Proteins column chromatography

Prymnesin (toxic protein from phytoflagellate Pyrymnesium parvum) [11025-94-8]. Purified by column chromatography, differential soln and pptn in solvent mixtures and differential partition between diphasic mixtures. The product has at least 6 components as observed by TLC. [Ulitzur and Shilo Biochim Biophys Acta 301 350 1970.]... 

Because temperature shifts may also influence the packing quality, the temperature should not be changed during the chromatographic step and the packing of the column should be done at the operation temperature. To prevent the denaturation of sensitive proteins, the chromatography is carried out in a cold chamber (or cabinet). For this purpose the column packing has to be performed at the same ambient temperature (store the gel before use at the same temperature ). 

Dornase alfa is produced by genetically engineered Chinese Hamster ovary cells containing DNA encoding for the native human protein deoxyribunuclease I. It is purified by tangential flow filtration and column chromatography. 

The enzyme was purified to 277 fold with very high specific activity, 1387 unit/mg protein by hydroxyapatite column chromatography (Figure 3). 

McCartney, J. E., Rapid purification of a recombinant protein using tandem radial flow ion-exchange column chromatography, BioTechniques, 11,648,1991. 

Figure 18 Very-high-speed gradient anion exchange chromatography of proteins. Column 0.46 x 3.5 cm ZipSep AX, 3 p. Eluent Tris-HCl, pH 8.0, operated on a gradient from 0-0.5 M NaCl. Flow rate 2ml/min. Detection UV absorbance at 280 nm. (1) Ribonuclease A, (2) carbonic anhydrase, (3) conalbumin, (4) bovine serum albumin. (Reproduced from Hatch, R. G., J. Chromatogr. Sci., 31, 469,1993. By permission of Preston Publications, A Division of Preston Industries, Inc.)...

Walhagen, A., Edholm, L.E. (1989). Coupled-column chromatography on immobilized protein phases for direct separation and determination of drug enantiomers in plasma. J. Chromatogr. 473, 371-379. 

Once the protein is recovered from its producer source and concentrated it must be purified to homogeneity. In other words, all contaminant proteins and other potential contaminants of potential medical significance (discussed in Chapter 7) must be removed. Purification is generally achieved by column chromatography. 

Serum samples from some animals fed the four dietary regimens were pooled and subjected to fractionation of the lipoproteins by agarose column chromatography. The total amounts of lipoproteins found in the sera from the four groups (ug/ml) were casein, 904 soy protein, 807 casein/arginine, 1,130 and, soy/lysine, 672. 

Buchacher et al. [43] discussed the continuous separation of protein polymers from monomers by continuous annular size exclusion chromatography. The P-CAC used for the experiments was a laboratory P-CAC type 3 as described in Table 1. The results were compared to conventional batch column chromatography in regard to resolution, recovery, fouling, and productivity. The protein used in the studies was an IgG preparation rich in aggregates. Under the conditions used, the polymers could be separated from the monomers, although no baseline separation could be achieved in either the continuous or the batch mode. The... 

Figure 4.8 Cation-exchange liquid chromatography of basic proteins. Column, Asahipak ES502C eluent, 20 min linear gradient of sodium chloride from 0 to 500 mM in 50 mM sodium phosphate buffer pH 7.0 flow rate, 1 ml min-1 temperature, 30 °C detection, UV 280 nm. Peaks 1, myoglobin from horse skeletal muscle (Mr 17 500, pi 6.8-7.3) 2, ribonuclease from bovine pancreas (Mr 13 700, pi 9.5-9.6) 3, a-chymotrypsinogen A from bovine pancreas (Mr 257 000, pi 9.5) and 4, lysozyme from egg white (Mr 14 300, pi 11.0-11.4). (Reproduced by permission from Asahikasei data)...

Proteins were precipitated with 170 mL of methanol and removed by centrifugation at 8000 rpm for 15 min. Then, 10 mL of Dowex 50WX8 H" " form resin was added, stirred for 30 min and removed by filtration. 10 mL of Dowex 1X8 HC03 form resin was added to the filtrate and stirred for 30 min. After filtration, the solution was concentrated and crude 5-thio-D-xylulofuranose was purified by flash column chromatography using 8/2 CH2Cl2/MeOH. 

After the production of the solution of proteins, the purification of the protein requires six different steps. Silica gel powder is added to the solution and filtered off, to adsorb large quantities of cytochrome C3. Several steps of column chromatography follow. 

Hydroxylapatite column chromatography relies on the selective adsorption of the protein onto the surface of calcium phosphate. The final stage of purification, on a high-performance DEAE column, is carried out just prior to crystallization of the protein. 

Work in solution is an absolute prerequisite for further studies of enzyme-substrate intermediates in the crystalline state. According to the Arrhenius relationship, k = A exp(—E IRT), which relates the rate constant k to the temperature, reactions normally occurring in the second to minute ranges might be sufficiently decreased in rate at subzero temperatures to permit intermediates to be stabilized, and occasionally purified by column chromatography if reactions are carried out in fluid solvent mixtures. Therefore, the first problem is to find a suitable cryoprotective solvent for the protein in question. 

Interest in the nature of interactions between shortchain organic surfactants and large molecular weight macromolecules and ions with hydroxyapatite extends to several fields. In the area of carles prevention and control, surfactant adsorption plays an important role in the Initial states of plaque formation (1-5) and in the adhesion of tooth restorative materials ( ). Interaction of hydroxyapatite with polypeptides in human urine is important in human biology as hydroxyapatite has been found as a major or minor component in a majority of kidney stones ( 7). Hydroxyapatite is used in column chromatography as a material for separating proteins (8-9). The flotation separation of apatite from... 

Reverse micelles of CTAB in octane with hexanol as cosurfactant were reported to be able to lyse whole cells quickly and accommodate the liberated enzyme rapidly into the water pool of surfactant aggregates [50,51]. In another case a periplasmic enzyme, cytochrome c553, was extracted from the periplasmic fraction using reverse micelles [52]. The purity achieved in one separation step was very close to that achieved with extensive column chromatography. These results show that reverse micelles can be used for the extraction of intracellular proteins. 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

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