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Recombinant Proteins, Protein Folding and Drug Discovery
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Recombinant Proteins, Protein Folding and Drug Discovery

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

Deadline for manuscript submissions: 20 June 2025 | Viewed by 5729

Special Issue Editor

Prof. Dr. Mohamed Raafat El-Gewely

E-Mail Website
Guest Editor
Department of Medical Biology, Faculty of Health Sciences, UiT Arctic University of Norway, Tromsø, Norway
Interests: molecular biology; genetic and protein engineering; noncoding RNAs
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Recombinant proteins are used in diagnosis, therapeutics, and drug discovery, and they pose continuous challenges.

The development of recombinant DNA technology in the early 1970s has revolutionized the field of medicine in both diagnostics and therapeutics, as well in other fields such as agriculture. This technology and its applications are being continuously improved. For example, this technology aided the development of recombinant therapeutic proteins, with recombinant human insulin being the first on the list, in October 1982. This revolutionary product, named Humulin as an alternative to the prevalent animals’ insulin, was developed by Eli Lilly in collaboration with Genentech. The fact that the recombinant human insulin gene was first cloned and constructed for expression earlier in 1978 by a team of scientists led by Herbert Boyer and Arthur Riggs at Genentech reflects the difficulties at the time to produce and isolate an active recombinant protein ready for the clinic and for patients. Currently, there are hundreds of therapeutic recombinant proteins on the market.

The encountered problems are many, including misfolding, aggregation, degradation, and a low yield of recombinant proteins. In addition to therapeutic recombinant proteins and special enzymes, antibodies, and viral antigens for diagnostics, producing a validated protein target for drug discovery.

Solutions for encountered problems include changing the expression host, codon optimization, lowering the expression temperature, and the co-expression of foldases and chaperons. In addition, there was always the option of purifying the misfolded and aggregated protein and solubilizing it in vitro.

In this Special Issue, we will focus on the applications and innovations in this important field of genetic engineering and its further application in medicine. We invite your contributions in the form of an article or a review.

Prof. Dr. Mohamed Raafat El-Gewely
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

  • heterologous hosts for gene expression
  • recombinant therapeutic proteins, antigens, and antibodies
  • large scale production of recombinant proteins
  • recombinant protein bioreactor
  • downstream processing of recombinant proteins
  • codon optimization for protein expression
  • refolding of misfolded recombinant proteins
  • solubilization of aggregated and inclusion bodies of recombinant proteins
  • protein target validation for drug discovery and development
  • protein target expression for drug discovery and development
  • metabolic engineering of recombinant protein production
  • regulatory noncoding RNAs to boost the expression of protein
  • the use of foldases & conformases in recombinant protein expression
  • delivery of therapeutic proteins
  • therapeutic protein Vs mRNA therapeutics

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Published Papers (3 papers)

