Purification and Characterization of Proteins

Methods of Protein Fractionation and Characterization Differential Centrifugation Divides a Sample into Two Fractions 

Column Procedures Are the Most Versatile and Productive Purification Methods Electrophoresis Is Used for Resolving Mixtures Sedimentation and Diffusion Are Used for Size and Shape Determination Protein Purification Procedures 

Proteins can be isolated and characterized according to their size, shape, and charge. 

I n the previous three chapters we described the structures of amino acids and proteins, and in two cases we examined how these structures relate to their function. Some of the methods for structure determination were also discussed (e.g., sequence analysis in chapter 3 and x-ray diffraction in chapter 4). To analyze the structure of a protein we must isolate it from the complex mixture in which it exists in whole cells. The primary object of this chapter is to acquaint you with techniques used for protein purification. Because these procedures are often used for protein characterization as well, they will add to your repertoire of methods for protein characterization. 

In the first part of this chapter methods for protein fractionation are discussed in isolation. Success in protein purification depends on picking a number of procedures and combining them in an effective order. Two examples are given in which trains of procedures are combined to purify specific proteins from crude whole-cell extracts. 

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Molecular biologists are utilizing hplc for characterization and purification of proteins, peptides, and antibodies. 

Functional Diversity of Proteins 101 Methods for Characterization and Purification of Proteins 118... 

As is the case with other biotechnological products, the extent of protein purification depends on the final intended application of the product. There are applications where the plant tissue can be directly used, and hence purification is not needed. In other situations particularly pharmaceutical products administered parenterally, there are stringent purity requirements, necessitating complete removal of viral particles, endotoxins and other contaminants. There are very few published reports that make quantitative and characterize the extraction and purification of proteins from transgenic plants. Furthermore, there are practically none dealing with the economics of their downstream processing. 

The nse of aptamers for the separation, pnriflcation, and quantification of analytes in chromatography, electrochromatography, and capillary electrophoresis techniqnes in general is described in detail and with examples by Ravelet et al. (2006). DNA and RNA aptamers have been nsed for the separation and purification of proteins and the separation of small molecnles and enantiomers. In capillary electrophoresis, aptamers are nsed primarily for the separation of species and the characterization of affinity interactions. Ravelet et al. (2006) state the hnge potential of these molecular tools in the separation science field. However, for big separation units, a large amonnt of aptamer is necessary, making it more expensive than other separation materials. Therefore, the nse of aptamers for separation units is limited primarily to miniatnrized systems. 

Nemere, I., Yazzie-Atkinson, D., Johns, D.O., Larsson, D. 2002. Biochemical characterization and purification of a binding protein for 24,25-dihydroxyvitamin D3 from chick intestine. 7. Endocrinol. 172(1) 211-9. 

Ahmed, H. 2005. Principles and Reactions of Protein Extraction, Purification and Characterization. CRC Press. 

Turner DC, Stadtman TC. 1973. Purification of protein components of the clostridial glycine reductase system and characterization of protein A as a selenoprotein. Arch Biochem Biophys 154 366-81. 

Traditionally protein-protein interactions studies have been performed in vitro after isolation and purification of individual proteins. While some in vivo or in situ protein-protein interaction studies can be performed by traditional methods using microinjection of purified proteins into oocytes, technical complexities limit the number of proteins that can be studied. Furthermore, many putative proteins of interest, predicted by genomic analysis, are not characterized and cannot be used in such studies. Some of the limitations posed by traditional methods have been overcome by use of yeast two-hybrid systems. These systems allow studies of many recombinant test proteins... 

Whether the goal is purification of a large-scale batch of protein for biocatalytic purposes or purification of an analytical amount to homogeneity for biochemical characterization of the protein, there is going to be a conflict between yield and purity. Despite the high salt levels required, ammonium sulfate precipitation remains one of the most effective methods for initial purification of proteins after fermentations, especially if an overexpressed protein has to be isolated even a purification factor of 2 increases purity significantly. Typical yields for this step should be expected to fall between 60 and 80%. 

Lige B, Ma S, Zhao D, Van Huystee RB. Cationic plant peroxidase Expression and characterization in transgenic tobacco and purification of the histidine-tagged protein. Plant Sci 1998 136(2) 159—168. 

Steroid hormones achieve their effects on target tissues through intracellular receptor proteins. According to recent views, oestrogen and progestin receptors are localized in the nuclear compartment of the cells, whereas glucocorticoid receptors may reside in both the cytoplasm and the nucleus. Determination of the intracellular localization of androgen receptors awaits the development of (monoclonal) antibodies which will enable immunohistochemical studies. The molecular aspects of the mechanism of action of steroid hormones will be covered in other chapters [1-3] in this volume. The present chapter deals with the characterization, assay and purification of steroid receptors. 

At present identification and quantification of selenoamino acids in seafood relies solely on matching retention time of peaks with available standards. To unambiguously assign peaks, the use of HPLC coupled to a mass spectrometer is required, as for the identification of As species [158, 159]. However, at present the low concentrations of Se species, the low detection power, and the lack of knowledge about Se species does not make this possible [131, 135, 136, 163], Preparative scale isolation and purification of Se proteins as done for bacterial proteins is required to allow Se proteins and selenoamino acids to be characterized by mass spectrometry [164], Preparative gel and two-dimensional electrophoresis offers promise to obtain pure Se protein fractions. Selenium-containing... 

The ability to inhibit the growth of ice has potential medical, industrial and commercial applications. Unfortunately, many of these applications have not been fully realized. One reason for this is that the isolation and purification of AFGP is a laborious and costly process often resulting in mixtures, making characterization difficult (5). Additional reasons include the fact that the AFGP mechanism of action is not understood at the molecular level and the nature of the protein-ice interface remains in question (6). 

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