2018
DOI: 10.1038/s41598-018-25192-3
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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

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 transloc… Show more

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Cited by 27 publications
(32 citation statements)
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“…We were able to screen these TIR LIBRARIES as we fused signal peptides to the β-lactamase protein, which confers resistance to β-lactam antibiotics. For proteins where no simple screening assay is available it is possible to directly couple [18], or translationally-couple β-lactamase to the recombinant protein [40][41][42]. It is also possible to use the pET28a-based TIRs EVOLVED , which we identified in this study and which improved production yields of a single chain antibody fragment, a hormone and another recombinant protein in Escherichia coli.…”
Section: Discussionmentioning
confidence: 95%
See 2 more Smart Citations
“…We were able to screen these TIR LIBRARIES as we fused signal peptides to the β-lactamase protein, which confers resistance to β-lactam antibiotics. For proteins where no simple screening assay is available it is possible to directly couple [18], or translationally-couple β-lactamase to the recombinant protein [40][41][42]. It is also possible to use the pET28a-based TIRs EVOLVED , which we identified in this study and which improved production yields of a single chain antibody fragment, a hormone and another recombinant protein in Escherichia coli.…”
Section: Discussionmentioning
confidence: 95%
“…Signal peptides have unpredictable effects on the production yields of recombinant proteins. For example, a signal peptide that supports a high-level of protein synthesis and secretion for one recombinant protein often supports a low-level of protein synthesis and secretion for another [3,4,18]. Herein we refer to this phenomenon as signal peptide performance.…”
Section: Introductionmentioning
confidence: 99%
See 1 more Smart Citation
“…It employs a novel polymerase with increased processivity, allowing efficient read through consecutive base-pair mismatches. EpPCR has been successfully adopted for library generation in various platforms, including phage [219], E. coli [220], and ribosome [221] display.…”
Section: Random Mutagenesismentioning
confidence: 99%
“…In this way RP is translated more slowly enabling in turn more efficient folding and therefore a higher proportion of soluble, functional RP (Sevastsyanovich et al 2009). Stress minimisation approaches have been successfully used to optimise production of cytoplasmic model proteins (Sevastsyanovich et al 2009; Wyre and Overton 2014), cytoplasmic biotherapeutics (Selas Castiñeiras et al 2018b) and periplasmically-targeted antibody fragments (Hsu et al 2016; Selas Castiñeiras et al 2018a) in fed-batch fermentation processes.…”
Section: Introductionmentioning
confidence: 99%