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Special Issue "Recombinant Proteins"

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

Deadline for manuscript submissions: closed (30 November 2017).

Special Issue Editors

Guest Editor
Prof. Karen A. McDonald

Department of Chemical Engineering, University of California, Davis, CA 95616, USA
Website | E-Mail
Interests: plant-based recombinant protein production; bioprocess engineering; techno-economic analyses
Guest Editor
Prof. Somen Nandi

Department of Chemical Engineering and Global HealthShare® Initiative, University of California, Davis, CA 95616, USA
Website | E-Mail
Interests: recombinant protein expression; drug development; glycan modification; protein process engineering; process economics

Special Issue Information

Dear Colleagues,

By simple definition, a recombinant protein is a protein encoded by a heterologous or synthetic gene(s)—recombinant DNA—that has been cloned in a system that supports expression of the gene(s) to produce messenger RNA (mRNA) followed by translation to protein and possibly post-translational modification and subcellular targeting. Modification of the gene by recombinant DNA technology can lead to expression of many proteins. Potential applications of these recombinant protein products include human and animal therapeutics, subunit vaccines, diagnostics, industrial enzymes, food additives or processing agents, crop protectant biologics, biopolymers, and reagents, to name a few. Although there are a variety of potential hosts and manufacturing strategies that are currently available or are being developed, each application has different constraints in terms of capital and manufacturing cost, production scale needed, quality and purity attributes, and regulatory requirements.

The focus of this Special Issue of the International Journal of Molecular Sciences (IJMS), entitled Recombinant Proteins, is the science and engineering of recombinant protein production, particularly production of complex and hard to produce proteins (membrane proteins, ones with solubility issues or requiring extensive post-translational modifications), transient production of recombinant proteins in a variety of hosts for rapid response, novel expression systems, host engineering and stability, secretion, localization and post-translational modifications, protein engineering including enzyme engineering, fusion proteins and biobetter biologics.

Prof. Karen A. McDonald
Prof. Somen Nandi
Guest Editors

Manuscript Submission Information

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Keywords

  • Recombinant Proteins
  • Recombinant DNA
  • Rapid (e.g., transient) production
  • Scale-up of production platforms
  • New hosts for rapid production
  • Novel technologies to treat or prevent pathogens

Published Papers (16 papers)