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Research

30 pages, 6186 KiB  
Article
Discovery of PPAR Alpha Lipid Pathway Modulators That Do Not Bind Directly to the Receptor as Potential Anti-Cancer Compounds
by Arwa Al Subait, Raghad H. Alghamdi, Rizwan Ali, Amani Alsharidah, Sarah Huwaizi, Reem A. Alkhodier, Aljawharah Saud Almogren, Barrak A. Alzomia, Ahmad Alaskar and Mohamed Boudjelal
Int. J. Mol. Sci. 2025, 26(2), 736; https://doi.org/10.3390/ijms26020736 - 16 Jan 2025
Cited by 1 | Viewed by 1389
Abstract
Peroxisome proliferator-activated receptors (PPARs) are considered good drug targets for breast cancer because of their involvement in fatty acid metabolism that induces cell proliferation. In this study, we used the KAIMRC1 breast cancer cell line. We showed that the PPARE-Luciferase reporter gets highly [...] Read more.
Peroxisome proliferator-activated receptors (PPARs) are considered good drug targets for breast cancer because of their involvement in fatty acid metabolism that induces cell proliferation. In this study, we used the KAIMRC1 breast cancer cell line. We showed that the PPARE-Luciferase reporter gets highly activated without adding any exogenous ligand when PPAR alpha is co-transfected, and the antagonist GW6471 can inhibit the activity. Using this reporter system, we screened 240 compounds representing kinase inhibitors, epigenetic modulators, and stem cell differentiators and identified compounds that inhibit the PPARα-activated PPARE-Luciferase reporter in the KAIMRC1 cell. We selected 11 compounds (five epigenetic modulators, two stem cell differentiators, and four kinase inhibitors) that inhibited the reporter by at least 40% compared to the controls (DMSO-treated cells). We tested them in a dose-dependent manner and measured the KAIMRC1 cell viability after 48 h. All 11 compounds induced the cell killing at different IC50 values. We selected two compounds, PHA665752 and NSC3852, to dissect how they kill KAIMRC1 cells compared to the antagonist GW6741. First, molecular docking and a TR-FRET PPARα binding assay showed that compared to GW6471, these two compounds could not bind to PPARα. This means they inhibit the PPARα pathway independently rather than binding to the receptor. We further confirmed that PHA665752 and NSC3852 induce cell killing depending on the level of PPARα expression, and as such, their potency for killing the SW620 colon cancer cell line that expresses the lowest level of PPARα was less potent than for the KAIMRC1 and MDA-MB-231 cell lines. Further, using an apoptosis array and fatty acid gene expression panel, we found that both compounds regulate the PPARα pathway by controlling the genes involved in the fatty acid oxidation process. Our findings suggest that these two compounds have opposite effects involving fatty acid oxidation in the KAIMRC1 breast cancer cell line. Although we do not fully understand their mechanism of action, our data provide new insights into the potential role of these compounds in targeting breast cancer cells. Full article
(This article belongs to the Special Issue Recombinant Proteins, Protein Folding and Drug Discovery)
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18 pages, 3326 KiB  
Article
Efficient Production of Recombinant Human Brain-Derived Neurotrophic Factor in Escherichia coli Through the Engineering of Its Pro-Region
by Elisa Spaccapaniccia, Tiziano Cazzorla, Daniela Rossetti, Lucio De Simone, Maria Irene Antonangeli, Andrea Antonosante, Francesca Galli, Franca Cattani, Mariano Maffei and Franck Martin
Int. J. Mol. Sci. 2024, 25(24), 13425; https://doi.org/10.3390/ijms252413425 - 14 Dec 2024
Viewed by 1248
Abstract
Thus far, no manufacturing process able to support industrialization has been reported for the recombinant human brain-derived neurotrophic factor (rhBDNF). Here, we described the setup of a new protocol for its production in Escherichia coli (E. coli) and its purification to [...] Read more.
Thus far, no manufacturing process able to support industrialization has been reported for the recombinant human brain-derived neurotrophic factor (rhBDNF). Here, we described the setup of a new protocol for its production in Escherichia coli (E. coli) and its purification to homogeneity. A synthetic gene, codifying for the neurotrophin precursor, was inserted into an E. coli expression vector and transformed into BL21 (DE3) strain. The recombinant protein accumulates, at high yields, into inclusion bodies. With the developed strategy, more than 50% of the precursor can be refolded. The protein is successively digested by trypsin and the rhBDNF mature form is finally purified by two additional chromatographic steps If the wild-type precursor can be efficiently obtained by the proposed methodology, its pro-peptide remotion, through enzymatic digestion, is however problematic. To circumvent this difficulty, the precursor hinge region, containing the natural furin recognition site, was engineered to be more specifically cleaved by trypsin. Notwithstanding the substitution of three residues in the pro-region carboxyterminal, the precursor correctly refolds and is efficiently cleaved to generate a biologically active mature rhBDNF. This efficient high-yield process fills the current need of a scalable protocol to produce GMP-grade material and unlocks the rhBDNF employment in future clinical investigations. Full article
(This article belongs to the Special Issue Recombinant Proteins, Protein Folding and Drug Discovery)
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13 pages, 5016 KiB  
Article
Cloning, Expression, Purification, and Characterization of Lactate Dehydrogenase from Plasmodium knowlesi: A Zoonotic Malaria Parasite
by Jae-Won Choi, Min-Ji Choi, Yeon-Jun Kim and So Yeon Kim
Int. J. Mol. Sci. 2024, 25(11), 5615; https://doi.org/10.3390/ijms25115615 - 22 May 2024
Viewed by 2160
Abstract
Plasmodium knowlesi is the only Plasmodium that causes zoonotic disease among the Plasmodium that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate dehydrogenase (LDH), an enzyme that catalyzes the final step of glycolysis, [...] Read more.
Plasmodium knowlesi is the only Plasmodium that causes zoonotic disease among the Plasmodium that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate dehydrogenase (LDH), an enzyme that catalyzes the final step of glycolysis, is a biomarker for diagnosing infection by Plasmodium spp. parasite. Therefore, this study aimed to efficiently produce the soluble form of P. knowlesi LDH (PkLDH) using a bacterial expression system for studying malaria caused by P. knowlesi. Recombinant pET-21a(+)-PkLDH plasmid was constructed by inserting the PkLDH gene into a pET-21a(+) expression vector. Subsequently, the recombinant plasmid was inserted into the protein-expressing Escherichia coli Rosetta(DE3) strain, and the optimal conditions for overexpression of the PkLDH protein were established using this strain. We obtained a yield of 52.0 mg/L PkLDH from the Rosetta(DE3) strain and confirmed an activity of 483.9 U/mg through experiments. This methodology for high-efficiency PkLDH production can be utilized for the development of diagnostic methods and drug candidates for distinguishing malaria caused by P. knowlesi. Full article
(This article belongs to the Special Issue Recombinant Proteins, Protein Folding and Drug Discovery)
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