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Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems 2.0

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Microbiology".

Deadline for manuscript submissions: closed (30 April 2022) | Viewed by 9414

Special Issue Editors

Dr. Neus Ferrer-Miralles

E-Mail Website
Guest Editor
1. Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain
2. Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain
3. Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain
4. CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain
5. Plataforma de Producción de Proteínas, CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) and Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain
Interests: recombinant protein; protein engineering; nanobiotechnology; microbiology; protein nanoparticles; protein aggregation; functional amyloids; cell-targeted delivery
Special Issues, Collections and Topics in MDPI journals
Dr. Joaquin Seras-Franzoso

E-Mail Website
Co-Guest Editor
Drug Delivery and Targeting Group, Molecular Biology and Biochemistry Research Centre for Nanomedicine (CIBBIM-Nanomedicine), Vall d'Hebron Institut de Recerca, Universitat Autònoma de Barcelona, Barcelona, Spain
Interests: drug delivery; cancer stem cells; bacterial amyloids; bacterial inclusion bodies; protein products

Special Issue Information

Dear Colleagues,

Many proteins of interest are produced in recombinant prokaryotic or eukaryotic expression systems. Among the prokaryotic expression systems, bacterial hosts are widely used for the production of recombinant proteins. Under overexpression conditions, the overproduced heterologous protein in recombinant bacteria can partition into two separate fractions: soluble and insoluble (also known as inclusion bodies). Often, the proteins of interest are difficult to express and, as a consequence, the final yields are unacceptable. In other cases, the proteins are prone to aggregation, making it challenging or impossible to obtain protein from the soluble fraction. In light of these issues, much of the research effort during the last few decades has gone towards the development of strategies to increase the efficiency of the production process for those difficult-to-obtain proteins. Thus, this Special Issue of IJMS will cover recent research activity towards the development of novel strategies used to obtain optimal yields of difficult-to-produce heterologous proteins which use bacterial expression systems as cell factories.

Dr. Neus Ferrer-Miralles
Guest Editor
Dr. Joaquin Seras-Franzoso
Co-Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • recombinant proteins
  • heterologous proteins
  • difficult-to-produce proteins
  • bacterial expression systems
  • strategies for protein production
  • strategies for protein purification

Published Papers (4 papers)

