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Membrane-based inverse transition cycling: an improved means for purifying plant-derived recombinant protein-elastin-like polypeptide fusions.

Phan HT, Conrad U - Int J Mol Sci (2011)

Bottom Line: The presence of the ELP tag enhanced the accumulation of the heterologous proteins in the tobacco leaves.An effective membrane-based Inverse Transition Cycling was developed to recover the ELPylated antigens and antibodies from plant material.The functionality of both the ELPylated neuraminidase and an ELPylated nanobody was demonstrated.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, Gatersleben 06466, Germany; E-Mail: hoang@ipk-gatersleben.de.

ABSTRACT
Elastin-like peptide (ELP) was fused to two different avian flu H5N1 antigens and expressed in transgenic tobacco plants. The presence of the ELP tag enhanced the accumulation of the heterologous proteins in the tobacco leaves. An effective membrane-based Inverse Transition Cycling was developed to recover the ELPylated antigens and antibodies from plant material. The functionality of both the ELPylated neuraminidase and an ELPylated nanobody was demonstrated.

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The binding behavior of Ntanti-hTNFα-VHH-ELP to human TNFα, as assessed by competitive ELISA. Ntanti-hTNFα-VHH-ELP was mixed with various concentrations of hTNFα. The solid phase binding of c-myc tagged Ntanti-hTNFα-VHH-ELP to hTNFα was quantified. Binding without competition from free hTNFα was set to 100%. Standard deviations shown as bars.
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f6-ijms-12-02808: The binding behavior of Ntanti-hTNFα-VHH-ELP to human TNFα, as assessed by competitive ELISA. Ntanti-hTNFα-VHH-ELP was mixed with various concentrations of hTNFα. The solid phase binding of c-myc tagged Ntanti-hTNFα-VHH-ELP to hTNFα was quantified. Binding without competition from free hTNFα was set to 100%. Standard deviations shown as bars.

Mentions: Purification procedures need to be optimized not only with respect to their recovery efficiency, but also to maintain biological activity. The three ELPylated proteins expressed in planta and purified by mITC were highly soluble even at relatively high concentrations. The enzymatic activity of the NA, assessed on the basis of its ability to cleave 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid, was over four fold that of the cITC purified equivalent (Table 3). The binding behavior of the mITC purified nanobody Ntanti-hTNFα-VHH-ELP was investigated by a competitive ELISA, in which it exhibited a dissociation constant of ∼4.5 nM (Figure 6), comparable to the behavior of its cITC purified equivalent (4 nM, [19]).


Membrane-based inverse transition cycling: an improved means for purifying plant-derived recombinant protein-elastin-like polypeptide fusions.

Phan HT, Conrad U - Int J Mol Sci (2011)

The binding behavior of Ntanti-hTNFα-VHH-ELP to human TNFα, as assessed by competitive ELISA. Ntanti-hTNFα-VHH-ELP was mixed with various concentrations of hTNFα. The solid phase binding of c-myc tagged Ntanti-hTNFα-VHH-ELP to hTNFα was quantified. Binding without competition from free hTNFα was set to 100%. Standard deviations shown as bars.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3116158&req=5

f6-ijms-12-02808: The binding behavior of Ntanti-hTNFα-VHH-ELP to human TNFα, as assessed by competitive ELISA. Ntanti-hTNFα-VHH-ELP was mixed with various concentrations of hTNFα. The solid phase binding of c-myc tagged Ntanti-hTNFα-VHH-ELP to hTNFα was quantified. Binding without competition from free hTNFα was set to 100%. Standard deviations shown as bars.
Mentions: Purification procedures need to be optimized not only with respect to their recovery efficiency, but also to maintain biological activity. The three ELPylated proteins expressed in planta and purified by mITC were highly soluble even at relatively high concentrations. The enzymatic activity of the NA, assessed on the basis of its ability to cleave 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid, was over four fold that of the cITC purified equivalent (Table 3). The binding behavior of the mITC purified nanobody Ntanti-hTNFα-VHH-ELP was investigated by a competitive ELISA, in which it exhibited a dissociation constant of ∼4.5 nM (Figure 6), comparable to the behavior of its cITC purified equivalent (4 nM, [19]).

Bottom Line: The presence of the ELP tag enhanced the accumulation of the heterologous proteins in the tobacco leaves.An effective membrane-based Inverse Transition Cycling was developed to recover the ELPylated antigens and antibodies from plant material.The functionality of both the ELPylated neuraminidase and an ELPylated nanobody was demonstrated.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, Gatersleben 06466, Germany; E-Mail: hoang@ipk-gatersleben.de.

ABSTRACT
Elastin-like peptide (ELP) was fused to two different avian flu H5N1 antigens and expressed in transgenic tobacco plants. The presence of the ELP tag enhanced the accumulation of the heterologous proteins in the tobacco leaves. An effective membrane-based Inverse Transition Cycling was developed to recover the ELPylated antigens and antibodies from plant material. The functionality of both the ELPylated neuraminidase and an ELPylated nanobody was demonstrated.

Show MeSH
Related in: MedlinePlus