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Research

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Open AccessArticle
A Recombinant Affinity Reagent Specific for a Phosphoepitope of Akt1
Int. J. Mol. Sci. 2018, 19(11), 3305; https://doi.org/10.3390/ijms19113305
Received: 17 August 2018 / Revised: 10 October 2018 / Accepted: 16 October 2018 / Published: 24 October 2018
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Abstract
The serine/threonine-protein kinase, Akt1, plays an important part in mammalian cell growth, proliferation, migration and angiogenesis, and becomes activated through phosphorylation. To monitor phosphorylation of threonine 308 in Akt1, we developed a recombinant phosphothreonine-binding domain (pTBD) that is highly selective for the Akt1 [...] Read more.
The serine/threonine-protein kinase, Akt1, plays an important part in mammalian cell growth, proliferation, migration and angiogenesis, and becomes activated through phosphorylation. To monitor phosphorylation of threonine 308 in Akt1, we developed a recombinant phosphothreonine-binding domain (pTBD) that is highly selective for the Akt1 phosphopeptide. A phage-display library of variants of the Forkhead-associated 1 (FHA1) domain of yeast Rad53p was screened by affinity selection to the phosphopeptide, 301-KDGATMKpTFCGTPEY-315, and yielded 12 binding clones. The strongest binders have equilibrium dissociation constants of 160–180 nanomolar and are phosphothreonine-specific in binding. The specificity of one Akt1-pTBD was compared to commercially available polyclonal antibodies (pAbs) generated against the same phosphopeptide. The Akt1-pTBD was either equal to or better than three pAbs in detecting the Akt1 pT308 phosphopeptide in ELISAs. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle
The Production of Human β-Glucocerebrosidase in Nicotiana benthamiana Root Culture
Int. J. Mol. Sci. 2018, 19(7), 1972; https://doi.org/10.3390/ijms19071972
Received: 18 April 2018 / Revised: 25 June 2018 / Accepted: 1 July 2018 / Published: 6 July 2018
Cited by 2 | PDF Full-text (2275 KB) | HTML Full-text | XML Full-text
Abstract
Gaucher disease is caused by a deficiency of the enzyme glucocerebrosidase (GCase). Currently, enzyme-replacement therapy using recombinant GCase produced in mammalian cells is considered the most effective treatment. Plants are an attractive alternative host for recombinant protein production due to the low cost [...] Read more.
Gaucher disease is caused by a deficiency of the enzyme glucocerebrosidase (GCase). Currently, enzyme-replacement therapy using recombinant GCase produced in mammalian cells is considered the most effective treatment. Plants are an attractive alternative host for recombinant protein production due to the low cost of large-scale production and lack of risk of contamination by human pathogens. Compared to whole plants, root cultures can grow faster. Therefore, this study aimed to produce recombinant GCase in a Nicotiana benthamiana root culture. Root culture of a GCase-producing transgenic plant was induced by indole-3-acetic acid at the concentration of 1 mg/L. Recombinant GCase was successfully produced in roots as a functional protein with an enzyme activity equal to 81.40 ± 17.99 units/mg total protein. Crude proteins were extracted from the roots. Recombinant GCase could be purified by concanavalin A and phenyl 650C chromatography. The productivity of GCase was approximately 1 µg/g of the root. A N-glycan analysis of purified GCase was performed using nano LC/MS. The Man3XylFucGlcNAc2 structure was predominant in purified GCase with two plant-specific glycan residues. This study presents evidence for a new, safe and efficient system of recombinant GCase production that might be applied to other recombinant proteins. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle
Enhancement of Recombinant Protein Production in Transgenic Nicotiana benthamiana Plant Cell Suspension Cultures with Co-Cultivation of Agrobacterium Containing Silencing Suppressors
Int. J. Mol. Sci. 2018, 19(6), 1561; https://doi.org/10.3390/ijms19061561
Received: 7 March 2018 / Revised: 6 May 2018 / Accepted: 18 May 2018 / Published: 24 May 2018
Cited by 1 | PDF Full-text (2294 KB) | HTML Full-text | XML Full-text
Abstract
We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter (Cauliflower mosaic virus (CaMV) 35S) or [...] Read more.
We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter (Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium-mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle
Transient Recombinant Protein Production in Glycoengineered Nicotiana benthamiana Cell Suspension Culture
Int. J. Mol. Sci. 2018, 19(4), 1205; https://doi.org/10.3390/ijms19041205
