Recently, transformation of chloroplast genome has been used for the production of heterologous proteins. We transformed tobacco chloroplasts with two different versions of the storage protein of Phaseolus vulgaris, phaseolin (with or without signal peptide), in which a cysteine residue has been added to its C-terminal region. This modification allows for the formation of inter-chain disulfide bonds. The aim is to demonstrate the different ability of chloroplast compartments (stroma and thylakoids) in the formation of phaseolin polypeptides held together by disulfide bonds. The presence of the signal peptide should direct phaseolin into the thylakoid compartment, where the protein is able to form disulfide bridges and high molecular weight polymers[1]. It is then necessary to assess whether the extracted proteic matter consists of polymer, protein oligomers, or both. To verify the effect of the peptide modification and to quantify the polymer formation, we employed hollow-fiber flow field-flow fractionation coupled to UV and multi-angle laser scattering detection (HF5-UV-MALS). HF5 allows for the selective size-based separation of nano- and micro-sized particulate, while smaller species are filtered away in the pre-separation step[2]. Hence, HF5-UV-MALS showed its effectiveness both in sample purification and characterization of fractionated species in terms of spectroscopic behavior and molar mass. We observed that both stroma and thylakoid-derived extracts contained phaseolin polymer (with a molar mass above 1 MDa), whereas PBS proved to be a more efficient extraction solvent than alcoholic mixtures. The formation of phaseolin polymers in these plant compartments, not detected in P. vulgaris, could be very interesting for industrial purposes. Chloroplasts could be employed as a reactor to produce a biopolymer derived from an edible protein. A possible application is the production of biodegradable films.
Characterization of a single cystein-enriched phaseolin expressed in transplastomic tobacco plants
F De Marchis;M Bellucci;A Pompa
2018
Abstract
Recently, transformation of chloroplast genome has been used for the production of heterologous proteins. We transformed tobacco chloroplasts with two different versions of the storage protein of Phaseolus vulgaris, phaseolin (with or without signal peptide), in which a cysteine residue has been added to its C-terminal region. This modification allows for the formation of inter-chain disulfide bonds. The aim is to demonstrate the different ability of chloroplast compartments (stroma and thylakoids) in the formation of phaseolin polypeptides held together by disulfide bonds. The presence of the signal peptide should direct phaseolin into the thylakoid compartment, where the protein is able to form disulfide bridges and high molecular weight polymers[1]. It is then necessary to assess whether the extracted proteic matter consists of polymer, protein oligomers, or both. To verify the effect of the peptide modification and to quantify the polymer formation, we employed hollow-fiber flow field-flow fractionation coupled to UV and multi-angle laser scattering detection (HF5-UV-MALS). HF5 allows for the selective size-based separation of nano- and micro-sized particulate, while smaller species are filtered away in the pre-separation step[2]. Hence, HF5-UV-MALS showed its effectiveness both in sample purification and characterization of fractionated species in terms of spectroscopic behavior and molar mass. We observed that both stroma and thylakoid-derived extracts contained phaseolin polymer (with a molar mass above 1 MDa), whereas PBS proved to be a more efficient extraction solvent than alcoholic mixtures. The formation of phaseolin polymers in these plant compartments, not detected in P. vulgaris, could be very interesting for industrial purposes. Chloroplasts could be employed as a reactor to produce a biopolymer derived from an edible protein. A possible application is the production of biodegradable films.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
