Membrane-Based Inverse Transition Cycling: An Improved Means for Purifying Plant-Derived Recombinant 
											Protein-Elastin-Like Polypeptide Fusions

Phan, Hoang Trong; Conrad, Udo

doi:10.3390/ijms12052808

Cited by 39 publications

(48 citation statements)

References 39 publications

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“…In another study, Phan et al . 27 produced both ELP-HA and -NA fusion proteins in tobacco plants, and demonstrated their in vitro antigenic properties. Apart from influenza, ELP technology has also been extended to tuberculosis vaccine.…”

Section: Resultsmentioning

confidence: 99%

“…20,22–26 More recently, ELPylation, the process of recombinantly fusing proteins to ELP has been used to purify proteins including influenza antigens. 27,28 Mycobacterium tuberculosis antigens, 29 antibody fragments 30 and complete antibodies. 31 Protein purification is simply achieved by cycling the ELPylated protein solution above the T t to cause protein precipitation, centrifugation to remove the contaminated supernatant, and then lowering the temperature below T t to redissolve the ELPylated protein molecules in fresh buffer.…”

Section: Introductionmentioning

confidence: 99%

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Synthesis and Immunogenicity Assessment of Elastin-Like Polypeptide-M2e Construct as an Influenza Antigen

et al. 2014

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The 23 amino acid-long extracellular domain of the influenza virus transmembrane protein M2 (M2e) has remained highly conserved since the 1918 pandemic, and is thus considered a good candidate for development of a universal influenza A vaccine. However, M2e is poorly immunogenic. In this study we assessed the potential of increasing immunogenicity of M2e by constructing a nanoscale-designed protein polymer containing the M2e sequence and an elastin-like polypeptide (ELP) nanodomain consisting of alanine and tyrosine guest residues (ELP(A2YA2)24). The ELP nanodomain was included to increase antigen size, and to exploit the inherent thermal inverse phase transition behavior of ELPs to purify the protein polymer. The ELP(A2YA2)24 + M2e nanodomained molecule was recombinantly synthesized. Characterization of its inverse phase transition behavior demonstrated that attachment of M2e to ELP(A2YA2)24 increased its transition temperature compared to ELP(A2YA2)24. Using a dot blot test we determined that M2e conjugated to ELP is recognizable by M2e–specific antibodies, suggesting that the conjugation process does not adversely affect the immunogenic property of M2e. Further, upon vaccinating mice with ELP(A2YA2)24 + M2e it was found that indeed the nanodomained protein enhanced M2e–specific antibodies in mouse serum compared to free M2e peptide and ELP(A2YA2)24. The immune serum could also recognize M2 expressed on influenza virions. Overall, this data suggests the potential of using molecules containing M2e–ELP nano-domains to develop a universal influenza vaccine.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Synthesis and Immunogenicity Assessment of Elastin-Like Polypeptide-M2e Construct as an Influenza Antigen

et al. 2014

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“…The aims of heat treatment are the inactivation of proteases and the removal of HCPs by precipitation. Heat treatment has been successfully used for the purification of ELPylated hemagglutinin and ELPylated recombinant antibodies from leaves by mITC [40]. Heat treatment of plant leaves via blanching in a boiling solvent has been used to effectively remove HCPs during the purification of recombinant proteins produced in tobacco [35].…”

Section: Discussionmentioning

confidence: 99%

Low-Tech, Pilot Scale Purification of a Recombinant Spider Silk Protein Analog from Tobacco Leaves

Heppner

Weichert

Schierhorn

et al. 2016

IJMS

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Spider dragline is used by many members of the Araneae family not only as a proteinogenic safety thread but also for web construction. Spider dragline has been shown to possess high tensile strength in combination with elastic behavior. This high tensile strength can be attributed to the presence of antiparallel β-sheets within the thread; these antiparallel β-sheets are why the protein is classified as a silk. Due to the properties of spider silk and its technical and medical uses, including its use as a suture material and as a scaffold for tissue regeneration, spider dragline is a focus of the biotechnology industry. The production of sufficient amounts of spider silk is challenging, as it is difficult to produce large quantities of fibers because of the cannibalistic behavior of spiders and their large spatial requirements. In recent years, the heterologous expression of genes coding for spider silk analogs in various hosts, including plants such as Nicotiana tabacum, has been established. We developed a simple and scalable method for the purification of a recombinant spider silk protein elastin-like peptide fusion protein (Q-/K-MaSp1-100× ELP) after heterologous production in tobacco leaves involving heat and acetone precipitation. Further purification was performed using centrifugal Inverse Transition Cycling (cITC). Up to 400 mg of highly pure spider silk protein derivatives can be isolated from six kilograms of tobacco leaves, which is the highest amount of silk protein derivatives purified from plants thus far.

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“…Another substitution is to replace the centrifugation step by a microfiltration step in order to reduce any potential proteolysis due to mechanical cleavage, during the centrifugation step and to maintain high protein stability and activity (Ge et al, 2006). Using this modified approach, two avian flu H5N1 antigens have been recently purified from transgenic tobacco (Phan & Conrad, 2011). The attachment of cysteinerich and low-molecular mass hydrophobin family members is another method to facilitate the protein purification.…”

Section: Non-chromatography Methodsmentioning

confidence: 98%

Molecular farming on rescue of pharma industry for next generations

Moustafa

Makhzoum

Trémouillaux-Guiller

2015

Critical Reviews in Biotechnology

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Recombinant proteins expressed in plants have been emerged as a novel branch of the biopharmaceutical industry, offering practical and safety advantages over traditional approaches. Cultivable in various platforms (i.e. open field, greenhouses or bioreactors), plants hold great potential to produce different types of therapeutic proteins with reduced risks of contamination with human and animal pathogens. To maximize the yield and quality of plant-made pharmaceuticals, crucial factors should be taken into account, including host plants, expression cassettes, subcellular localization, post-translational modifications, and protein extraction and purification methods. DNA technology and genetic transformation methods have also contributed to great parts with substantial improvements. To play their proper function and stability, proteins require multiple post-translational modifications such as glycosylation. Intensive glycoengineering research has been performed to reduce the immunogenicity of recombinant proteins produced in plants. Important strategies have also been developed to minimize the proteolysis effects and enhance protein accumulation. With growing human population and new epidemic threats, the need for new medications will be paramount so that the traditional pharmaceutical industry will not be alone to answer medication demands for upcoming generations. Here, we review several aspects of plant molecular pharming and outline some important challenges that hamper these ambitious biotechnological developments.

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Membrane-Based Inverse Transition Cycling: An Improved Means for Purifying Plant-Derived Recombinant Protein-Elastin-Like Polypeptide Fusions

Cited by 39 publications

References 39 publications

Synthesis and Immunogenicity Assessment of Elastin-Like Polypeptide-M2e Construct as an Influenza Antigen

Synthesis and Immunogenicity Assessment of Elastin-Like Polypeptide-M2e Construct as an Influenza Antigen

Low-Tech, Pilot Scale Purification of a Recombinant Spider Silk Protein Analog from Tobacco Leaves

Molecular farming on rescue of pharma industry for next generations

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