One-Step Protein Purification: Use of a Novel Epitope Tag for Highly Efficient Detection and Purification of Recombinant Proteins

Rasche, Stefan

doi:10.2174/1874070701105010001

Cited by 16 publications

(21 citation statements)

References 18 publications

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“…To confirm that active cationic α‐helical antimicrobial peptides can be made to accumulate in transgenic plants, we developed a strategy based on the fusion of BP100 to the Discosoma spp. red fluorescent reporter protein DsRed (Matz et al ., ) and the epitope tag54 sequence for the detection of recombinant proteins using the specific antibody mAb54 (Rasche et al ., ).…”

Section: Resultsmentioning

confidence: 97%

The production of recombinant cationic α‐helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum

Nadal

Paz

Martinez

et al. 2013

Plant Biotechnology Journal

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Summary Synthetic linear antimicrobial peptides with cationic α‐helical structures, such as BP100, are valuable as novel therapeutics and preservatives. However, they tend to be toxic when expressed at high levels as recombinant peptides in plants, and they can be difficult to detect and isolate from complex plant tissues because they are strongly cationic and display low extinction coefficient and extremely limited immunogenicity. We therefore expressed BP100 with a C‐terminal tag which preserved its antimicrobial activity and demonstrated significant accumulation in plant cells. We used a fluorescent tag to trace BP100 following transiently expression in Nicotiana benthamiana leaves and showed that it accumulated in large vesicles derived from the endoplasmic reticulum (ER) along with typical ER luminal proteins. Interestingly, the formation of these vesicles was induced by BP100. Similar vesicles formed in stably transformed Arabidopsis thaliana seedlings, but the recombinant peptide was toxic to the host during latter developmental stages. This was avoided by selecting active BP100 derivatives based on their low haemolytic activity even though the selected peptides remained toxic to plant cells when applied exogenously at high doses. Using this strategy, we generated transgenic rice lines producing active BP100 derivatives with a yield of up to 0.5% total soluble protein.

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Section: Resultsmentioning

confidence: 97%

The production of recombinant cationic α‐helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum

Nadal

Paz

Martinez

et al. 2013

Plant Biotechnology Journal

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“…The sequence of the epitope tag, tag54, was c-terminally fused to the gene encoding phl p 1 to enable its detection via the tag54-specific monoclonal antibody mab54 [31]. The constructs used in the present study were introduced into A. tumefaciens GV3101 using a Multiporator (Eppendorf, Hamburg Germany) according to the manufacturer's instructions (Protocol No.…”

Section: Methodsmentioning

confidence: 99%

Tackling Heterogeneity: A Leaf Disc-Based Assay for the High-Throughput Screening of Transient Gene Expression in Tobacco

2012

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Transient Agrobacterium-mediated gene expression assays for Nicotiana tabacum (N. tabacum) are frequently used because they facilitate the comparison of multiple expression constructs regarding their capacity for maximum recombinant protein production. However, for three model proteins, we found that recombinant protein accumulation (rpa) was significantly influenced by leaf age and leaf position effects. The ratio between the highest and lowest amount of protein accumulation (max/min ratio) was found to be as high as 11. Therefore, construct-based impacts on the rpa level that are less than 11-fold will be masked by background noise. To address this problem, we developed a leaf disc-based screening assay and infiltration device that allows the rpa level in a whole tobacco plant to be reliably and reproducibly determined. The prototype of the leaf disc infiltration device allows 14 Agrobacterium-mediated infiltration events to be conducted in parallel. As shown for three model proteins, the average max/min rpa ratio was reduced to 1.4 using this method, which allows for a sensitive comparison of different genetic elements affecting recombinant protein expression.

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“…Unmodified mAbFL 6 and biotinylated mAbFL 6 were flushed over a gold surface functionalized with a RaM-Fc, and the change in reflectivity was monitored over time. The nonspecific monoclonal antibody mAb54k was captured in parallel as a negative control (49).…”

Section: Resultsmentioning

confidence: 99%

Simple and Portable Magnetic Immunoassay for Rapid Detection and Sensitive Quantification of Plant Viruses

Rettcher

Jungk

Kühn

et al. 2015

Appl Environ Microbiol

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c Plant pathogens cause major economic losses in the agricultural industry because late detection delays the implementation of measures that can prevent their dissemination. Sensitive and robust procedures for the rapid detection of plant pathogens are therefore required to reduce yield losses and the use of expensive, environmentally damaging chemicals. Here we describe a simple and portable system for the rapid detection of viral pathogens in infected plants based on immunofiltration, subsequent magnetic detection, and the quantification of magnetically labeled virus particles. Grapevine fanleaf virus (GFLV) was chosen as a model pathogen. Monoclonal antibodies recognizing the GFLV capsid protein were immobilized onto immunofiltration columns, and the same antibodies were linked to magnetic nanoparticles. GFLV was quantified by immunofiltration with magnetic labeling in a double-antibody sandwich configuration. A magnetic frequency mixing technique, in which a two-frequency magnetic excitation field was used to induce a sum frequency signal in the resonant detection coil, corresponding to the virus concentration within the immunofiltration column, was used for high-sensitivity quantification. We were able to measure GFLV concentrations in the range of 6 ng/ml to 20 g/ml in less than 30 min. The magnetic immunoassay could also be adapted to detect other plant viruses, including Potato virus X and Tobacco mosaic virus, with detection limits of 2 to 60 ng/ml.

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One-Step Protein Purification: Use of a Novel Epitope Tag for Highly Efficient Detection and Purification of Recombinant Proteins

Cited by 16 publications

References 18 publications

The production of recombinant cationic α‐helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum

The production of recombinant cationic α‐helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum

Tackling Heterogeneity: A Leaf Disc-Based Assay for the High-Throughput Screening of Transient Gene Expression in Tobacco

Simple and Portable Magnetic Immunoassay for Rapid Detection and Sensitive Quantification of Plant Viruses

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