Special Issue "State of the Art of Protein Expression Systems"

A special issue of Processes (ISSN 2227-9717). This special issue belongs to the section "Biological Processes and Systems".

Deadline for manuscript submissions: 20 September 2022 | Viewed by 4101

Special Issue Editors

Prof. Dr. Tzong-Yuan Wu
E-Mail Website
Guest Editor
Department of Bioscience Technology, Chung Yuan Christian University, Chungli 320, Taiwan
Interests: baculovirus expression system; virology, protein physical chemistry
Prof. Dr. Monique M. van Oers
E-Mail Website
Guest Editor
Laboratory of Virology, Department of Plant Sciences, Wageningen University, Wageningen, The Netherlands
Interests: insect viruses; virus-host interactions; baculovirus; virus evolution; virus taxonomy; antiviral defense; caterpillars; bees; dipteran insects
Special Issues, Collections and Topics in MDPI journals
Dr. Li-Fen Huang
E-Mail Website
Guest Editor
Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li 320, Taiwan
Interests: engineering protein expression system in rice cells for valuable medical protein production; DNA detection biosensor for GMO; Develop plant tissue culture systems for Chinese medical plants, snow lotus, snow lotus

Special Issue Information

Dear Colleagues,

One of the big social impacts of biotechnology is the production of recombinant proteins. Recombinant proteins have dramatically changed the art of medical treatments, drug discoveries, vaccine developments, as well as basic biological research studies. However, recombinant protein production is the basic challenge for ongoing medical applications and basic research studies. Thus, the protein expression system, as an interesting and fast-growing research area in biotechnology, might be an attractive tool to solve this challenge.

The Special Issue “State of the Art of Protein Expression System” covers original research papers or reviews on protein expression systems, e.g., Escherichia coli, Bacillus subtilis, Lactococcus lactis, yeast, insect cells, mammalian cells, animal, as well as plants.

This Special Issue aims to focus on recent progresses and advances in vectors design, secretion signals, recombinant protein purifications, oligomeric protein expression, virus-like particle production and subunit vaccines production.

Topics include, but are not limited to:

  • vector designs for protein expression system;
  • investigations on signal peptide for recombinant proteins production;
  • downstream process of recombinant proteins;
  • development of oligomeric recombinant expression vectors;
  • virus like particle production and preparations;
  • subunit vaccines production and preparations.

Prof. Dr. Tzong-Yuan Wu
Prof. Dr. Monique M. van Oers
Dr. Li-Fen Huang
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. Processes 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 2000 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

  • downstream process
  • protein expression system
  • recombinant proteins
  • vectors design
  • signal peptide
  • vaccine
  • virus-like particle

Published Papers (6 papers)

