Purification and characterization of the highly toxic lectin modeccin.

Olsnes, Sjur; Haylett, Thomas; Refsnes, Karin

doi:10.1016/s0021-9258(17)34658-6

Cited by 64 publications

(5 citation statements)

References 14 publications

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“…Accordingly, the combined pH and heat treatment may have unintentionally triggered the precipitation of FTH1 along with the HCPs, as previously shown for other proteins (Opdensteinen, Lobanov, et al, 2021). Previous studies have also shown that target proteins can coprecipitate with HCPs (Olsnes et al, 1978), and this could also explain the loss of FTH1 at high temperatures with long incubation times.…”

Section: Resultsmentioning

confidence: 87%

“…Comparative production cost analysis was difficult due to limited data availability, but suggested that FTH1 from plants would achieve a substantial sales margin. Plants are therefore suitable as a production platform to meet the growing demand for ferritin as a nanocarrier (Olsnes et al, 1978), for example, to deliver cancer therapeutics (Chiou & Connor, 2018) or antigen‐based vaccines, such as those indicated for COVID‐19 (http://ClinicalTrials.gov Identifier: NCT04784767) (Ma et al, 2020; Powell et al, 2021). The low‐cost production of untagged FTH1 in plants would facilitate clinical translation as well as the subsequent large‐scale, cGMP‐compliant production of ferritin (Buyel et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…The FTH1 protein can also be modified, for example, with additional peptides displayed on the outer surface of assembled particles (X. Zhang et al, 2019), to improve solubility, (thermo)stability (Kim et al, 2001), or immunogenicity by using in silico protein engineering approaches (Olsnes et al, 1978; X. Sun et al, 2021; X. Zhang et al, 2019) that can be tested in PCPs.…”

Section: Discussionmentioning

confidence: 99%

“…The assembled expression vectors (Supporting Information Table S3) were propagated in E. coli DH5a cells (New England Biolabs) in lysogeny broth (10 g L −1 tryptone, 5 g L −1 yeast extract, 10 g L −1 sodium chloride, 100 mg L −1 ampicillin, pH 7.0) at 37°C for 24 h, shaking at 160 rpm. Electrocompetent R. radiobacter ( A. tumefaciens ) strain GV3101:pMP90RK cells were transformed with purified plasmid DNA by electroporation as previously described (Olsnes et al, 1978).…”

Section: Methodsmentioning

confidence: 99%

“…Several antibodies, antibody–toxin fusions, enzymes, and vaccine candidates have been produced in plants according to current good manufacturing practices (cGMP), and some have reached late‐stage clinical development and approval (Knödler & Buyel, 2020; Komarova et al, 2011; Olsnes et al, 1978; Schillberg & Finnern, 2021; Ward et al, 2021). Similarly, the ability of plants to produce multimeric self‐assembling protein structures has been demonstrated for virus‐like particle vaccine candidates, which can also be adapted for the delivery of drugs and imaging reagents (Dickmeis et al, 2021; Marsian & Lomonossoff, 2016).…”

Section: Introductionmentioning

confidence: 99%

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Simple plant‐based production and purification of the assembled human ferritin heavy chain as a nanocarrier for tumor‐targeted drug delivery and bioimaging in cancer therapy

Knödler

Opdensteinen

Sankaranarayanan

et al. 2023

Biotech & Bioengineering

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Nanoparticles are used as carriers for the delivery of drugs and imaging agents.Proteins are safer than synthetic nanocarriers due to their greater biocompatibility and the absence of toxic degradation products. In this context, ferritin has the additional benefit of inherently targeting the membrane receptor transferrin 1, which is overexpressed by most cancer cells. Furthermore, this self-assembling multimeric protein can be loaded with more than 2000 iron atoms, as well as drugs, contrast agents, and other cargos. However, recombinant ferritin currently costs ~3.5 million € g −1 , presumably because the limited number of producers cannot meet demand, making it generally unaffordable as a nanocarrier. Because plants can produce proteins at very-large-scale, we developed a simple, proof-of-concept process for the production of the human ferritin heavy chain by transient expression in Nicotiana benthamiana. We optimized the protein yields by screening different compartments and 5′-untranslated regions in PCPs, and selected the bestperforming construct for production in differentiated plants. We then established a rapid and scalable purification protocol by combining pH and heat treatment before extraction, followed by an ultrafiltration/diafiltration size-based separation process. The optimized process achieved ferritin levels of ~40 mg kg −1 fresh biomass although depth filtration limited product recovery to ~7%. The purity of the recombinant product was >90% at costs ~3% of the current sales price. Our method therefore allows the production of affordable ferritin heavy chain as a carrier for

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Section: Resultsmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Simple plant‐based production and purification of the assembled human ferritin heavy chain as a nanocarrier for tumor‐targeted drug delivery and bioimaging in cancer therapy

Knödler

Opdensteinen

Sankaranarayanan

et al. 2023

Biotech & Bioengineering

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Site‐directed mutagenesis of ricin A‐chain and implications for the mechanism of action

Ready¹,

Kim²,

Robertus³

1991

Proteins

165

139

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Ricin A-chain is an N-glycosidase that attacks ribosomal RNA at a highly conserved adenine residue. The enzyme is representative of a large family of medically significant proteins used in the design of anticancer agents and in the treatment of HIV infection. The x-ray structure has been used as a guide to create several active site mutations by directed mutagenesis of the cloned gene. Glu177 is a key catalytic residue, and conversion to Gln reduces activity 180-fold. Asn209 is shown to participate in substrate binding by kinetic analysis. Conversion to Ser increases Km sixfold but has no effect on kcat. Conversion of Tyr80 and Tyr123 to Phe decreases activity by 15- and 7-fold respectively. A mechanism of action is proposed that involves binding of the substrate adenine in a syn configuration that resembles the transition state; the putative oxycarbonium ion is probably stabilized by interaction with Glu177.

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Toxic Lectins and Related Ribosome-inactivating Plant Proteins

Stirpe¹

1987

Selectivity and Molecular Mechanisms of Toxicity

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Purification and characterization of the highly toxic lectin modeccin.

Cited by 64 publications

References 14 publications

Simple plant‐based production and purification of the assembled human ferritin heavy chain as a nanocarrier for tumor‐targeted drug delivery and bioimaging in cancer therapy

Simple plant‐based production and purification of the assembled human ferritin heavy chain as a nanocarrier for tumor‐targeted drug delivery and bioimaging in cancer therapy

Site‐directed mutagenesis of ricin A‐chain and implications for the mechanism of action

Toxic Lectins and Related Ribosome-inactivating Plant Proteins

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