ArticleApplied and environmental microbiology2013
Combinatorial mutagenesis and selection of improved signal sequences and their application for high-level production of translocated heterologous proteins in Escherichia coli.
Article in Applied and environmental microbiology, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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17 citing papers in PubMed, 26 citations in OpenAlex.
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- Advancements in Escherichia coli secretion systems for enhanced recombinant protein production.World journal of microbiology & biotechnology · 2025Review
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- Barcoded bulk QTL mapping reveals highly polygenic and epistatic architecture of complex traits in yeast.eLife · 2022Article
- Genome-wide high-throughput signal peptide screening via plasmid pUC256E improves protease secretion in Lactiplantibacillus plantarum and Pediococcus acidilactici.BMC genomics · 2022Article
- Dual UTR-A novel 5' untranslated region design for synthetic biology applications.Synthetic biology (Oxford, England) · 2020Article
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- Development of a generic β-lactamase screening system for improved signal peptides for periplasmic targeting of recombinant proteins in Escherichia coli.Scientific reports · 2018Article
- Site-saturation mutagenesis of mutant L-asparaginase II signal peptide hydrophobic region for improved excretion of cyclodextrin glucanotransferase.Journal of industrial microbiology & biotechnology · 2017Article
- The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering.Microbial biotechnology · 2017Review
- Tracking Proteins Secreted by Bacteria: What's in the Toolbox?Frontiers in cellular and infection microbiology · 2017Review
- A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity.Microbial cell factories · 2016Article
- In vitro prediction of the evolution of GES-1 β-lactamase hydrolytic activity.Antimicrobial agents and chemotherapy · 2015Article
- Engineering cells to improve protein expression.Current opinion in structural biology · 2014Review
- Recombinant polypeptide production in E. coli: towards a rational approach to improve the yields of functional proteins.Microbial cell factories · 2013Review
- Design and optimization of short DNA sequences that can be used as 5' fusion partners for high-level expression of heterologous genes in Escherichia coli.Applied and environmental microbiology · 2013Article
- Article
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Authors and funding
7 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We previously designed the consensus signal peptide (CSP) and demonstrated that it can be used to strongly stimulate heterologous protein production in Escherichia coli. A comparative study using CSP and two bacterial signal sequences, pelB and ompA, showed that the effect of signal sequences on both expression level and translocation efficiency can be highly protein specific. We report here the generation of CSP mutant libraries by a combinatorial mutagenesis approach. Degenerated CSP oligonucleotides were cloned in frame with the 5' end of the bla gene, encoding the mature periplasmic β-lactamase released from its native signal sequence. This novel design allows for a direct selection of improved signal sequences that positively affect the expression level and/or translocation efficiency of β-lactamase, based on the ampicillin tolerance level of the E. coli host cells. By using this strategy, 61 different CSP mutants with up to 8-fold-increased ampicillin tolerance level and up to 5.5-fold-increased β-lactamase expression level were isolated and characterized genetically. A subset of the CSP mutants was then tested with the alternative reporter gene phoA, encoding periplasmic alkaline phosphatase (AP), resulting in an up to 8-fold-increased production level of active AP protein in E. coli. Moreover, it was demonstrated that the CSP mutants can improve the production of the medically important human interferon α2b under high-cell-density cultivations. Our results show that there is a clear potential for improving bacterial signal sequences by using combinatorial mutagenesis, and bioinformatics analyses indicated that the beneficial mutations could not be rationally predicted.
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