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Research

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Communication
Plum Pox Virus Genome-Based Vector Enables the Expression of Different Heterologous Polypeptides in Nicotiana benthamiana Plants
Processes 2022, 10(8), 1526; https://doi.org/10.3390/pr10081526 - 03 Aug 2022
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Abstract
Plant viral vectors have become a promising tool for the rapid and cost-effective production of recombinant proteins in plants. Among the numerous genera of viruses that have been used for heterologous expression, potyviruses offer several advantages, such as polyprotein expression strategy or a [...] Read more.
Plant viral vectors have become a promising tool for the rapid and cost-effective production of recombinant proteins in plants. Among the numerous genera of viruses that have been used for heterologous expression, potyviruses offer several advantages, such as polyprotein expression strategy or a broad host range. In our work, the expression vectors pAD/pAD-agro based on the plum pox virus (PPV) genome were used for the heterologous expression of different foreign polypeptides: alfalfa mosaic virus capsid protein (AMV CP), zucchini yellow mosaic virus capsid protein (ZYMV CP), the small heat-shock protein of Cronobacter sakazakii fused with hexahistidine (sHSP-his), a fragment of influenza A virus hemagglutinin (HA2-2), influenza A virus protein PB1-F2, SARS-CoV-2 nucleocapsid protein (CoN2-his), and its N- and C-terminal fragments (CoN-1-his and CoN3-his, respectively), each fused with a hexahistidine anchor. Particular proteins differed in their accumulation, tissue localization, stability, and solubility. The accumulation rate of produced polypeptides varied from low (N, hemagglutinin fragment) to relatively high (plant viral CPs, N-terminal fragment of N, PB1-F2). Some proteins preferentially accumulated in roots (sHSP, hemagglutinin fragment, PB1-F2), showing signs of proteolytic degradation in leaf tissues. Thus, each expression requires an individual approach and optimization. Here, we summarize our several-year experiments and discuss the usefulness of the pAD/pADep vector system. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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Article
Production of Bivalent Subunit Vaccine for Porcine via 2A-Like Sequence in Baculovirus Expression Vector System
Processes 2022, 10(5), 895; https://doi.org/10.3390/pr10050895 - 01 May 2022
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Abstract
Classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV2) have caused severe diseases in swine populations worldwide. Here, a polycistronic baculovirus vector was developed to express a bivalent vaccine, consisting of the CSFV-E2 and PCV2-Cap protein, and an immunomodulator protein derived [...] Read more.
Classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV2) have caused severe diseases in swine populations worldwide. Here, a polycistronic baculovirus vector was developed to express a bivalent vaccine, consisting of the CSFV-E2 and PCV2-Cap protein, and an immunomodulator protein derived from the Flammulina velutipes, FVE-FIP, as well as the selection marker, green fluorescent protein. The simultaneous expression of the CSFV-E2 and PCV2-Cap protein was mediated by the 2A-like sequence derived from the Perina nuda virus (PnV), while the expression of the FVE-FIP was driven by the internal ribosome entry site (IRES) element derived from the Rhophalosipum padi virus (RhPV). The Western blot analysis result suggested that the CSFV-E2, PCV2-Cap, and FVE-FIP protein were successfully co-expressed by the infected Spodoptera frugiperda IPBL-Sf21 (Sf21) cell line. The extracted cell lysate containing all three recombinant proteins was administered to Balb/C mice with or without the supplementation of Freund’s adjuvant. The ELISA analysis of the serum collected from all the immunized groups showed detectable antibodies against CSFV-E2 and PCV2-Cap. Furthermore, the immunized group without the adjuvant supplementation demonstrated a similar level of antibodies to the group with adjuvant supplementation, suggesting the efficiency of the FVE-FIP in enhancing the immune response. These results demonstrated the polycistronic baculovirus vector could be employed to develop bivalent vaccines for pigs. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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Article
Secreted Trimeric Chikungunya Virus Spikes from Insect Cells: Production, Purification, and Glycosylation Status
Processes 2022, 10(1), 162; https://doi.org/10.3390/pr10010162 - 14 Jan 2022
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Abstract
Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne virus that causes a severe febrile illness with long-lasting arthralgia in humans. As there is no vaccine to protect humans and limit CHIKV epidemics, the virus continues to be a global public health concern. The [...] Read more.
Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne virus that causes a severe febrile illness with long-lasting arthralgia in humans. As there is no vaccine to protect humans and limit CHIKV epidemics, the virus continues to be a global public health concern. The CHIKV envelope glycoproteins E1 and E2 are important immunogens; therefore, the aim of this study is to produce trimeric CHIKV spikes in insect cells using the baculovirus expression system. The CHIKV E1 and E2 ectodomains were covalently coupled by a flexible linker that replaces the 6K transmembrane protein. The C-terminal E1 transmembrane was replaced by a Strep-tag II for the purification of secreted spikes from the culture fluid. After production in Sf9 suspension cells (product yields of 5.8–7.6 mg/L), the CHIKV spikes were purified by Strep-Tactin affinity chromatography, which successfully cleared the co-produced baculoviruses. Bis(sulfosuccinimidyl)suberate cross-linking demonstrated that the spikes are secreted as trimers. PNGase F treatment showed that the spikes are glycosylated. LC–MS/MS-based glycoproteomic analysis confirmed the glycosylation and revealed that the majority are of the mannose- or hybrid-type N-glycans and <2% have complex-type N-glycans. The LC –MS/MS analysis also revealed three O-glycosylation sites in E1. In conclusion, the trimeric, glycosylated CHIKV spikes have been successfully produced in insect cells and are now available for vaccination studies. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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Article
Optimizing Recombinant Baculovirus Vector Design for Protein Production in Insect Cells
Processes 2021, 9(12), 2118; https://doi.org/10.3390/pr9122118 - 25 Nov 2021
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Abstract
Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before [...] Read more.
Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before the native ATG. Many transfer vectors also include a short sequence downstream of the ATG, in which case this sequence is mutated to ATT to abolish translation. However, the ATT sequence, or AUU in the mRNA, is known to be leaky. If a target-coding region is placed in the frame with the AUU, then some products will comprise a chimeric molecule with part of the polyhedrin protein. In this study, we showed that if AUU is placed in the frame with a Strep tag and eGFP coding region, we could identify a protein product with both sequences present. Further work examined if alternative codons in lieu of AUG might reduce translation initiation further. We found that AUA was used slightly more efficiently than AUU, whereas AUC was the least efficient at initiating translation. The use of this latter codon suggested that there might also be a slight improvement of protein yield if this is incorporated into expression vectors. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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Review

