Enhancing recombinant protein yields in the E. coli periplasm by combining signal. Peptide and production rate screening
Open accessAbstract
, disulfide bond containing recombinant proteins are often targeted to the periplasm by an N-terminal signal peptide that is removed once it passes through the Sec-translocon in the cytoplasmic membrane. Despite their conserved targeting function, signal peptides can impact recombinant protein production yields in the periplasm, as can the production rate. Here, we present a combined screen involving different signal peptides and varying production rates that enabled the identification of more optimal conditions for periplasmic production of recombinant proteins with disulfide bonds. The data was generated from two targets, a single chain antibody fragment (BL1) and human growth hormone (hGH), with four different signal peptides and a titratable rhamnose promoter-based system that enables the tuning of protein production rates. Across the screen conditions, the yields for both targets significantly varied, and the optimal signal peptide and rhamnose concentration differed for each protein. Under the optimal conditions, the periplasmic BL1 and hGH were properly folded and active. Our study underpins the importance of combinatorial screening approaches for addressing the requirements associated with the production of a recombinant protein in the periplasm.
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Karyolaimos and coworkers obtained a yield of around 0.2 g/L of a functional scFv fragment in the periplasm using the OmpA signal peptide and 100 μM rhamnose as inductor.
“Karyolaimos and coworkers have obtained a yield of around 0.2 g/L of a functional scFv fragment in the periplasm using the OmpA signal peptide and 100 μM rhamnose as inductor [68].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [68]
Combinatorial screening of different signal peptides in a titratable system that tunes protein production rates was a valuable and effective approach to enhance scFv recombinant production yields in the periplasm of E. coli.
“Karyolaimos and coworkers [68] have successfully demonstrated that a combinatorial screening of different signal peptides in a titratable system that tunes protein production rates was a valuable and effective approach to enhance scFv recombinant production yields in the periplasm of E. coli.”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [68]
The highest periplasmic production yield of an scFv was 0.2 g/L of culture, achieved using the OmpA signal peptide, LB medium and induction with 100 μM rhamnose at 30 °C in a shake flask.
“The highest periplasmic production yield of an scFv was 0.2 g/L of culture and was achieved using the OmpA signal peptide, LB medium and induction with 100 μM rhamnose at 30 °C in a shake flask [68].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [68]
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“Escherichia coli is a widely used expression platform because of its well-studied genetic background, short generation cycle, and cost-effective culture methods.([17,18]) In general, prokaryotic expression vectors contain the T7 promoter require bacteriophage T7 RNA polymerase for the transcription of target genes, wh…”
BackgroundA quantitative interpretation of oxidative protein folding activity in Escherichia coli
Rettenbacher, L. A., & von der Haar, T. (2022) · Microbial Cell Factories · doi:10.1186/s12934-022-01982-3 · Cited by 2 · see citing works
“investigated how different recombinant protein production rates affected steady-state expression levels of secreted single chain antibody (scFv) and human growth hormone (hGH), two recombinant proteins with differing disulfide bond patterns [25].”
BackgroundTang, D., Gunson, J., Tran, E., Lam, C., Shen, A., Snedecor, B., Barnard, G. C., & Misaghi, S. (2022) · Biotechnology progress (Print) · doi:10.1002/btpr.3290 · Cited by 2
Anshori, I., Nugroho, A. E., Solihin, A., Jessika, Yusuf, M., Hartati, Y., Sari, S. P., Tohari, T. R., Yuliarto, B., Gumilar, G., et al. (2021) · Italian National Conference on Sensors · doi:10.1109/sensors47087.2021.9639841 · Cited by 2
“Normally, the suspension of scFv has impurity agents, including the native proteins [31].”
BackgroundRecombinant Fab and scFv fragments of Tocilizumab: Microbial production and functional insights
Das, P., Panja, P., Veeranki, V. D., & Naidu, V. M. (2026) · Chemical Engineering Journal · doi:10.1016/j.cej.2026.172991 · Cited by 1 · see citing works
Deep learning guided mutagenesis of signal peptide in Escherichia coli
Hu, R.-E., Liu, H.-J., Chen, H.-C., & Ng, I.-S. (2025) · Process Biochemistry · doi:10.1016/j.procbio.2025.09.011 · Cited by 1
Exploit and elucidate chaperone assisted PET hydrolase for upcycling plastics
Ting, W.-W., Yu, J., Hsiang, C.-C., Tan, S.-I., Chang, C.-C., Huang, H.-L., Yu, C.-H., Hu, R.-E., Ma, H.-N., & Ng, I.-S. (2024) · Chemical Engineering Journal · doi:10.1016/j.cej.2024.157777 · Cited by 1 · see citing works
An ExbD Disordered Domain Peptide Inhibits TonB System ActivityPreprint
Kopp, D. R., & Postle, K. (2020) · bioRxiv · doi:10.1101/2020.01.05.895219 · Cited by 1 · see citing works
A biosensor toolbox for monitoring recombinant protein production in Escherichia coli
Mürköster, M., Bałka, M., Kamp, C. J., Källström, J., Karlsson, L., Aparicio, C. A. R., Tunäng, E., Ögren, H.-L., Theopold, S., Cumming, A. J., et al. (2026) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2026.1956994
“…simple approach is to optimize induction conditions, such as inducer concentration, temperature, and growth rate, to ensure that the rate of recombinant protein synthesis does not exceed the cellular capacity for protein folding and quality control (Karyolaimos et al., 2019; Horga et al., 2018).”