Received: 24 February 2018 / Revised: 6 April 2018 / Accepted: 9 April 2018 / Published: 16 April 2018
Cited by 3 | PDF Full-text (12398 KB) | HTML Full-text | XML Full-text
Abstract
Transient recombinant protein production is a promising alternative to stable transgenic systems, particularly for emergency situations in which rapid production of novel therapeutics is needed. In plants, Agrobacterium tumefaciens can be used as a gene delivery vector for transient expression. A potential barrier [...] Read more.
Transient recombinant protein production is a promising alternative to stable transgenic systems, particularly for emergency situations in which rapid production of novel therapeutics is needed. In plants, Agrobacterium tumefaciens can be used as a gene delivery vector for transient expression. A potential barrier for plant-based production of human therapeutics is that different glycosylation patterns are found on plant and mammalian proteins. Since glycosylation can affect the efficacy, safety and stability of a therapeutic protein, methods to control glycan structures and distributions in plant-based systems would be beneficial. In these studies, we performed Agrobacterium-mediated transient expression in glycoengineered plant cell suspension cultures. To reduce the presence of plant-specific glycans on the product, we generated and characterized cell suspension cultures from β-1,2-xylosyltransferase and α-1,3-fucosyltransferase knockdown Nicotiana benthamiana. An anthrax decoy fusion protein was transiently produced in these glycoengineered plant cell suspension cultures through co-culture with genetically engineered Agrobacterium. The mass ratio of Agrobacterium to plant cells used was shown to impact recombinant protein expression levels. N-glycosylation analysis on the anthrax decoy fusion protein produced in glycoengineered N. benthamiana showed a dramatic reduction in plant-specific N-glycans. Overall, the results presented here demonstrate the feasibility of a simple, rapid and scalable process for transient production of recombinant proteins without plant-specific glycans in a glycoengineered plant cell culture host. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessCommunication
Glycoform Modification of Secreted Recombinant Glycoproteins through Kifunensine Addition during Transient Vacuum Agroinfiltration
Int. J. Mol. Sci. 2018, 19(3), 890; https://doi.org/10.3390/ijms19030890
Received: 27 February 2018 / Revised: 14 March 2018 / Accepted: 15 March 2018 / Published: 17 March 2018
Cited by 2 | PDF Full-text (699 KB) | HTML Full-text | XML Full-text
Abstract
Kifunensine, a potent and selective inhibitor of class I α-mannosidases, prevents α-mannosidases I from trimming mannose residues on glycoproteins, thus resulting in oligomannose-type glycans. We report for the first time that through one-time vacuum infiltration of kifunensine in plant tissue, N-linked glycosylation of [...] Read more.
Kifunensine, a potent and selective inhibitor of class I α-mannosidases, prevents α-mannosidases I from trimming mannose residues on glycoproteins, thus resulting in oligomannose-type glycans. We report for the first time that through one-time vacuum infiltration of kifunensine in plant tissue, N-linked glycosylation of a recombinant protein transiently produced in whole-plants shifted completely from complex-type to oligomannose-type. Fc-fused capillary morphogenesis protein 2 (CMG2-Fc) containing one N-glycosylation site on the Fc domain, produced in Nicotiana benthamiana whole plants, served as a model protein. The CMG2-Fc fusion protein was produced transiently through vacuum agroinfiltration, with and without kifunensine at a concentration of 5.4 µM in the agroinfiltration suspension. The CMG2-Fc N-glycan profile was determined using LC-MS/MS with a targeted dynamic multiple reaction monitoring (MRM) method. The CMG2-Fc expression level in the infiltrated plant tissue and the percentage of oligomannose-type N-glycans for kifunensine treated plants was 874 mg/kg leaf fresh weight (FW) and 98.2%, respectively, compared to 717 mg/kg leaf FW and 2.3% for untreated plants. Oligomannose glycans are amenable to in vitro enzymatic modification to produce more human-like N-glycan structures that are preferred for the production of HIV-1 viral vaccine and certain monoclonal antibodies. This method allows glycan modifications using a bioprocessing approach without compromising protein yield or modification of the primary sequence, and could be expanded to other small molecule inhibitors of glycan-processing enzymes. For recombinant protein targeted for secretion, kifunensine treatment allows collection of glycoform-modified target protein from apoplast wash fluid (AWF) with minimal plant-specific complex N-glycan at higher starting purity and concentration than in whole-leaf extract, thus simplifying the downstream processing. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle
Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii
Int. J. Mol. Sci. 2018, 19(2), 585; https://doi.org/10.3390/ijms19020585
Received: 21 November 2017 / Revised: 10 February 2018 / Accepted: 13 February 2018 / Published: 16 February 2018
Cited by 3 | PDF Full-text (4543 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of [...] Read more.
Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a “plant-like” algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT) and Gallium-immobilized metal affinity chromatography (Ga-IMAC) were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle
Implementation of Glycan Remodeling to Plant-Made Therapeutic Antibodies
Int. J. Mol. Sci. 2018, 19(2), 421; https://doi.org/10.3390/ijms19020421
Received: 2 December 2017 / Revised: 9 January 2018 / Accepted: 27 January 2018 / Published: 31 January 2018
Cited by 3 | PDF Full-text (2674 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment [...] Read more.
N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment that changes with scale and production batches leading to glycosylation heterogeneity and inconsistency. On the other hand, in vitro chemoenzymatic glycan remodeling has been successful in producing homogeneous pre-defined protein glycoforms, but needs to be combined with a cost-effective and scalable production method. An efficient chemoenzymatic glycan remodeling technology using a plant expression system that combines in vivo deglycosylation with an in vitro chemoenzymatic glycosylation is described. Using the monoclonal antibody rituximab as a model therapeutic protein, a uniform Gal2GlcNAc2Man3GlcNAc2 (A2G2) glycoform without α-1,6-fucose, plant-specific α-1,3-fucose or β-1,2-xylose residues was produced. When compared with the innovator product Rituxan®, the plant-made remodeled afucosylated antibody showed similar binding affinity to the CD20 antigen but significantly enhanced cell cytotoxicity in vitro. Using a scalable plant expression system and reducing the in vitro deglycosylation burden creates the potential to eliminate glycan heterogeneity and provide affordable customization of therapeutics’ glycosylation for maximal and targeted biological activity. This feature can reduce cost and provide an affordable platform to manufacture biobetter antibodies. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle
A Simple Method to Reduce both Lactic Acid and Ammonium Production in Industrial Animal Cell Culture
Int. J. Mol. Sci. 2018, 19(2), 385; https://doi.org/10.3390/ijms19020385
Received: 23 December 2017 / Revised: 16 January 2018 / Accepted: 23 January 2018 / Published: 28 January 2018
Cited by 3 | PDF Full-text (2464 KB) | HTML Full-text | XML Full-text
Abstract
Fed-batch animal cell culture is the most common method for commercial production of recombinant proteins. However, higher cell densities in these platforms are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to [...] Read more.
Fed-batch animal cell culture is the most common method for commercial production of recombinant proteins. However, higher cell densities in these platforms are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to nutrient feeds and base additions to control pH. To partly overcome these factors, we investigated a simple method to reduce both ammonium and lactic acid production—termed Lactate Supplementation and Adaptation (LSA) technology—through the use of CHO cells adapted to a lactate-supplemented medium. Using this simple method, we achieved a reduction of nearly 100% in lactic acid production with a simultaneous 50% reduction in ammonium production in batch shaker flasks cultures. In subsequent fed-batch bioreactor cultures, lactic acid production and base addition were both reduced eight-fold. Viable cell densities of 35 million cells per mL and integral viable cell days of 273 million cell-days per mL were achieved, both among the highest currently reported for a fed-batch animal cell culture. Investigating the benefits of LSA technology in animal cell culture is worthy of further consideration and may lead to process conditions more favorable for advanced industrial applications. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle
Characterization and Oral Delivery of Proinsulin-Transferrin Fusion Protein Expressed Using ExpressTec
Int. J. Mol. Sci. 2018, 19(2), 378; https://doi.org/10.3390/ijms19020378
Received: 26 November 2017 / Revised: 23 January 2018 / Accepted: 24 January 2018 / Published: 26 January 2018
