Strategies to Optimize Protein Expression in <i>E. coli</i>

Francis, Dana M.; Page, Rebecca

doi:10.1002/0471140864.ps0524s61

Cited by 174 publications

(145 citation statements)

References 188 publications

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“…Expression of soluble proteins can be regulated through many factors that the host cell normally use in controlling of toxic protein expression [6,8,[12][13][14] .…”

Section: Tight Control Of the E Coli Cellular Milieumentioning

confidence: 99%

“…It also has very strong reversible binding attributes allowing for rapid single-step purification. Polyhistidine tags can be attached on either the N-or C-termini of recombinant proteins, but the optimal location depends on the folding and biochemical characteristics of the adjacent recombinant protein [14,20,26,27] .…”

Section: Refolding Of Solubilized and Unfolded Proteinsmentioning

confidence: 99%

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Strategies for production of active eukaryotic proteins in bacterial expression system

Khow

Suntrarachun

2012

Asian Pacific Journal of Tropical Biomedicine

113

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“…Expression of soluble proteins can be regulated through many factors that the host cell normally use in controlling of toxic protein expression [6,8,[12][13][14] .…”

Section: Tight Control Of the E Coli Cellular Milieumentioning

confidence: 99%

Section: Refolding Of Solubilized and Unfolded Proteinsmentioning

confidence: 99%

Strategies for production of active eukaryotic proteins in bacterial expression system

Khow

Suntrarachun

2012

Asian Pacific Journal of Tropical Biomedicine

113

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“…Failure to achieve native folding in a timely manner can prompt the proteins to aggregate before folding (Esposito andChatterjee 2006, Francis andPage 2010). 116 Various methods have been used to maximise production of soluble recombinant protein in E. coli.…”

Section: Results Inmentioning

confidence: 99%

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

Anggraeni¹

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A promising alternative to replace the current egg-or cell culture-based technology for vaccine production from live viruses is virus-like particle (VLP) technology based on a microbial platform. VLPs are macromolecular assemblies of viral capsid proteins, which have been shown to tolerate insertion of antigen modules via genetic recombinant technology, yielding modular VLPs. Many studies on modular VLPs presume that when a peptide antigen element is taken out from the intact proteins and then modularised on VLPs, it is unable to fold into its native structure. However, until now, presentation of a peptide antigen element on a VLP and the impact of the display strategy to present the antigen element on the quality of the resulting antibodies (i.e. the ability of the antibodies to recognise the intact protein) are not fully understood. This thesis aims to understand the underlying fundamentals regarding modularisation of peptide antigen elements on VLPs for induction of high-quality antibodies. A hypervariable receptor-binding domain, Helix 190 (H190), from the hemagglutinin protein of influenza A virus was used as a model for modularisation on VLPs from murine polyomavirus (MuPyV) VP1 protein. Four major findings are presented. Firstly, two display strategies, i.e. arraying of H190 in tandem repeats and the use of helix promoter elements, were shown to display H190 in its immunogenic form equally. However, modularisation using tandem repeat display induced antibodies of a higher quality than modularisation using helix promoter elements.Secondly, the quality of antibodies induced by the tandem repeat display bearing two copies of H190 was optimum, thus no significant improvement was observed following the use of adjuvant or increasing the copy number of H190. Additionally, the increase in the copy number of H190 was shown to reduce the assembly capability and solubility of modular VP1 in an environment that was optimised for wild-type VP1. Thirdly, this thesis shows the novel finding in the use of flanking ionic elements to stabilise VLP precursors, termed as capsomeres, bearing two copies of H190 containing a hydrophobic stretch, which caused aggregation. Fourthly, the first steps towards obtaining the atomic crystal structure of presented H190 on a modular protein were performed, i.e. a mild and satisfactory laboratory process was developed to achieve high-purity modular VP1 capsomeres, unattainable using previously established expression and purification process of wild-type MuPyV VP1. This thesis shows a step forward towards understanding the presentation of a peptide antigen element on a VLP that enables induction of highquality antibodies, and towards VLP engineering to manipulate the aggregation and solubility of modular VP1. VLP technology based on a microbial platform presented here is iii a potentially safe and effective alternative vaccine candidate that targets a hypervariable peptide antigen element. The speed of the microbial platform allows a rapid response to the hypervariability of the pe...

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“…Thus, after bioinformatics evaluations, we designed a truncated fragment of Pta passenger domain (amino acid residues 207-730) which implied a conserved, stable and cell-surface-exposed fragment which was expressed and purified consequently. On the other hand, we showed that the truncated Pta was expressed robustly in pET28a-BL21 [23,24] and could be purified with high quality and concentration (2 mg/ml). By comparing the yield of the purified truncated form of Pta in this study with its full length yield, achieved by Alamuri et al [8] (1 mg/ml), we could find the advantage of truncation of Pta protein in the yield of purification as compared to the full length of Pta.…”

Section: Discussionmentioning

confidence: 98%

Bioinformatics analysis and expression of a truncated form of Proteus mirabilis Pta protein as a novel vaccine target against urinary tract infection

Choubini

Karam

Kazemi

et al. 2016

vacres

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Introduction: Pathogenic strains of Proteus mirabilis have important roles in urinary tract infection. Proteus toxic agglutinin (Pta) is amongst the most important virulence factors of P. mirabilis. This protein has a conserved sequence present in all the strains which could be evaluated as a novel vaccine target against them. The aims of the current study were the expression, purification and characterization of a truncated Pta protein of P. mirabilis strain HI4320 as well as the bioinformatics analysis of the truncated protein. Methods: The passenger domain of pta genes in P. mirabilis was evaluated by bioinformatics studies. The selected domain (residues 207-730) was amplified by PCR and cloned into pET28a expression vector. The Pta was expressed in BL21 (DE3) host and purified by Ni-NTA resin. The analyses of the purified protein were performed by SDS-PAGE and Western blotting. Results: The bioinformatics studies predicted the appropriateness of the passenger domain of Pta protein in terms of conservation, stability and cell-surface exposure. The length of PCR fragment of truncated form of pta gene was ~1500 bp. The cloning and expression of the truncated pta gene was successfully performed using pET28a-BL21 (DE3) system. Analyses of the purified Pta by SDS-PAGE and Western blotting confirmed the purification of a ~60 kDa His-tagged polypeptide. Conclusion: The high frequency of P. mirabilis infection, especially in patients with abnormalities in their urinary tracts and also the rising of antibiotic resistance among the strains of this pathogen point to the need for effective controlling measures against them. In this regard, the passenger domain of Pta could be considered as a vaccine target. The efficacy and in-vivo immunogenicity of this purified protein is currently under study.

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Strategies to Optimize Protein Expression in E. coli

Cited by 174 publications

References 188 publications

Strategies for production of active eukaryotic proteins in bacterial expression system

Strategies for production of active eukaryotic proteins in bacterial expression system

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

Bioinformatics analysis and expression of a truncated form of Proteus mirabilis Pta protein as a novel vaccine target against urinary tract infection

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