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Cited by 32Bacterial Genetics and BiotechnologyProtein purification and stabilityFungal and yeast genetics research

Development of a generic β-lactamase screening system for improved signal peptides for periplasmic targeting of recombinant proteins in Escherichia coli

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Selas Castiñeiras, Tania, Williams, Steven G., Hitchcock, Antony, Cole, Jeffrey A., Smith, Daniel C., Overton, Tim W.·2018·Scientific Reports
DOI 10.1038/s41598-018-25192-3PMC PMC5934370
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Abstract

Targeting of recombinant proteins to the Escherichia coli periplasm is a desirable industrial processing tool to allow formation of disulphide bonds, aid folding and simplify recovery. Proteins are targeted across the inner membrane to the periplasm by an N-terminal signal peptide. The sequence of the signal peptide determines its functionality, but there is no method to predict signal peptide function for specific recombinant proteins, so multiple signal peptides must be screened for their ability to translocate each recombinant protein, limiting throughput. We present a screening system for optimising signal peptides for translocation of a single chain variable (scFv) antibody fragment employing TEM1 β-lactamase (Bla) as a C-terminal reporter of periplasmic localisation. The Pectobacterium carotovorum PelB signal peptide was selected as the starting point for a mutagenic screen. β-lactamase was fused to the C-terminal of scFv and β-lactamase activity was correlated against scFv translocation. Signal peptide libraries were generated and screened for β-lactamase activity, which correlated well to scFv::Bla production, although only some high activity clones had improved periplasmic translocation of scFv::Bla. Selected signal peptides were investigated in fed-batch fermentations for production and translocation of scFv::Bla and scFv without the Bla fusion. Improved signal peptides increased periplasmic scFv activity by ~40%.

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  • Two new peptides selected from signal peptide libraries improved the periplasmic production of active 13R4 by approximately 40% compared to expression obtained using wild type pelB.

    “According to the signal related to β-lactamase activities, two new peptides were selected from the libraries as improving the periplasmic production of active 13R4 by ~40% compared to the expression obtained using the wild type pelB [98].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [98]

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Authors
Selas Castiñeiras, T., Williams, S. G., Hitchcock, A., Cole, J. A., Smith, D. C., & Overton, T. W.
Affiliations
University of Birmingham (GB)
Corresponding author
Overton, Tim W.
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2018-05-03
Volume
8
Issue
1
Pages
6986-6986
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-018-25192-3
PMC
PMC5934370
OpenAlex
W2801113769
Cited by
32
References
43
Citations by year
2026: 1 · 2025: 3 · 2024: 2 · 2023: 6 · 2022: 5 · 2021: 7 · 2020: 4 · 2019: 4
FWCI
2.29
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • University of Birmingham Research Portal (University of Birmingham) (submittedVersion, cc-by)
Funders
  • Research Councils UK
  • Directorate for Biological Sciences
  • Biotechnology and Biological Sciences Research Council
  • Innovate UK
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
Keywords
periplasmic targeting, scFv antibody fragments, pelB signal peptide, Erwinia carotovora, Escherichia coli periplasm, protein translocation, fed-batch fermentation, recombinant protein expression
MeSH terms
beta-Lactamases, Escherichia coli, Genetic Testing, Polysaccharide-Lyases, Recombinant Fusion Proteins, Mutagenesis, Pectobacterium carotovorum, Genes, Reporter, Periplasm, Protein Transport, Protein Sorting Signals, Single-Chain Antibodies, Metabolic Engineering

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