Purification of protein

The purification process of proteins in biological fluids governs the production cost of the proteins in pharmaceutical and food industries. Therefore, there is a constant demand for highly efficient recovery of 

Highly efficient recovery is defined as the fulfillment of the following three requirements high rate, high capacity, and repeated use. These requirements are satisfied by a novel functional porous membrane that can be prepared by grafting of functional polymer chains onto a microfiltration membrane. 

The key to repeated use is the coexistence of hydrophilic groups and functional groups. The surface of protein has hydrophobic patches. Therefore, proteins easily adsorb nonselectively onto the surface of a hydrophobic polymeric support. In order to realize repeated adsorption and elution of protein using functional porous membranes, alcoholic hydroxyl groups are appended along with the functional groups of the polymer chains grafted onto the pore surface. This idea is similar to that on which Svec s work [68] was based. 

Similar permeation experiments were performed using the hoUow fiber with various DEA-group mole fractions of the graft chain. The DEA-group mole fraction is defined by DEA-group mole fraction of the graft chain (- ) 

Second, the degree of multilayer binding of protein onto the pore surface, as defined by equation (3), can be determined by dividing Qe by the theoretical monolayer binding capacity, Qj. 

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Crystallisation. The ultimate in purification of proteins or nucleic acids is crystallisation. This involves very specialised procedures and techniques and is best left to the experts in the field of X-ray crystallography who provide a complete picture of the structure of these large molecules. [A. Ducruix and R. Gieg6 eds. Crystallisation of Nucleic Acids and Proteins A Practical Approach, 2nd Edition, 2000,... 

This chapter lists some representative examples of biochemicals and their origins, a brief indication of key techniques used in their purification, and literature references where further details may be found. Simpler low molecular weight compounds, particularly those that may have been prepared by chemical syntheses, e.g. acetic acid, glycine, will be found in Chapter 4. Only a small number of enzymes and proteins are included because of space limitations. The purification of some of the ones that have been included has been described only briefly. The reader is referred to comprehensive texts such as the Methods Enzymol (Academic Press) series which currently runs to more than 344 volumes and The Enzymes (3rd Edn, Academic Press) which runs to 22 volumes for methods of preparation and purification of proteins and enzymes. Leading referenees on proteins will be found in Advances in Protein Chemistry (59 volumes. Academic Press) and on enzymes will be found in Advances in Enzymology (72 volumes, then became Advances in Enzymology and Related Area of Molecular Biology, J Wiley Sons). The Annual Review of Biochemistry (Annual Review Inc. Patio Alto California) also is an excellent source of key references to the up-to-date information on known and new natural compounds, from small molecules, e.g. enzyme cofactors to proteins and nucleic acids. 

Dextran can be produced in a range of molecular weights and crossed-linked or substituted with a variety of functional groups. These products (Sephadex) are routinely used in the purification of proteins and pharmaceutical and other medically important compounds. 

Fig. 17.1. Conventional downstream processing scheme for the purification of proteins.

Knuth, M. W. and Burgess, R. R., purification of proteins in the denatured state, in Protein Purification — Micro to Macro, Burgess, R., Ed., Alan R. Liss, New York, 1988, 279. 

Figure 7.6. Purification of protein from pooled yeast strains. Each yeast ORF was cloned as a fusion to glutathione-S-transferase in a protein expression vector to create 6144 yeast strains. The individual strains were pooled in groups of 96 to create a set of 64 pools. Each pool was grown and the 96 fusion proteins are purified in batch. Each pool was then assayed for a biochemical function (Martzen et al., 1999). Pools positive for function were then deconvoluted using smaller pools consisting of strains from rows and columns of a 96-well plate.

Tejero-Diez, P., Rodriguez-Sanchez, P., Diez-Guerra, F.J. (1999). Microscale purification of proteins exhibiting anomalous electrophoretic migration application to the analysis of GAP-43 phosphorylation. Anal. Biochem. 274, 278-282. 

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. 

Bergseid, M., Baytan, A.R., Wiley, J.P., Andener, W.M., Stolowitz, M.L., Hughes, K.A., and Chesnut, J.D. (2000) Small molecule-based chemical affinity system for the purification of proteins. BioTechniques 29, 1126-1133. 

Sulkowski, E. (1985) Purification of proteins by IMAC. Trends Biotechnol. 3, 1-7. 

HL Ball, P Mascagni. Chemical synthesis and purification of proteins a methodology. Int J Pept Prot Res 48, 31, 1996. 

The pore size of porous titania can be up to 2000 A. Titania is used for the purification of proteins and as a support for bound enzymes. The purification of /1-lactoglobulin from cheese whey, of protease from pineapple, /5-lactamase, and amylase can be achieved with titania. The latter two purifications are impossible on alumina. Titania is also used as a support in peptide synthesis. The separation of plasmid DNA is shown in Figure 3.24. 

SEC is a useful tool for monitoring enzyme reactions, as seen in Figure 4.21, where the speed of decomposition of /Mactoglobulin by a-chymotrypsin is shown. SEC is widely used for purification of proteins, but the separation is due only to the difference in molecular mass. Therefore, ion-exchange liquid chromatography is combined with SEC to improve the selectivity. 

Walker PA, Leong LE, Ng PW, Tan SH, Waller S, Murphy D, Porter AG. 1994. Efficient and rapid affinity purification of proteins using recombinant fusion proteases. Biotechnology 12 601-605. 

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. 

His first work in the Laboratory of Biological Chemistry was on the purification and properties of potato phosphorylase, but he soon changed his subject to the study of amino-containing sugars. It was not well understood at that time whether there were impurities in the products of separation and purification of proteins. Consequently, Onodera made up his mind to devote his whole life to identifying the chemical nature of amino-containing sugars. 

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