Cited by 1 | PDF Full-text (4064 KB) | HTML Full-text | XML Full-text
Abstract
Proinsulin-transferrin fusion protein (ProINS-Tf) has been designed and successfully expressed from the mammalian HEK293 cells (HEK-ProINS-Tf). It was found that HEK-ProINS-Tf could be converted into an activated form in the liver. Furthermore, HEK-ProINS-Tf was demonstrated as an extra-long acting insulin analogue with liver-specific [...] Read more.
Proinsulin-transferrin fusion protein (ProINS-Tf) has been designed and successfully expressed from the mammalian HEK293 cells (HEK-ProINS-Tf). It was found that HEK-ProINS-Tf could be converted into an activated form in the liver. Furthermore, HEK-ProINS-Tf was demonstrated as an extra-long acting insulin analogue with liver-specific insulin action in streptozotocin (STZ)-induced type 1 diabetic mice. However, due to the low production yield from transfected HEK293 cells, there are other interesting features, including the oral bioavailability, which have not been fully explored and characterized. To improve the protein production yield, an alternative protein expression system, ExpressTec using transgenic rice (Oryza sativa L.), was used. The intact and active rice-derived ProINS-Tf (ExpressTec-ProINS-Tf) was successfully expressed from the transgenic rice expression system. Our results suggested that, although the insulin-like bioactivity of ExpressTec-ProINS-Tf was slightly lower in vitro, its potency of in vivo blood glucose control was considerably stronger than that of HEK-ProINS-Tf. The oral delivery studies in type 1 diabetic mice demonstrated a prolonged control of blood glucose to near-normal levels after oral administration of ExpressTec-ProINS-Tf. Results in this report suggest that ExpressTec-ProINS-Tf is a promising insulin analog with advantages including low cost, prolonged and liver targeting effects, and most importantly, oral bioactivity. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle
Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana
Int. J. Mol. Sci. 2018, 19(1), 115; https://doi.org/10.3390/ijms19010115
Received: 30 November 2017 / Revised: 24 December 2017 / Accepted: 30 December 2017 / Published: 1 January 2018
Cited by 1 | PDF Full-text (1437 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish (Armoracia rusticana); however, this crop is only available seasonally, yields are variable [...] Read more.
Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish (Armoracia rusticana); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens-mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle
Simple Purification of Nicotiana benthamiana-Produced Recombinant Colicins: High-Yield Recovery of Purified Proteins with Minimum Alkaloid Content Supports the Suitability of the Host for Manufacturing Food Additives
Int. J. Mol. Sci. 2018, 19(1), 95; https://doi.org/10.3390/ijms19010095
Received: 28 November 2017 / Revised: 20 December 2017 / Accepted: 22 December 2017 / Published: 29 December 2017
Cited by 3 | PDF Full-text (1173 KB) | HTML Full-text | XML Full-text
Abstract
Colicins are natural non-antibiotic bacterial proteins with a narrow spectrum but an extremely high antibacterial activity. These proteins are promising food additives for the control of major pathogenic Shiga toxin-producing E. coli serovars in meats and produce. In the USA, colicins produced in [...] Read more.
Colicins are natural non-antibiotic bacterial proteins with a narrow spectrum but an extremely high antibacterial activity. These proteins are promising food additives for the control of major pathogenic Shiga toxin-producing E. coli serovars in meats and produce. In the USA, colicins produced in edible plants such as spinach and leafy beets have already been accepted by the U. S. Food and Drug Administration (FDA) and U. S. Department of Agriculture (USDA) as food-processing antibacterials through the GRAS (generally recognized as safe) regulatory review process. Nicotiana benthamiana, a wild relative of tobacco, N. tabacum, has become the preferred production host plant for manufacturing recombinant proteins—including biopharmaceuticals, vaccines, and biomaterials—but the purification procedures that have been employed thus far are highly complex and costly. We describe a simple and inexpensive purification method based on specific acidic extraction followed by one chromatography step. The method provides for a high recovery yield of purified colicins, as well as a drastic reduction of nicotine to levels that could enable the final products to be used on food. The described purification method allows production of the colicin products at a commercially viable cost of goods and might be broadly applicable to other cost-sensitive proteins. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle
