Protein isolation and purification

The traditional methodology for the isolation of proteins involves biochemical separation techniques, whereby the protein of interest is isolated from all the other proteins based on its unique physical and chemical properties. This includes molecular weight (and hence size), shape (for example, globular versus rod), hydrophobicity, and net electric charge. 

In the case of enzymes involved in biochemical pathways, the isolation is often based on activity assays. The nature of the activity assay depends on the enzymatic reaction and can involve, for example, the detection of a product on a thin-layer chromatography (TLC) plate (see Chapter 4, Section 1.2.1), the appearance or disappearance of a specific absorbance in a spectrophotometric assay, or a coupled assay involving the oxidation or reduction of a co-factor such as nicotinamide dinucleotide (NAD(H)), which can be measured by changes in fluorescence. 

The isolation procedure starts with the preparation of a cell extract in which the enzyme activity can be detected. This typically involves grinding the tissue in an extraction buffer so that the cell contents, including the proteins, become accessible. Protease inhibitors, such as phenylmethyl 

Hydrophobic interaction chromatography relies on hydrophobic interactions between apolar amino acid residues in the proteins and a resin containing hydrophobic groups, such as /7-octyl or phenyl groups. After the protein mixture is applied to the column, an elution buffer with decreasing ionic strength is used. Hydrophilic proteins will elute first, whereas hydrophobic proteins elute last. 

While there are no set protocols for the specific order in which the different chromatographic separations are carried out, ion exchange and hydrophobic interaction chromatography typically precede gel filtration and bio-affinity chromatography. The latter can involve expensive resins and is often performed after various contaminants have been removed during earlier purification steps. 

---

The importance of zinc to growth and development in all forms of life was first established through zinc deficiency studies of microorganisms followed by those in plants and animals see Nutritional Aspects of Metals Trace Elements) The involvement of zinc in a wide variety of metabolic processes including carbohydrate, lipid, protein, and nucleic acid synthesis and degradation paralleled the technical advances in analytical methods that could detect the presence of zinc in minute amounts coupled with advances in the methodology for protein isolation and purification. 

Ulber R, Plate K, Reif OW et al. (2003) Membranes for protein isolation and purification. In Hatti-Kaul R, Mattiasson B (eds). Isolation and purification of proteins. CRC Press, Boca Raton, pp... 

Its fluorescence showed two emission peaks, 564 nm and 626 nm the excitation peaks for the former emission peak were at 305 nm and 374 nm, and those for the latter were at 332 nm and 392 nm, indicating the presence of two different components in the preparation. It appears that the characteristics of the fluorescent proteins were altered by isolation and purification. 

In a more recent paper, Roy et al. reported on the synthesis and biological pro -perties of mannosylated Starburst dendrimers [46]. In addition to the presence of good biological properties in ligand- and inhibitor-tests, these dendrimers were shown to constitute novel biochromatography materials of high affinity for the rapid and easy isolation and purification of carbohydratebinding proteins from crude mixtures. 

Due to the importance of this technology for protein expression, manufacturers Sigma, Serotec, Abeam and many others offer a wide range of products for the detection, isolation and purification of tagged proteins. These vendors also provide monoclonal and polyclonal antibodies specific for the most commonly... 

The practical achievement of this goal was held up for 18 years, primarily because of the great difficulty in isolation and purification of single-species proteins from the immune repertoire. During that time, many attempts to elicit catalysis by inhomogeneous (i.e. polyclonal) mixtures of antibodies were made and failed (e.g. Raso and Stollar, 1975 Summers, 1983). The problem was resolved in 1976 by Kbhler and Milstein s development of hybridoma technology, which has made it possible today both to screen rapidly the complete immune repertoire and to produce in vitro relatively large amounts of one specific monoclonal antibody species (Kohler and Milstein, 1975 Kohler et al., 1976). 

The enzyme catalyzing the formation of retinal 2 by means of central cleavage of P-carotene 1 has been known to exist in many tissues for quite some time. Only recently, however, the active protein was identified in chicken intestinal mucosa (3) following an improvement of a novel isolation and purification protocol and was cloned in Escherichia coli and BHK cells (4,5). Iron was identified as the only metal ion associated with the (overexpressed) protein in a 1 1 stoichiometry and since a chromophore is absent in the protein heme coordination and/or iron complexation by tyrosine can be excluded. The structure of the catalytic center remains to be elucidated by X-ray crystallography but from the information available it was predicted that the active site contains a mononuclear iron complex presumably consisting of histidine residues. This suggestion has been confirmed by... 

Medronho RA (2002) Solid-liquid separation. In Mattiasson B, Hatti-Kaul R (eds) Isolation and purification of proteins. Marcel Dekker Inc, New York (in press)... 

In contrast to the isolation of small molecules, the isolation and purification of microbial proteins is tedious and often involves a number of expensive large-scale chromatographic operations. 

Rhizosecretion is easy to scale up and very cost effective with respect to isolation and purification. However, the bioreactor systems used for hairy root cultures differ from those used for plant cell suspensions. Traditional bioreactor systems have recently been adapted for root culture, and this technology is now being taken to commercial scales. The most traditional system is the airlift bioreactor used for microorganisms or plant cells. This system is adapted for the culturing of roots in liquid medium. Mist culture systems have also been developed. For this technology, the volume of the culture medium is reduced and the concentration of the secreted therapeutic protein is increased. If the protein to be produced is known to be quite stable, then a less expensive hydroponic culture can be designed in a manner suitable for scale-up. 

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... 

Affinity adsorbents having carbohydrate ligands have been used for the isolation and purification of many types of macromolecular substances. To illustrate, several types of antibodies, enzymes, lectins, and myeloma proteins have been obtained in highly purified form. All of the substances that have been purified by this procedure exhibit specificity for a particular carbohydrate moiety. It is the purpose of this article to assemble information on the methods for preparing affinity adsorbents having carbohydrate ligands, and to illustrate the use of these adsorbents for the purification of representative, macromolecular substances. 

Affinity chromatography can be applied to the isolation and purification of virtually all biological macromolecules. It has been used to purify nucleic acids, enzymes, transport proteins, antibodies, hormone receptor proteins, drug-binding proteins, neurotransmitter proteins, and many others. 

---