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Biomolecules
Special Issues
Recombinant Enzymes/Proteins in Biotechnology
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Recombinant Enzymes/Proteins in Biotechnology

A special issue of Biomolecules (ISSN 2218-273X). This special issue belongs to the section "Enzymology".

Deadline for manuscript submissions: closed (31 August 2022) | Viewed by 24431

Special Issue Editors

Dr. Stanislav Stuchlík

E-Mail Website
Guest Editor
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia
Interests: recombinant protein expression and purification; recombinant enzymes in biotransformation, recombinant therapeuticals; genomics; proteomics
Dr. Zdenko Levarski

E-Mail Website
Guest Editor
Science Park, Comenius University in Bratislava, Slovakia
Interests: recombinant protein production; recombinant therapeuticals and enzymes; natural compound biotransformation; strain development; protein chromatography

Special Issue Information

Dear Colleagues,

The production of recombinant enzymes and proteins has been one of the main pillars of modern biotechnology, spreading throughout industries from food and agriculture to modern biopharmaceuticals. Robust design of production host strains and processes ensure sustainability of production in the modern sense and allow generation of high-value, high-impact molecules with decreased environmental impact. From simple peptides and proteins produced in E. coli to complex, multi-domain proteins produced in eukaryotic cell lines, the same basic principle remains—production of pure and functional target molecules that are applicable in the subsequent processes. This Special Issue is intended to cover broader aspects of recombinant protein production and their application in multiple aspects of both—the industry and life sciences. Potential topics include but are not limited to:

  • Novel bacterial and/or higher organism production strains;
  • Application of synthetic biology in strain development and process optimization;
  • Production of enzymes for industrial application;
  • Application of enzymes in biotransformation of compounds.

We welcome the submission of original manuscripts, short communications and reviews. 

Dr. Stanislav Stuchlík
Dr. Zdenko Levarski
Guest Editors

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. Biomolecules is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). 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 enzymes and therapeuticals
  • Protein production
  • Strain development
  • Biotransformation

Published Papers (5 papers)

