Use of a design of experiments approach to optimise production of a recombinant antibody fragment in the periplasm of Escherichia coli: Selection of signal peptide and optimal growth conditions
Open accessAbstract
Production of recombinant proteins such as antibody fragments in the periplasm of the bacterium Escherichia coli has a number of advantages, including the ability to form disulphide bonds, aiding correct folding, and the relative ease of release and subsequent capture and purification. In this study, we employed two N-terminal signal peptides, PelB and DsbA, to direct a recombinant scFv antibody (single-chain variable fragment), 13R4, to the periplasm via the Sec and SRP pathways respectively. A design of experiments (DoE) approach was used to optimise process conditions (temperature, inducer concentration and induction point) influencing bacterial physiology and the productivity, solubility and location of scFv. The DoE study indicated that titre and subcellular location of the scFv depend on the temperature and inducer concentration employed, and also revealed the superiority of the PelB signal peptide over the DsbA signal peptide in terms of scFv solubility and cell physiology. Baffled shake flasks were subsequently used to optimise scFv production at higher biomass concentrations. Conditions that minimised stress (low temperature) were shown to be beneficial to production of periplasmic scFv. This study highlights the importance of signal peptide selection and process optimisation for the production of scFv antibodies, and demonstrates the utility of DoE for selection of optimal process parameters.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The pelB signal sequence resulted largely superior over the DsbA signal peptide in terms of scFv solubility and cell physiology.
“The pelB signal sequence resulted largely superior over the DsbA signal peptide in terms of scFv solubility and cell physiology [67].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [67]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
Protein export through the bacterial Sec pathway
Tsirigotaki, A., De Geyter, J., Šoštaric´, N., Economou, A., & Karamanou, S. (2017) · Nature Reviews Microbiology · doi:10.1038/nrmicro.2016.161 · Cited by 447 · see citing works
Flagged first, then by citations
Research works printed but not linked: 39
- Beacham IR Periplasmic enzymes in gram-negative bacteria Int J Biochem 1979 10 877 883 10.1016/0020-711X(79)90117-4 389690 · DOI
- Bürk J Weiche B Wenk M Boy D Nestel S Heimrich B Koch HG Depletion of the signal recognition particle receptor inactivates ribosomes in Escherichia coli J Bacteriol 2009 191 7017 7026 10.1128/JB.00208-09 19749044 PMC2772476 · DOI
- de Marco A Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli Microb Cell Fact 2009 8 26 10.1186/1475-2859-8-26 19442264 PMC2689190 · DOI
- Du Plessis DJF Nouwen N Driessen AJM The sec translocase Biochim Biophys Acta Biomembr 2011 1808 851 865 10.1016/j.bbamem.2010.08.016 20801097 · DOI
- Fluman N Navon S Bibi E Pilpel Y mRNA-programmed translation pauses in the targeting of E. coli membrane proteins Elife 2014 3 1 19 10.7554/eLife.03440 PMC4359368 25135940 · DOI
- Freudl R Signal peptides for recombinant protein secretion in bacterial expression systems Microb Cell Fact 2018 17 52 10.1186/s12934-018-0901-3 29598818 PMC5875014 · DOI · Paper page
- Guzman LM Belin D Carson MJ Beckwith J Tight regulation, modulation, and high-level expression by vectors containing the arabinose P(BAD) promoter J Bacteriol 1995 177 4121 4130 10.1128/jb.177.14.4121-4130.1995 7608087 PMC177145 · DOI
- Heggeset TMB Kucharova V Naerdal I Valla S Sletta H Ellingsen TE Brautaset T Combinatorial mutagenesis and selection of improved signal sequences and their application for high-level production of translocated heterologous proteins in Escherichia coli Appl Environ Microbiol 2013 79 559 568 10.1128/AEM.02407-12 23144128 PMC3553763 · DOI
