Protein Purification Procedures

The source of a protein is generally tissue or microbial cells. The first step in any protein purification procedure is to break open these cells, releasing their proteins into a solution called a crude extract. If necessary, differential centrifugation can be used to pre-... 

During a protein purification procedure there are two measurements that need to be made, preferably for each fraction. Measurements of the amount of total protein and of the amount (usually bioactivity) of the desired protein both must be made. It is not possible to isolate a protein without a method of determining whether it is present an assay, either quantitative or at least semiquantitative, indicating which fraction contains the most of the desired protein is essential. 

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... 

Purification protocols should always aim for brevity, to minimize complexity and cost. The goal of the protein purification procedure, besides a pure protein, is a purification table listing all the operations undertaken, with results on overall yield, specific activity, and purification factor. An assay for both protein function and protein concentration is required at every step. 

The lectin from Phaseolus vulgaris L. (var. red), isolated by conventional protein-purification procedures, was shown to be localized in the cytoplasm of the cotyledon and embryo of the seed.243e A glycoprotein (6.6% carbohydrate), the lectin had molecular weight 128,000, and contained high proportions of aspartic acid, serine, and tryptophan, but no cysteine.243e... 

The introduction of hexahistidine tag (Hise-tag) to the N-terminus of chimeric protein significantly simplify the protein purification procedure, enabling isolation of target protein directly from crude cell extract (Efremenko et al, 2006). There were two main tasks in this work was i) to obtain the genetic construct encoding synthesis of fusion protein N-Hise-X-OPH, where X = superecliptic-pH-sensitive fluorine ii) to reveal conditions (host-strain, temperature and inductor concentration) favorable for construct expression in E.coli cells. 

Extraction of G proteins from membranes usually enhances ADP-ribosylation. In addition, small amounts of detergents like SDS, CHAPS or Lubrol PX facilitate the toxin s enzymatic activity (see above). For detection and identification of G proteins during G protein purification procedures, aliquots are also subjected to PT-catalyzed ADP-ribosylation for detection and identification (Rosenthal... 

It is the intention of this article to survey the literature on hemicellulases from 1950 to 1973, and to deal, in detail, only with those enzyme preparations that have, by the usual criteria of purity employed in protein purification procedures, been shown to be homogeneous. The article will, therefore, exclude most of the work published on hemicellulases in which crude or partially purified enzyme prepara-... 

The ability of polyelectrolytes to remove oppositely charged proteins from solutions has been exploited through the incorporation of protein-polymer precipitation steps into a variety of protein purification procedures, wherein precipitated proteins are recovered from the insoluble complex aggregate via redissolution by pH or ionic strength adjustment (14-16). Furthermore, preferential complexation of polyelectrolytes with specific proteins has been substantiated (13). Although there are reports of the optimization of bulk complexation yield through the adjustment of solution parameters such as pH and ionic strength (17), very little has been accomplished in the optimization of the selectivity of complex formation. 

The last class of selenoproteins contains the selenium binding proteins. This is an operational class defined by Sunde (1990) as "selenoproteins with selenium bound tightly enough so that the selenium remains attached during standard protein purification procedures that produce discrete selenium labeled species." This class contains selenoproteins that have not been fully characterized. 

Many of the techniques used in protein purification procedures have also been adapted for use with nucleic acids. For example, several types of chromatography (e.g., ion-exchange, gel filtration, and affinity) have been used in several stages of nucleic acid purification and in the isolation of individual nucleic acid sequences. Because of its speed, HPLC has replaced many slower chromatographic separation techniques when small samples are involved. 

Commercially, SEC has been used for desalting proteins and as one step in protein purification procedures.The equipment for desalting is similar to LC equipment and is described in detail elsewhere. The SEC desalting and buffer exchange of human blood plasma is shown in Fig. 14.2-5.Repeated injections of the feed are illustrated. Size exclusion chromatography is used in blood banks for purification of various factors from human plasma either in conjunction with ion-exchange chromatography (see Section... 

PicoLiNic Acid. The oxidation of 3-hydroxykynurenine by preparations of the oxidase from liver was found to result in the formation of picolinic acid as well as quinolinic acid (SSS, 334). The formation of pico-linic acid is enzymic, whereas that of quinolinic acid is nonenzymic. This enzyme was demonstrated in extracts of fresh or frozen liver of various species, the most consistent activity being found in guinea pig liver. The enzyme was concentrated about seventyfold from guinea pig liver by the usual protein purification procedures. The product was identified to be picolinic acid from its chromatographic behavior and identity of infrared spectra. Activity of the enzyme that forms picolinic acid in liver is quite weak. 

The most active source of histidinol dehydrogenase was obtained from a histidinol-adapted soil organism Arthrobacter histidindovorana). Extracts of this was purified about twentyfold. Subsequently an enzyme preparation from brewers yeast was purified about a hundredfold by the usual protein purification procedures. Even so the bacteria] preparation had twice the qiecific activity of the yeast enz3une. The dehydrogenase was found to be present in various bacteria and in yeast, but was absent in E. coli mutants unable to convert histidinol to histidine. 

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