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Research

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26 pages, 5731 KiB  
Article
Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells
by Guillaume Gerez, Jerome Martinez, Christophe Steinbrugger, Sandra Bouanich, Johanna Dimino, Corine Piegay, Maxime Combe, Franck Berthier and Soizic Daniel
Biomolecules 2022, 12(9), 1170; https://doi.org/10.3390/biom12091170 - 24 Aug 2022
Cited by 2 | Viewed by 1929
Abstract
SARS-CoV-2 receptor-binding domain (RBD) is a major target for the development of diagnostics, vaccines and therapeutics directed against COVID-19. Important efforts have been dedicated to the rapid and efficient production of recombinant RBD proteins for clinical and diagnostic applications. One of the main [...] Read more.
SARS-CoV-2 receptor-binding domain (RBD) is a major target for the development of diagnostics, vaccines and therapeutics directed against COVID-19. Important efforts have been dedicated to the rapid and efficient production of recombinant RBD proteins for clinical and diagnostic applications. One of the main challenges is the ongoing emergence of SARS-CoV-2 variants that carry mutations within the RBD, resulting in the constant need to design and optimise the production of new recombinant protein variants. We describe here the impact of naturally occurring RBD mutations on the secretion of a recombinant Fc-tagged RBD protein expressed in HEK 293 cells. We show that mutation E484K of the B.1.351 variant interferes with the proper disulphide bond formation and folding of the recombinant protein, resulting in its retention into the endoplasmic reticulum (ER) and reduced protein secretion. Accumulation of the recombinant B.1.351 RBD-Fc fusion protein in the ER correlated with the upregulation of endogenous ER chaperones, suggestive of the unfolded protein response (UPR). Overexpression of the chaperone and protein disulphide isomerase PDIA2 further impaired protein secretion by altering disulphide bond formation and increasing ER retention. This work contributes to a better understanding of the challenges faced in producing mutant RBD proteins and can assist in the design of optimisation protocols. Full article
(This article belongs to the Special Issue Recombinant Enzymes/Proteins in Biotechnology)
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12 pages, 2249 KiB  
Article
Optimization of an Inclusion Body-Based Production of the Influenza Virus Neuraminidase in Escherichia coli
by Sabina Lipničanová, Barbora Legerská, Daniela Chmelová, Miroslav Ondrejovič and Stanislav Miertuš
Biomolecules 2022, 12(2), 331; https://doi.org/10.3390/biom12020331 - 19 Feb 2022
Cited by 5 | Viewed by 2415
Abstract
Neuraminidase (NA), as an important protein of influenza virus, represents a promising target for the development of new antiviral agents for the treatment and prevention of influenza A and B. Bacterial host strain Escherichia coli BL21 (DE3)pLysS containing the NA gene of the [...] Read more.
Neuraminidase (NA), as an important protein of influenza virus, represents a promising target for the development of new antiviral agents for the treatment and prevention of influenza A and B. Bacterial host strain Escherichia coli BL21 (DE3)pLysS containing the NA gene of the H1N1 influenza virus produced this overexpressed enzyme in the insoluble fraction of cells in the form of inclusion bodies. The aim of this work was to investigate the effect of independent variables (propagation time, isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration and expression time) on NA accumulation in inclusion bodies and to optimize these conditions by response surface methodology (RSM). The maximum yield of NA (112.97 ± 2.82 U/g) was achieved under optimal conditions, namely, a propagation time of 7.72 h, IPTG concentration of 1.82 mM and gene expression time of 7.35 h. This study demonstrated that bacterially expressed NA was enzymatically active. Full article
(This article belongs to the Special Issue Recombinant Enzymes/Proteins in Biotechnology)
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9 pages, 1157 KiB  
Article
Robust Recombinant Expression of Human Placental Ribonuclease Inhibitor in Insect Cells
by Beáta Flachner, Krisztina Dobi, Anett Benedek, Sándor Cseh, Zsolt Lőrincz and István Hajdú
Biomolecules 2022, 12(2), 273; https://doi.org/10.3390/biom12020273 - 08 Feb 2022
Cited by 1 | Viewed by 2500
Abstract
Ribonuclease inhibitors (RIs) are an indispensable biotechnological tool for the detection and manipulation of RNA. Nowadays, due to the outbreak of COVID-19, highly sensitive detection of RNA has become more important than ever. Although the recombinant expression of RNase inhibitors is possible in [...] Read more.
Ribonuclease inhibitors (RIs) are an indispensable biotechnological tool for the detection and manipulation of RNA. Nowadays, due to the outbreak of COVID-19, highly sensitive detection of RNA has become more important than ever. Although the recombinant expression of RNase inhibitors is possible in E. coli, the robust expression is complicated by maintaining the redox potential and solubility by various expression tags. In the present paper we describe the expression of RI in baculovirus-infected High Five cells in large scale utilizing a modified transfer vector combining the beneficial properties of Profinity Exact Tag and pONE system. The recombinant RI is expressed at a high level in a fusion form, which is readily cleaved during on-column chromatography. A subsequent anion exchange chromatography was used as a polishing step to yield 12 mg native RI per liter of culture. RI expressed in insect cells shows higher thermal stability than the commercially available RI products (mainly produced in E. coli) based on temperature-dependent RNase inhibition studies. The endotoxin-free RI variant may also be applied in future therapeutics as a safe additive to increase mRNA stability in mRNA-based vaccines. Full article
(This article belongs to the Special Issue Recombinant Enzymes/Proteins in Biotechnology)
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22 pages, 6016 KiB  
Article
High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells
by Živa Švab, Luca Braga, Corrado Guarnaccia, Ivan Labik, Jeremias Herzog, Marco Baralle, Mauro Giacca and Nataša Skoko
Biomolecules 2021, 11(8), 1125; https://doi.org/10.3390/biom11081125 - 30 Jul 2021
Cited by 3 | Viewed by 2403
Abstract
CHO is the cell line of choice for the manufacturing of many complex biotherapeutics. The constant upgrading of cell productivity is needed to meet the growing demand for these life-saving drugs. Manipulation of small non-coding RNAs—miRNAs—is a good alternative to a single gene [...] Read more.
CHO is the cell line of choice for the manufacturing of many complex biotherapeutics. The constant upgrading of cell productivity is needed to meet the growing demand for these life-saving drugs. Manipulation of small non-coding RNAs—miRNAs—is a good alternative to a single gene knockdown approach due to their post-transcriptional regulation of entire cellular pathways without posing translational burden to the production cell. In this study, we performed a high-throughput screening of 2042-human miRNAs and identified several candidates able to increase cell-specific and overall production of Erythropoietin and Etanercept in CHO cells. Some of these human miRNAs have not been found in Chinese hamster cells and yet were still effective in them. We identified miR-574-3p as being able, when overexpressed in CHO cells, to improve overall productivity of Erythropoietin and Etanercept titers from 1.3 to up to 2-fold. In addition, we validated several targets of miR-574-3p and identified p300 as a main target of miR-574-3p in CHO cells. Furthermore, we demonstrated that stable CHO cell overexpressing miRNAs from endogenous CHO pri-miRNA sequences outperform the cells with human pri-miRNA sequences. Our findings highlight the importance of flanking genomic sequences, and their secondary structure features, on pri-miRNA processing offering a novel, cost-effective and fast strategy as a valuable tool for efficient miRNAs engineering in CHO cells. Full article
(This article belongs to the Special Issue Recombinant Enzymes/Proteins in Biotechnology)
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Review

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25 pages, 7004 KiB  
Review
Glucose Oxidase, an Enzyme “Ferrari”: Its Structure, Function, Production and Properties in the Light of Various Industrial and Biotechnological Applications
by Jacob A. Bauer, Monika Zámocká, Juraj Majtán and Vladena Bauerová-Hlinková
Biomolecules 2022, 12(3), 472; https://doi.org/10.3390/biom12030472 - 19 Mar 2022
Cited by 62 | Viewed by 13036
Abstract
Glucose oxidase (GOx) is an important oxidoreductase enzyme with many important roles in biological processes. It is considered an “ideal enzyme” and is often called an oxidase “Ferrari” because of its fast mechanism of action, high stability and specificity. Glucose oxidase catalyzes the [...] Read more.
Glucose oxidase (GOx) is an important oxidoreductase enzyme with many important roles in biological processes. It is considered an “ideal enzyme” and is often called an oxidase “Ferrari” because of its fast mechanism of action, high stability and specificity. Glucose oxidase catalyzes the oxidation of β-d-glucose to d-glucono-δ-lactone and hydrogen peroxide in the presence of molecular oxygen. d-glucono-δ-lactone is sequentially hydrolyzed by lactonase to d-gluconic acid, and the resulting hydrogen peroxide is hydrolyzed by catalase to oxygen and water. GOx is presently known to be produced only by fungi and insects. The current main industrial producers of glucose oxidase are Aspergillus and Penicillium. An important property of GOx is its antimicrobial effect against various pathogens and its use in many industrial and medical areas. The aim of this review is to summarize the structure, function, production strains and biophysical and biochemical properties of GOx in light of its various industrial, biotechnological and medical applications. Full article
(This article belongs to the Special Issue Recombinant Enzymes/Proteins in Biotechnology)
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