- Hoffmann D Ebrahimi M Gerlach D Salzig D Czermak P Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins Crit Rev Biotechnol 2018 38 729 744 10.1080/07388551.2017.1398134 29124949 · DOI
- Hsu C-C Thomas ORT Overton TW Periplasmic expression in and release of Fab fragments from Escherichia coli using stress minimization J Chem Technol Biotechnol 2016 91 815 822 10.1002/jctb.4672 · DOI
- Inaba K Disulfide bond formation system in Escherichia coli J Biochem 2009 146 591 597 10.1093/jb/mvp102 19567379 · DOI
- Katsui N Tsuchido T Hiramatsu R Fujikawa S Takano M Shibasaki I Heat-induced blebbing and vesiculation of the outer membrane of Escherichia coli J Bacteriol 1982 151 1523 1531 7050091 PMC220434
- Kraemer J Harris C Afshar A Ruder LR Davies R Thomas ORT Dafforn TR Styrene maleic acid copolymers mediate selective release of protein targets from the periplasm of E. coli New Biotechnol 2016 33S S50 10.1016/j.nbt.2016.06.899 · DOI
- Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 1970 227 680 685 10.1038/227680a0 5432063 · DOI · Paper page
- Lei SP Lin HC Wang SS Callaway J Wilcox G Characterization of the Erwinia carotovora pelB gene and its product pectate lyase J Bacteriol 1987 169 4379 4383 10.1128/jb.169.9.4379-4383.1987 3040692 PMC213756 · DOI · Paper page
- Lorenz R Bernhart SH zu Siederdissen CH Tafer H Flamm C Stadler PF Hofacker IL ViennaRNA package 2.0 Algorithms Mol Biol 2011 6 26 10.1186/1748-7188-6-26 22115189 PMC3319429 · DOI · Paper page
- Martineau P Jones P Winter G Expression of an antibody fragment at high levels in the bacterial cytoplasm J Mol Biol 1998 280 117 127 10.1006/jmbi.1998.1840 9653035 · DOI
- Meyer AS Baker TA Proteolysis in the Escherichia coli heat shock response: a player at many levels Curr Opin Microbiol 2011 14 194 199 10.1016/j.mib.2011.02.001 21353626 PMC3118458 · DOI
- Naglak TJ Wang HY Recovery of a foreign protein from the periplasm of Escherichia coli by chemical permeabilization Enzyme Microb Technol 1990 12 603 611 10.1016/0141-0229(90)90134-C 1366783 · DOI
- Natale P Brüser T Driessen AJM Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane—distinct translocases and mechanisms Biochim Biophys Acta Biomembr 2008 1778 1735 1756 10.1016/j.bbamem.2007.07.015 17935691 · DOI
- Nebe-Von-Caron G Stephens PJ Hewitt CJ Powell JR Badley RA Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting J Microbiol Methods 2000 42 97 114 10.1016/S0167-7012(00)00181-0 11000436 · DOI
- Nelson AL Antibody fragments: hope and hype MAbs 2010 2 77 83 10.4161/mabs.2.1.10786 20093855 PMC2828581 · DOI
- Neu HC Heppel LA The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts J Biol Chem 1965 240 3685 3692 4284300
- Ng DTW Sarkar CA Engineering signal peptides for enhanced protein secretion from Lactococcus lactis Appl Environ Microbiol 2013 79 347 356 10.1128/AEM.02667-12 23124224 PMC3536070 · DOI
- Overton TW Recombinant protein production in bacterial hosts Drug Discov Today 2014 19 590 601 10.1016/j.drudis.2013.11.008 24246684 · DOI
- Plückthun A Skerra A Expression of functional antibody Fv and Fab fragments in Escherichia coli Methods Enzymol 1989 178 497 515 10.1016/0076-6879(89)78036-8 2689841 · DOI
- Running JA Bansal K Oxygen transfer rates in shaken culture vessels from Fernbach flasks to microtiter plates Biotechnol Bioeng 2016 113 1729 1735 10.1002/bit.25938 26806816 · DOI
- Sanchez-Garcia L Martín L Mangues R Ferrer-Miralles N Vázquez E Villaverde A Recombinant pharmaceuticals from microbial cells: a 2015 update Microb Cell Fact 2016 15 33 10.1186/s12934-016-0437-3 26861699 PMC4748523 · DOI
