyme activity increased about 2-fold compared to the LP4 clone. To get insight on the interaction of the enzyme with the substrate and establish possible strategies to improve its activity, the structure of Xyl4 was predicted, refined, and docked with xylohexaose. Our data unveiled, for the first time, the relevance of the amino acids Glu133 and Glu238 for catalysis, and a close inspection of the catalytic site suggested that the replacement of Phe316 by a bulkier Trp may improve Xyl4 activity. Our current findings contribute to enhancing the catalytic performance of Xyl4 towards industrial applications. Key points • A GH10 endo-1,4-β-xylanase (Xyl4) was isolated from a compost metagenomic library • MATLAB’s in-house functions were developed to identify the xylanase-producing clones • Computational analysis showed that Glu133 and Glu238 are crucial residues for catalysis Graphical abstract

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Section: Discussionmentioning

confidence: 67%

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library

Sousa,

Santos-Pereira,

Gomes

et al. 2024

Appl Microbiol Biotechnol

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“…However, nanobodies expressed in the cytosol of E. coli face several problems including the formation of insoluble inclusion bodies, and the inability to form disulfide bonds correctly (17) . Both will ultimately lead to toxicity to the host cell (18) . As data from our lab showed several proteins could be secreted into the culture medium by fusing it with the pelB signal peptide (Figure S1 HdeA, Spy, Im7 secretion).…”

Section: Resultsmentioning

confidence: 99%

A method for rapid nanobody screening with no bias of the library diversity

Tao

Wang

et al. 2023

Preprint

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Nanobody refers to the variable domain of heavy-chain only antibodies. The distinctive advantages of nanobodies including small size, feasible expression in Escherichia coli (E. coli), and superior stability make them promising tools for applications in scientific research and therapies. So far, the screening and expression of nanobodies are mainly following similar methods used for conventional antibodies, suffering from amplification-caused losses of the diversity of libraries and requirements of subcloning of interests into the expression vector. Here, based on the unique properties of nanobodies, we developed a new high-throughput (>105) method to screen and express nanobodies simultaneously, simplifying the production of specific nanobodies significantly. Fusing pelB signal peptide on the N-terminus, functional nanobodies were secreted into the culture medium, facilitating the direct screening of nanobody libraries and the subsequent purification of the target nanobody. Nanobodies specifically binding with target proteins were enriched through cell splitting and regrown steps in a way following the Poisson distribution to ensure no positive clones were dismissed, while the population of positive clones increased by one order of magnitude upon each round of cell splitting. In the end, 5 different nanobodies against the death domain receptor 5 (DR5) and 5 different nanobodies against the Pyrococcus furiosus (Pfu) DNA polymerase were produced directly out of their immunized libraries respectively. Our approach offers an integrated strategy for rapid, and low-cost nanobody screening and expression with no loss of the library diversity, demonstrating general applicability in the routine production of monoclonal nanobodies for diverse biomedical applications.

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“…Studies have shown that this imbalance can be controlled by regulating heterologous gene expression by considering the copy number of a plasmid and the adjusting promoter strength (Lozano Terol et al 2021 ). pET-based expression plasmids were widely used for research and commercial production (Zhang et al 2022 ). This plasmid has a T7 promoter that drives the heterologous expression of recombinant proteins.…”

Section: Essential Components Of E Coli -Based Exp...mentioning

confidence: 99%

“…Several reasons such as lacking post-translational modifications (PTMs), misfolding, lower solubility capacity, and host metabolic burden are involved in creating IBs (Zhang et al 2022 ). One of the strategies to overcome the creation of IBs during the over-expression of recombinant proteins is the co-expression of the target proteins with the chaperone molecules.…”

Section: Molecular Chaperonesmentioning

confidence: 99%

Factors involved in heterologous expression of proteins in E. coli host

Pouresmaeil

Azizi-Dargahlou

2023

Arch Microbiol

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The production of recombinant proteins is one of the most significant achievements of biotechnology in the last century. These proteins are produced in the eukaryotic or prokaryotic heterologous hosts. By increasing the omics data especially related to different heterologous hosts as well as the presence of new amenable genetic engineering tools, we can artificially engineer heterologous hosts to produce recombinant proteins in sufficient quantities. Numerous recombinant proteins have been produced and applied in various industries, and the global recombinant proteins market size is expected to be cast to reach USD 2.4 billion by 2027. Therefore, identifying the weakness and strengths of heterologous hosts is critical to optimize the large-scale biosynthesis of recombinant proteins. E. coli is one of the popular hosts to produce recombinant proteins. Scientists reported some bottlenecks in this host, and due to the increasing demand for the production of recombinant proteins, there is an urgent need to improve this host. In this review, we first provide general information about the E. coli host and compare it with other hosts. In the next step, we describe the factors involved in the expression of the recombinant proteins in E. coli . Successful expression of recombinant proteins in E. coli requires a complete elucidation of these factors. Here, the characteristics of each factor will be fully described, and this information can help to improve the heterologous expression of recombinant proteins in E. coli .

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Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity

Cited by 31 publications

References 151 publications

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library

A method for rapid nanobody screening with no bias of the library diversity

Factors involved in heterologous expression of proteins in E. coli host

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