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Review
Current Strategies to Improve Yield of Recombinant Protein Production in Rice Suspension Cells
Processes 2022, 10(6), 1120; https://doi.org/10.3390/pr10061120 - 03 Jun 2022
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Abstract
A plant cell-based recombinant glucocerebrosidase was approved by the FDA in 2012 for the treatment of human inherited Gaucher disease, indicating that plant suspension cells have advantages in biosafety and a low production cost as a commercial pharmaceutical recombinant protein expression system. A [...] Read more.
A plant cell-based recombinant glucocerebrosidase was approved by the FDA in 2012 for the treatment of human inherited Gaucher disease, indicating that plant suspension cells have advantages in biosafety and a low production cost as a commercial pharmaceutical recombinant protein expression system. A low allergenic rice suspension cell-based recombinant protein expression system controlled by the αAmy3/RAmy3D promoter has been shown to result in relatively high protein yields in plant cell-based systems. Although several recombinant proteins have been produced in rice suspension cell-based systems, yields must be improved to compete with the current commercial protein expression systems. Different strategies were performed and showed successful improvements in recombinant protein yields in this rice system. The review updates and highlights strategies for potential improvements of the αAmy3-based rice suspension cell-based system. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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Review
Recombinant Protein Technology in the Challenging Era of Coronaviruses
Processes 2022, 10(5), 946; https://doi.org/10.3390/pr10050946 - 10 May 2022
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Abstract
Coronaviruses have caused devastation in both human and animal populations, affecting both health and the economy. Amidst the emergence and re-emergence of coronaviruses, humans need to surmount the health and economic threat of coronaviruses through science and evidence-based approaches. One of these approaches [...] Read more.
Coronaviruses have caused devastation in both human and animal populations, affecting both health and the economy. Amidst the emergence and re-emergence of coronaviruses, humans need to surmount the health and economic threat of coronaviruses through science and evidence-based approaches. One of these approaches is through biotechnology, particularly the heterologous production of biopharmaceutical proteins. This review article briefly describes the genome, general virion morphology, and key structural proteins of different coronaviruses affecting animals and humans. In addition, this review paper also presents the different systems in recombinant protein technology such as bacteria, yeasts, plants, mammalian cells, and insect/insect cells systems used to express key structural proteins in the development of countermeasures such as diagnostics, prophylaxis, and therapeutics in the challenging era of coronaviruses. Full article
(This article belongs to the Special Issue State of the Art of Protein Expression Systems)
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