- Schierle CF Berkmen M Huber D Kumamoto C Boyd D Beckwith J The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway J Bacteriol 2003 185 5706 5713 10.1128/JB.185.19.5706-5713.2003 13129941 PMC193964 · DOI
- Schlegel S Rujas E Ytterberg AJ Zubarev RA Luirink J de Gier JW Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels Microb Cell Fact 2013 12 24 10.1186/1475-2859-12-24 23497240 PMC3605120 · DOI
- Schneider CA Rasband WS Eliceiri KW NIH Image to ImageJ: 25 years of image analysis Nat Methods 2012 9 671 675 10.1038/nmeth.2089 22930834 PMC5554542 · DOI · Paper page
- Selas Castiñeiras T Williams SG Hitchcock A Cole JA Smith DC Overton TW Development of a generic β-lactamase screening system for improved signal peptides for periplasmic targeting of recombinant proteins in Escherichia coli Sci Rep 2018 8 6986 10.1038/s41598-018-25192-3 29725125 PMC5934370 · DOI · Paper page
- Selas Castiñeiras T Williams SG Hitchcock A Cole JA Smith DC Overton TW Optimizing host cell physiology and stress avoidance for the production of recombinant human tumour necrosis factor α in Escherichia coli Microbiology 2018 164 440 452 10.1099/mic.0.000622 29458685 · DOI
- Sevastsyanovich Y Alfasi S Overton T Hall R Jones J Hewitt C Cole J Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins FEMS Microbiol Lett 2009 299 86 94 10.1111/j.1574-6968.2009.01738.x 19686347 · DOI
- Vera A González-Montalbán N Arís A Villaverde A The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures Biotechnol Bioeng 2007 96 1101 1106 10.1002/bit.21218 17013944 · DOI
- Villaverde A Carrió MM Protein aggregation in recombinant bacteria: biological role of inclusion bodies Biotechnol Lett 2003 25 1385 1395 10.1023/A:1025024104862 14514038 · DOI
- Walsh G Biopharmaceutical benchmarks 2014 Nat Biotechnol 2014 32 992 1000 10.1038/nbt.3040 25299917 · DOI · Paper page
- Wickström D Wagner S Baars L Ytterberg AJ Klepsch M Van Wijk KJ Luirink J De Gier JW Consequences of depletion of the signal recognition particle in Escherichia coli J Biol Chem 2011 286 4598 4609 10.1074/jbc.M109.081935 20923772 PMC3039323 · DOI
- Wyre C Overton TW Use of a stress-minimisation paradigm in high cell density fed-batch Escherichia coli fermentations to optimise recombinant protein production J Ind Microbiol Biotechnol 2014 41 1391 1404 10.1007/s10295-014-1489-1 25056840 · DOI · Paper page
Cited by
Sorted by citations
Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments
Sandomenico, A., Sivaccumar, J. P., & Ruvo, M. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21176324 · Cited by 134 · see citing works
“The pelB signal sequence resulted largely superior over the DsbA signal peptide in terms of scFv solubility and cell physiology [67].”
BackgroundMethodInfluential
- Authors
- Kasli, I. M., Thomas, O. R. T., & Overton, T. W.
- Affiliations
- University of Birmingham (GB)
- Corresponding author
- Overton, Tim W.
- Venue
- AMB Express
- Published
- 2019-01-07
- Volume
- 9
- Issue
- 1
- Pages
- 5-5
- ISSN
- 2191-0855 (ISSN-L)
- PMC
- PMC6323050
- OpenAlex
- W2917769110
- Cited by
- 31
- References
- 1
- Citations by year
- 2026: 4 · 2025: 5 · 2024: 5 · 2023: 2 · 2022: 2 · 2021: 5 · 2020: 5 · 2019: 3
- FWCI
- 1.24
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- AMB Express (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- 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)
- University of Birmingham Research Portal (University of Birmingham) (submittedVersion, other-oa)
- Funders
- Biotechnology and Biological Sciences Research Council (PhD studentship)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Keywords
- recombinant antibody fragments, scFv, Escherichia coli, DsbA, Sec pathway, process optimization, temperature, inducer concentration, protein solubility, disulfide bond formation
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
