Improving production of N-glycosylated recombinant proteins by leaky Escherichia coli

Ding, Ning; Ruan, Yao; Fu, Xin; Lin, Yue; Yu, Hongyou; Han, Lichi; Fu, Changzhen; Zhang, Jianing; Hu, Xuejun

doi:10.1007/s13205-019-1830-5

Cited by 8 publications

(3 citation statements)

References 29 publications

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“…Finally, di-N-diacetylbacillosamine is a prokaryotic monosaccharide not found in eukaryotes. Despite technologies to install N-glycosylation pathways in bacteria being in their nascent stages, proof-of-principle experiments ( Figure 7A ) coupled with continuing robust efforts to improve prokaryote glycosylation ( Ding et al, 2019 ; Wayman et al, 2019 ; Pratama et al, 2021 ; Yates et al, 2021 ) provide hope that in the future, additional therapeutic proteins will be manufactured in bacterial hosts.…”

Section: Considerations For the Design And Production Of Glycoenginee...mentioning

confidence: 99%

Strategies for Glycoengineering Therapeutic Proteins

et al. 2022

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Almost all therapeutic proteins are glycosylated, with the carbohydrate component playing a long-established, substantial role in the safety and pharmacokinetic properties of this dominant category of drugs. In the past few years and moving forward, glycosylation is increasingly being implicated in the pharmacodynamics and therapeutic efficacy of therapeutic proteins. This article provides illustrative examples of drugs that have already been improved through glycoengineering including cytokines exemplified by erythropoietin (EPO), enzymes (ectonucleotide pyrophosphatase 1, ENPP1), and IgG antibodies (e.g., afucosylated Gazyva®, Poteligeo®, Fasenra™, and Uplizna®). In the future, the deliberate modification of therapeutic protein glycosylation will become more prevalent as glycoengineering strategies, including sophisticated computer-aided tools for “building in” glycans sites, acceptance of a broad range of production systems with various glycosylation capabilities, and supplementation methods for introducing non-natural metabolites into glycosylation pathways further develop and become more accessible.

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Section: Considerations For the Design And Production Of Glycoenginee...mentioning

confidence: 99%

Strategies for Glycoengineering Therapeutic Proteins

et al. 2022

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“…Meanwhile, leaky expression—i.e., expression that occurs in the absence of proper induction—is another major concern in the use of the E. coli expression system. Whereas leaky expression can be optimized and is beneficial in some cases (e.g., production of toxic proteins, technical enzymes, and biopharmaceutical products) (Ding et al 2019 ), it can also reduce growth rates, cause cell death, or result in plasmid instability, which is an issue for large-scale industrial production (Saida et al 2006 ).…”

Section: Introductionmentioning

confidence: 99%

Omics-guided bacterial engineering of Escherichia coli ER2566 for recombinant protein expression

Zhou

Wang

et al. 2022

Appl Microbiol Biotechnol

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The goal of bacterial engineering is to rewire metabolic pathways to generate high-value molecules for various applications. However, the production of recombinant proteins is constrained by the complexity of the connections between cellular physiology and recombinant protein synthesis. Here, we used a rational and highly efficient approach to improve bacterial engineering. Based on the complete genome and annotation information of the Escherichia coli ER2566 strain, we compared the transcriptomic profiles of the strain under leaky expression and low temperature-induced stress. Combining the gene ontology (GO) enrichment terms and differentially expressed genes (DEGs) with higher expression, we selected and knocked out 36 genes to determine the potential impact of these genes on protein production. Deletion of bluF , cydA , mngR , and udp led to a significant decrease in soluble recombinant protein production. Moreover, at low-temperature induction, 4 DEGs ( gntK , flgH , flgK , flgL ) were associated with enhanced expression of the recombinant protein. Knocking out several motility-related DEGs (ER2666- ΔflgH-ΔflgL-ΔflgK ) simultaneously improved the protein yield by 1.5-fold at 24 °C induction, and the recombinant strain had the potential to be applied in the expression studies of different exogenous proteins, aiming to improve the yields of soluble form to varying degrees in comparison to the ER2566 strain. Totally, this study focused on the anabolic and stress-responsive hub genes of the adaptation of E. coli to recombinant protein overexpression on the transcriptome level and constructs a series of engineering strains increasing the soluble protein yield of recombinant proteins which lays a solid foundation for the engineering of bacterial strains for recombinant technological advances. Key points • Comparative transcriptome analysis shows host responses with altered induction stress . • Deletion of bluF, cydA, mngR, and udp genes was identified to significantly decrease the soluble recombinant protein productions . • Synchronal knockout of flagellar genes in E. coli can enhance recombinant protein yield up to ~ 1.5-fold at 24 °C induction . • Non-model bacterial strains can be re-engineered for recombinant protein expression . Supplementary Information The online version contains supplementary material available at 10.1007/s00253-022-12339-6.

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“…FN3 lacks cysteine residues, suggesting high-level expression in the cytoplasm of bacteria, such as Escherichia coli . In previous studies, we efficiently expressed monobodies and receptor proteins based on FN3 as a protein scaffold in E. coli ( Ding et al, 2019 ; Zhu et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens

Ruan

Chao

et al. 2021

Front. Mol. Biosci.

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Construction of substitute antigens based on alternative scaffold proteins is a promising strategy in bioassay technology. In this study, we proposed a strategy for constructing substitute antigens derived from 10th human fibronectin type III (FN3) using two peptide epitopes of terminal pro-brain natriuretic peptide (NT-proBNP) as an example. The base sequences encoding the two antigenic epitopes of NT-proBNP were recombined into the FG loop region and the C-terminus of FN3, fused by 4 GS or polyN linker. The fusion proteins (named FN3-epitopes-4GS and FN3-epitopes-polyN, respectively) were expressed and purified cost-effectively using an Escherichia coli expression system. The immunoreactivity of recombinant substitutes was preliminarily confirmed by western blot analysis using epitope-specific antibodies. The sandwich enzyme-linked immunosorbent assay demonstrated that either FN3-epitopes-polyN or FN3-epitopes-4GS was highly sensitive, and FN3-epitopes-polyN exhibited better kinetics to specific antibodies than FN3-epitopes-4GS, showing a linear dose-response relationship in the concentration range of 0.06–12.85 ng/ml, which suggest that the polyN linker was more suitable for constructing the FN3-based substitute antigens compared to the 4 GS linker. Furthermore, the serum stability test and differential scanning calorimetry analysis showed that the recombinant FN3-epitopes-polyN maintained the original stability of FN3. Therefore, it was confirmed that FN3 could be engineered to construct a stable biomacromolecular substitute for displaying double epitopes of antigen proteins, such as NT-proBNP. In summary, a cost-effective strategy to produce NT-proBNP substitute antigens with good immunoreactivity and physicochemical stability was established in this work, which may provide potential uses for the production of other substitute antigens in the future.

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Improving production of N-glycosylated recombinant proteins by leaky Escherichia coli

Cited by 8 publications

References 29 publications

Strategies for Glycoengineering Therapeutic Proteins

Strategies for Glycoengineering Therapeutic Proteins

Omics-guided bacterial engineering of Escherichia coli ER2566 for recombinant protein expression

FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens

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