Recombinant Zika NS1 Protein Secreted from Vero Cells Is Efficient for Inducing Production of Immune Serum Directed against NS1 Dimer
Int. J. Mol. Sci. 2018, 19(1), 38; https://doi.org/10.3390/ijms19010038
Received: 2 November 2017 / Revised: 19 December 2017 / Accepted: 21 December 2017 / Published: 23 December 2017
Cited by 3 | PDF Full-text (5263 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Zika virus (ZIKV) is a mosquito-borne flavivirus that recently emerged in the South Pacific, Americas, and Caribbean islands, where the larger epidemics were documented. ZIKV infection in humans is responsible for neurological disorders and microcephaly. Flavivirus NS1 is a non-structural glycoprotein that is [...] Read more.
Zika virus (ZIKV) is a mosquito-borne flavivirus that recently emerged in the South Pacific, Americas, and Caribbean islands, where the larger epidemics were documented. ZIKV infection in humans is responsible for neurological disorders and microcephaly. Flavivirus NS1 is a non-structural glycoprotein that is expressed on the cell surface and secreted as a hexameric lipoprotein particle. Intracellular NS1 exists as a dimer that is required for viral replication, whereas the secreted NS1 hexamer interacts with host factors, leading to pathophysiological conditions. In an effort to dispose of specific anti-ZIKV NS1 immune serum, Vero cells were transduced with a lentiviral vector containing the NS1 gene from an epidemic strain of ZIKV. We showed that stably transduced Vero/ZIKV NS1 cell clone was efficient in the secretion of recombinant NS1 oligomer. Immunization of adult rat with purified extracellular NS1 developed anti-ZIKV antibodies that specifically react with the NS1 dimer produced in human cells infected with African and Asian strains of ZIKV. The rat antibody against ZIKV NS1 dimer is a reliable biological tool that enables the immunological detection of secreted NS1 from host-cells infected with ZIKV. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle
Rational Design of Recombinant Papain-Like Cysteine Protease: Optimal Domain Structure and Expression Conditions for Wheat-Derived Enzyme Triticain-α
Int. J. Mol. Sci. 2017, 18(7), 1395; https://doi.org/10.3390/ijms18071395
Received: 23 May 2017 / Revised: 21 June 2017 / Accepted: 23 June 2017 / Published: 29 June 2017
Cited by 3 | PDF Full-text (2912 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Triticain-α is a papain-like cysteine protease from wheat (Triticum aestivum L.) that possesses activity towards toxic gluten-derived peptides, and was thus proposed as a novel therapeutic tool for celiac disease. We report an original approach employing rational design of domain architecture of [...] Read more.
Triticain-α is a papain-like cysteine protease from wheat (Triticum aestivum L.) that possesses activity towards toxic gluten-derived peptides, and was thus proposed as a novel therapeutic tool for celiac disease. We report an original approach employing rational design of domain architecture of Triticain-α and selection of the appropriate expression system for development of cheap and efficient protocol yielding active recombinant enzyme. The segregated catalytic domain of Triticain-α did not adopt native structure in bacteria, neither being expressed as a single protein nor upon conjugation or co-expression with extrinsic chaperones. Meanwhile, its attachment to prodomain of the enzyme resulted in generation of insoluble (inclusion bodies) product that can be transformed into active protease upon refolding in vitro. The estimated yield of the product was affected by affinity six-histidine tag required for its single-step purification with the preferable N-terminal position of the tag. Expression of the two-domain Triticain-α construct in yeast (Pichia pastoris) strain GS115 and bacterial (Escherichia coli) strain Rosetta gami B (DE3) led to the accumulation of a soluble protein, which underwent autocatalytic maturation during expression (in yeast)/purification (in bacteria) procedures and exhibited pronounced protease activity. Furthermore, expression and solubility of such construct in Rosetta gami B (DE3) cells was improved by reducing the temperature of the bacterial growth yielding more active enzyme than yeast counterpart presumably due to facilitated formation of a characteristic disulfide bond critical for maintaining the catalytic site. We suggest that these findings are helpful for obtaining active Triticain-α preparations for scientific or medical applications, and can be employed for the design and production of beneficial recombinant products based on other papain-like cysteine proteases. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Review

