Protein purification, from plants

Witte, C. P., Noel, L. D., Gielbert, J., Parker, J. E. and Romeis, T. (2004). Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope. Plant Mol. Biol. 55, 135-47. 

Proteins produced in plant cells can remain within the cell or are secreted into the apoplast via the bulk transport (secretory) pathway. In whole plants, because levels of protein accumulated intracellularly, e. g. using the KDEL sequence to ensure retention in the endoplasmic reticulum, are often higher than when the product is secreted [58], foreign proteins are generally not directed for secretion. However, as protein purification from plant biomass is potentially much more difficult and expensive than protein recovery from culture medium, protein secretion is considered an advantage in tissue culture systems. For economic harvesting from the medium, the protein should be stable once secreted and should accumulate to high levels in the extracellular environment. 

The extraction and purification of proteins from organisms or biological tissue can be a laborious and expensive process, and often represents the principal reason why vaccines and other therapeutic agents reach costs that become unattainable for many. Downstream processing also can be a major obstacle with respect to cost for large-scale protein manufacturing in plants. However, purification from plant tissues, while still costly, is in general less expensive than purification from their mammalian and bacterial counterparts. Indeed, some plant-derived biopharmaceuticals, such as topically applied monoclonal antibodies, may require only partial purification and thus be even less intensive in terms of labor and cost. 

Rohila JS, Chen M, Cerny R, Fromm ME (2004) Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. Plant J 38(1) 172-181.doi 10.1111/j.l365-313X.2004.02031.x... 

Bonk, M. et al.. Purification and characterization of chaperonin 60 and heat-shock protein 70 from chromoplast of Narcissus pseudonarcissus involvement of heat-shock protein 70 in a soluble protein complex containing phytoene desaturase. Plant Physiol. Ill, 931, 1996. 

LSBC has a long history of process development, scale-up and manufacturing experience, since its foundation back in 1987. The company has developed proprietary, industrial-scale extraction processes for the purification of proteins, peptides, and other biochemicals from plant biomass. It has also developed commercial methods for the extraction and purification of secreted plant proteins. 

Extensive research has been carried out into the molecular aspects of foreign protein production in whole plants to enhance the yield, quality and stability of the product and to facilitate protein separation and purification from the biomass [3, 6, 9], In contrast, comparatively little research has been undertaken to investigate the... 

Spider Silk and Spider Silk-ELP Fusion Proteins from Plants Expression, Purification and Applications... 

Spider silk proteins from plants remain soluble at high temperatures, allowing them to be enriched by boiling [26]. In order to enrich the spider silk-ELP fusion protein, we therefore exposed tobacco leaf extracts to heat treatment at 95°C for 60 min and then cleared the supernatant by centrifugation. In further steps, the reversible precipitation behavior of ELP fusion proteins was exploited to develop a suitable purification strategy. For the selective precipitation of SOl-lOOxELP, NaCl was added at a final concentration of 2 M and the temperature was increased to 60 °C. In this man-... 

Fig. 11.3 Purification ofSOl-lOOxELP-proteins from transgenic tobacco plants by inverse transition cycling and analysis by SDS-PAGE. 1 15 pg of total soluble leaf protein extracted in raw extract buffer 2 cleared supernatant of original 15 pg total soluble leaf protein after heat treatment (60 min, 95 °C) 3 cleared supernatant of original 300 pg leaf protein after heat treatment 4 cleared supernatant of original 300 pg leaf protein after heat treatment (60 min, 60 °C) with 2 M NaCI 5 redissolved spider silk-elastin protein pellet from original 300 pg of total soluble leaf protein after heat treatment (60 min, 60 °C) with 2 M NaCI.

Direct injection API-Electrospray MS is capable of analyzing much larger and less volatile substances than either EI/MS or CI/MS. As a result, this method is often used to provide structural information on peptides, proteins, and polymers derived from both natural and synthetic processes it is also useful in the analysis of many natural compounds including molecules such as saponins and flavonol glycosides, derived from plants. When using direct injection API-electrospray, partial purification and EC preparation are performed elsewhere and a collected fraction is dissolved in an appropriate solvent and injected as a bolus into the mass spectrometer (flow or direct injection or syringe infusion). This has an advantage, as the mass... 

Preparation of Other Antigenic Materials. Cotton plant tissues (stem, leaf, burr), cotton gin trash, baled cotton, clean cotton lint, both hand picked in the field and from plants grown in the greenhouse, cottonseed proteins, cottonseed hulls, house dust, and flax, soft hemp, sisal, and jute fibers, were extracted with deionized water. The purification process was, however, stopped to correspond to f-3 (see Figure 1). 

Protein purification has a >200-year history the first attempts at isolating substances from plants having similar properties to egg albumen, or egg white, were reported in 1789 by Fourcroy. Many proteins from plants were purified in the nineteenth century, though most would not be considered pure by modern standards. A century later, ovalbumin was the first crystalline protein obtained (by Hofmeister in 1889). The year 1989 may not go down in history as a milestone in protein chemistry, but since then there has been a resurgence of interest in proteins after more than a decade of gene excitement. 

Comparison of the predicted amino acid sequence of the protein encoded by pTOM 13 with the sequence of flavanone 3-hydroxylase showed considerable similarity (A. Prescott and C. Martin, personal communication, cited in Hamilton etal., 1990). This is particularly interesting, since it has been suggested that the conversion of ACC to ethylene might involve a hydroxylation reaction (Yang, 1985). Armed with this information and using conditions developed for extraction and purification of flavanone 3-hydroxylase, Ververidis John (1991) showed that it is possible to solubilise the ethylene forming enzyme from plants and retain full... 

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