Limits...
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.

Show MeSH

Related in: MedlinePlus

mITC purification, as demonstrated by Western blot analysis. (a) NtN1-ELP; (b) NtHA1-ELP; (c) Ntanti-hTNFα-VHH-ELP. RE: Raw extract; Sm: Supernatant raw extract passed through a 0.2 μm cellulose acetate membrane at room temperature; Pm: Proteins eluted from the membrane by ice-cold water. Arrows indicate the target recombinant proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-ijms-12-02808: mITC purification, as demonstrated by Western blot analysis. (a) NtN1-ELP; (b) NtHA1-ELP; (c) Ntanti-hTNFα-VHH-ELP. RE: Raw extract; Sm: Supernatant raw extract passed through a 0.2 μm cellulose acetate membrane at room temperature; Pm: Proteins eluted from the membrane by ice-cold water. Arrows indicate the target recombinant proteins.

Mentions: To overcome the proteolytic cleavage and/or partial denaturation of the ELPylated protein which affected the cITC purification (probably caused by a higher than optimal temperature regime), we adapted the filter-based ITC method described by Ge et al. [21]. For each of the three transgenic products (NtHA1-ELP, NtN1-ELP and Ntanti-hTNFα-VHH-ELP), the expected size molecule was successfully purified (Figure 3). Losses due to filtration at room temperature and the presence of 2 M NaCl were either insignificant (Figure 3a,b) or, at worst, minimal (Figure 3c). Since the re-solubilization rate was very high, the mITC purification efficiency was excellent (Table 2). For NA in particular, the procedure led to a large improvement in the degree of purity achieved (Figure 4a). For hemagglutinin, some proteolytic cleavage still occurred, but most of the product remained intact (Figure 4b). The robustness of the mITC method was proven by repeating the procedure four times for each target (Figure 5).


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)

mITC purification, as demonstrated by Western blot analysis. (a) NtN1-ELP; (b) NtHA1-ELP; (c) Ntanti-hTNFα-VHH-ELP. RE: Raw extract; Sm: Supernatant raw extract passed through a 0.2 μm cellulose acetate membrane at room temperature; Pm: Proteins eluted from the membrane by ice-cold water. Arrows indicate the target recombinant proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-ijms-12-02808: mITC purification, as demonstrated by Western blot analysis. (a) NtN1-ELP; (b) NtHA1-ELP; (c) Ntanti-hTNFα-VHH-ELP. RE: Raw extract; Sm: Supernatant raw extract passed through a 0.2 μm cellulose acetate membrane at room temperature; Pm: Proteins eluted from the membrane by ice-cold water. Arrows indicate the target recombinant proteins.
Mentions: To overcome the proteolytic cleavage and/or partial denaturation of the ELPylated protein which affected the cITC purification (probably caused by a higher than optimal temperature regime), we adapted the filter-based ITC method described by Ge et al. [21]. For each of the three transgenic products (NtHA1-ELP, NtN1-ELP and Ntanti-hTNFα-VHH-ELP), the expected size molecule was successfully purified (Figure 3). Losses due to filtration at room temperature and the presence of 2 M NaCl were either insignificant (Figure 3a,b) or, at worst, minimal (Figure 3c). Since the re-solubilization rate was very high, the mITC purification efficiency was excellent (Table 2). For NA in particular, the procedure led to a large improvement in the degree of purity achieved (Figure 4a). For hemagglutinin, some proteolytic cleavage still occurred, but most of the product remained intact (Figure 4b). The robustness of the mITC method was proven by repeating the procedure four times for each target (Figure 5).

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