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Open AccessReview
Expression of Lectins in Heterologous Systems
Int. J. Mol. Sci. 2018, 19(2), 616; https://doi.org/10.3390/ijms19020616
Received: 4 December 2017 / Revised: 24 December 2017 / Accepted: 27 December 2017 / Published: 21 February 2018
Cited by 1 | PDF Full-text (2361 KB) | HTML Full-text | XML Full-text
Abstract
Lectins are proteins that have the ability to recognize and bind in a reversible and specific way to free carbohydrates or glycoconjugates of cell membranes. For these reasons, they have been extensively used in a wide range of industrial and pharmacological applications. Currently, [...] Read more.
Lectins are proteins that have the ability to recognize and bind in a reversible and specific way to free carbohydrates or glycoconjugates of cell membranes. For these reasons, they have been extensively used in a wide range of industrial and pharmacological applications. Currently, there is great interest in their production on a large scale. Unfortunately, conventional techniques do not provide the appropriate platform for this purpose and therefore, the heterologous production of lectins in different organisms has become the preferred method in many cases. Such systems have the advantage of providing better yields as well as more homogeneous and better-defined properties for the resultant products. However, an inappropriate choice of the expression system can cause important structural alterations that have repercussions on their biological activity since the specificity may lay in their post-translational processing, which depends largely on the producing organism. The present review aims to examine the most representative studies in the area, exposing the four most frequently used systems (bacteria, yeasts, plants and animal cells), with the intention of providing the necessary information to determine the strategy to follow in each case as well as their respective advantages and disadvantages. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessReview
Fusion Proteins of NKG2D/NKG2DL in Cancer Immunotherapy
Int. J. Mol. Sci. 2018, 19(1), 177; https://doi.org/10.3390/ijms19010177
Received: 30 November 2017 / Revised: 22 December 2017 / Accepted: 3 January 2018 / Published: 7 January 2018
Cited by 4 | PDF Full-text (695 KB) | HTML Full-text | XML Full-text
Abstract
NKG2D (natural killer group 2, member D) is an important activating receptor in natural killer (NK) cells and some T cells. NKG2D ligands (NKG2DLs) are specifically expressed on most tumor cells. The engagement of these ligands on tumor cells to NKG2D on NK [...] Read more.
NKG2D (natural killer group 2, member D) is an important activating receptor in natural killer (NK) cells and some T cells. NKG2D ligands (NKG2DLs) are specifically expressed on most tumor cells. The engagement of these ligands on tumor cells to NKG2D on NK cells will induce cell-mediated cytotoxicity and have target cells destroyed. This gives NKG2D/NKG2DLs great potential in cancer therapeutic application. The creation of NKG2D/NKG2DL-based multi-functional fusion proteins is becoming one of the most promising strategies in immunotherapy for cancer. Antibodies, cytokines, and death receptors have been fused with NKG2D or its ligands to produce many powerful fusion proteins, including NKG2D-based chimeric antigen receptors (CARs). In this article, we review the recent developments of the fusion proteins with NKG2D/NKG2DL ligands in cancer immunotherapy. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessReview
Development of Antibody Therapeutics against Flaviviruses
Int. J. Mol. Sci. 2018, 19(1), 54; https://doi.org/10.3390/ijms19010054
Received: 30 November 2017 / Revised: 20 December 2017 / Accepted: 22 December 2017 / Published: 25 December 2017
Cited by 8 | PDF Full-text (1925 KB) | HTML Full-text | XML Full-text
Abstract
Recent outbreaks of Zika virus (ZIKV) highlight the urgent need to develop efficacious interventions against flaviviruses, many of which cause devastating epidemics around the world. Monoclonal antibodies (mAb) have been at the forefront of treatment for cancer and a wide array of other [...] Read more.
Recent outbreaks of Zika virus (ZIKV) highlight the urgent need to develop efficacious interventions against flaviviruses, many of which cause devastating epidemics around the world. Monoclonal antibodies (mAb) have been at the forefront of treatment for cancer and a wide array of other diseases due to their specificity and potency. While mammalian cell-produced mAbs have shown promise as therapeutic candidates against several flaviviruses, their eventual approval for human application still faces several challenges including their potential risk of predisposing treated patients to more severe secondary infection by a heterologous flavivirus through antibody-dependent enhancement (ADE). The high cost associated with mAb production in mammalian cell cultures also poses a challenge for the feasible application of these drugs to the developing world where the majority of flavivirus infection occurs. Here, we review the current therapeutic mAb candidates against various flaviviruses including West Nile (WNV), Dengue virus (DENV), and ZIKV. The progress of using plants for developing safer and more economical mAb therapeutics against flaviviruses is discussed within the context of their expression, characterization, downstream processing, neutralization, and in vivo efficacy. The progress of using plant glycoengineering to address ADE, the major impediment of flavivirus therapeutic development, is highlighted. These advancements suggest that plant-based systems are excellent alternatives for addressing the remaining challenges of mAb therapeutic development against flavivirus and may facilitate the eventual commercialization of these drug candidates. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
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