A self-assembled and covalently stabilized FcRn-binding albumin fragment-payload assembly.
Cho, Y. J., Lee, G.-J., Park, J.-H., Lim, S.-J., Shin, S.-M., An, Y.-J., Shin, S. O., & Lim, S.-I. (2026) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2026.154984
Engineering PET surface colonization with a synthetic bacterial consortium
Arab, B., Wortman, J., Zhao, B.-X., Chou, C. P., & Liu, Y.-L. (2026) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2026.1951515
Guo, Z.-T., Ye, K., Shi, Z.-Y., Li, J.-Y., Zhang, S.-Y., Li, B.-L., & Liu, F. (2026) · Comprehensive Reviews in Food Science and Food Safety · doi:10.1111/1541-4337.70618
Siavashi, S., Kojouri, P. S., Aboutalebi, F., & Dormiani, K. (2026) · World Journal of Microbiology & Biotechnology · doi:10.1007/s11274-026-05076-9
“…using periplasmic expression to facilitate oxidative folding and introduce correct disulfide bonds into target proteins, and applying solubility tags to enhance protein solubility can help overcome these complications (Upad-hyay et al. 2012 ; Karyolaimos et al. 2019 ; Emamipour et al. 2019 ).”
Kang, Y.-H., Ryu, Y.-K., Lee, J., Jo, E., Gang, Y., Kim, T., Park, G.-H., & Oh, C. (2026) · Microbial Cell Factories · doi:10.1186/s12934-026-02961-8
Kusuma, S., Fatinah, Y., Fadhlillah, M., Parwati, I., Rukayadi, Y., & Subroto, T. (2025) · Preparative Biochemistry & Biotechnology · doi:10.1080/10826068.2025.2551369
“[47,48] In this study, optimal secretion of recombinant MPT64 was achieved at 4 mM rhamnose.”
Pourani, Z., Keramati, M., Komijani, S., Golkar, M., Cohan, R. A., Mohseni, N., & Valizadeh, V. (2024) · Protein Expression and Purification · doi:10.1016/j.pep.2024.106618
Lénon, M., Ke, N., Szady, C., Sakhtah, H., Ren, G.-P., Manta, B., Causey, B., & Berkmen, M. (2020) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-020-10920-5
“Yet, periplasmic expression requires extensive optimization of conditions (Baumgarten et al. 2018) to avoid secretion blockage (Schlegel et al. 2012) and optimization of targeting signal peptides (Karyolaimos et al. 2019; Mirzadeh et al. 2020).”
- Authors
- Karyolaimos, A., Ampah-Korsah, H., Hillenaar, T., Mestre Borras, A., Dolata, K. M., Sievers, S., Riedel, K., Daniels, R., & de Gier, J.-W.
- Affiliations
- Stockholm University (SE)
- Universität Greifswald (DE)
- ORCID
- Venue
- Frontiers in Microbiology
- Publisher
- Frontiers Media SA
- Published
- 2019-07-23
- Volume
- 10
- Pages
- 1511-1511
- ISSN
- 1664-302X (ISSN-L)
- PMC
- PMC6664373
- OpenAlex
- W2964298233
- Cited by
- 66
- References
- 45
- Citations by year
- 2026: 9 · 2025: 11 · 2024: 10 · 2023: 5 · 2022: 8 · 2021: 8 · 2020: 14 · 2019: 1
- FWCI
- 4.67
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Frontiers in Microbiology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Vetenskapsrådet (2015-05288, K2015-57-21980-04-4)
- Stiftelsen för Strategisk Forskning (642863)
- Horizon 2020 (642863)
- Carl Tryggers Stiftelse för Vetenskaplig Forskning (CTS17:111, CTS17:114)
- National Institutes of Health (HHSN272201400005C)
- 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
- recombinant protein expression, Escherichia coli periplasm, signal peptide, protein production rate, Sec translocon, single-chain antibody fragments, human growth hormone, protein folding, combinatorial screening, periplasmic targeting
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