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Research

15 pages, 3914 KiB  
Article
Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH–DR5-B with Dual VEGFR2 and DR5 Receptor Specificity
by Anne V. Yagolovich, Artem A. Artykov, Alina A. Isakova, Yekaterina V. Vorontsova, Dmitry A. Dolgikh, Mikhail P. Kirpichnikov and Marine E. Gasparian
Int. J. Mol. Sci. 2022, 23(11), 5860; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23115860 - 24 May 2022
Cited by 3 | Viewed by 1872
Abstract
In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. [...] Read more.
In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. To enhance the efficacy of TRAIL, we designed the HRH–DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH. Initially low expression of HRH–DR5-B was enhanced by the substitution of E. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. However, the HRH–DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. The replacement of His by Ser at position P2 immediately after the initiator Met dramatically minimized degradation, allowing more than 20 mg of protein to be obtained from 200 mL of cell culture. The resulting SRH–DR5-B fusion bound the VEGFR2 and DR5 receptors with high affinity and showed increased cytotoxic activity in 3D multicellular tumor spheroids. SRH–DR5-B can be considered as a promising candidate for therapeutic applications. Full article
(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems 2.0)
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21 pages, 6159 KiB  
Article
Optimised Heterologous Expression and Functional Analysis of the Yersinia pestis F1-Capsular Antigen Regulator Caf1R
by Dharmender K. Gahlot, Gyles Ifill and Sheila MacIntyre
Int. J. Mol. Sci. 2021, 22(18), 9805; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22189805 - 10 Sep 2021
Cited by 3 | Viewed by 2538
Abstract
The bacterial pathogen, Yersinia pestis, has caused three historic pandemics and continues to cause small outbreaks worldwide. During infection, Y. pestis assembles a capsule-like protective coat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to [...] Read more.
The bacterial pathogen, Yersinia pestis, has caused three historic pandemics and continues to cause small outbreaks worldwide. During infection, Y. pestis assembles a capsule-like protective coat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to its clinical value in plague diagnostics and anti-plague vaccine development. Expression of F1 is tightly regulated by a transcriptional activator, Caf1R, of the AraC/XylS family, proteins notoriously prone to aggregation. Here, we have optimised the recombinant expression of soluble Caf1R. Expression from the native and synthetic codon-optimised caf1R cloned in three different expression plasmids was examined in a library of E. coli host strains. The functionality of His-tagged Caf1R was demonstrated in vivo, but insolubility was a problem with overproduction. High levels of soluble MBP-Caf1R were produced from codon optimised caf1R. Transcriptional-lacZ reporter fusions defined the PM promoter and Caf1R binding site responsible for transcription of the cafMA1 operon. Use of the identified Caf1R binding caf DNA sequence in an electrophoretic mobility shift assay (EMSA) confirmed correct folding and functionality of the Caf1R DNA-binding domain in recombinant MBP-Caf1R. Availability of functional recombinant Caf1R will be a valuable tool to elucidate control of expression of F1 and Caf1R-regulated pathophysiology of Y. pestis. Full article
(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems 2.0)
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14 pages, 1589 KiB  
Article
Evolving Escherichia coli Host Strains for Efficient Deuterium Labeling of Recombinant Proteins Using Sodium Pyruvate-d3
by Vinardas Kelpšas, Anna Leung and Claes von Wachenfeldt
Int. J. Mol. Sci. 2021, 22(18), 9678; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22189678 - 07 Sep 2021
Viewed by 2076
Abstract
Labeling of proteins with deuterium (2H) is often necessary for structural biology techniques, such as neutron crystallography, NMR spectroscopy, and small-angle neutron scattering. Perdeuteration in which all protium (1H) atoms are replaced by deuterium is a costly process. Typically, expression hosts are grown [...] Read more.
Labeling of proteins with deuterium (2H) is often necessary for structural biology techniques, such as neutron crystallography, NMR spectroscopy, and small-angle neutron scattering. Perdeuteration in which all protium (1H) atoms are replaced by deuterium is a costly process. Typically, expression hosts are grown in a defined medium with heavy water as the solvent, which is supplemented with a deuterated carbon source. Escherichia coli, which is the most widely used host for recombinant protein production, can utilize several compounds as a carbon source. Glycerol-d8 is often used as a carbon source for deuterium labelling due to its lower cost compered to glucose-d7. In order to expand available options for recombinant protein deuteration, we investigated the possibility of producing a deuterated carbon source in-house. E. coli can utilize pyruvate as a carbon source and pyruvate-d3 can be made by a relatively simple procedure. To circumvent the very poor growth of E. coli in minimal media with pyruvate as sole carbon source, adaptive laboratory evolution for strain improvement was applied. E. coli strains with enhanced growth in minimal pyruvate medium was subjected to whole genome sequencing and the genetic changes were revealed. One of the evolved strains was adapted for the widely used T7 RNA polymerase overexpression systems. Using the improved strain E. coli DAP1(DE3) and in-house produced deuterated carbon source (pyruvic acid-d4 and sodium pyruvate-d3), we produce deuterated (>90%) triose-phosphate isomerase, at quantities sufficient enough for large volume crystal production and subsequent analysis by neutron crystallography. Full article
(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems 2.0)
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11 pages, 1435 KiB  
Article
Soluble Expression and Efficient Purification of Recombinant Class I Hydrophobin DewA
by Sang-Oh Ahn, Ho-Dong Lim, Sung-Hwan You, Dae-Eun Cheong and Geun-Joong Kim
Int. J. Mol. Sci. 2021, 22(15), 7843; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22157843 - 22 Jul 2021
Cited by 3 | Viewed by 2141
Abstract
Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic–hydrophobic interfaces, which make them useful for a wide variety [...] Read more.
Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic–hydrophobic interfaces, which make them useful for a wide variety of industrial fields spanning protein immobilization to surface functionalization. However, the industrial use of recombinant hydrophobins has been hampered due to low yield from inclusion bodies owing to the complicated process, including an auxiliary refolding step. Herein, we report the soluble expression of a recombinant class I hydrophobin DewA originating from Aspergillus nidulans, and its efficient purification from recombinant Escherichia coli. Soluble expression of the recombinant hydrophobin DewA was achieved by a tagging strategy using a systematically designed expression tag (ramp tag) that was fused to the N-terminus of DewA lacking the innate signal sequence. Highly expressed recombinant hydrophobin DewA in a soluble form was efficiently purified by a modified aqueous two-phase separation technique using isopropyl alcohol. Our approach for expression and purification of the recombinant hydrophobin DewA in E. coli shed light on the industrial production of hydrophobins from prokaryotic hosts. Full article
(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems 2.0)
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