Cellulose affinity chromatography

TDTase can be purified by means of various conventional chromatographies (40), oligo(dT)7-cellulose affinity chromatography (6), and an anti-TDTase IgG-Sepharose immunoadsorbent column chromatography (46,55). TDTase is quite stable at pH 4.5, thus providing one of the most useful steps in the purification. 

Avidin (from egg white) [1405-69-2] Mr -70,000. Purified by chromatography of an ammonium acetate soln on CM-cellulose [Green Biochem J 101 774 1966]. Also purified by affinity chromatography on 2-iminobiotin-6-aminohexyl-Sepharose 4B [Orr 7 Bio/C/iew 256 761 1981]. It is a biotin binding protein. 

Pituitary Growth Factor (from human pituitary giand) [336096-71-0]. Purified by heparin and copper affinity chromatography, followed by chromatography on carboxymethyl cellulose (Whatman 52). [Rowe et al. Biochemistry 25 6421 1986.]... 

Reverse transcriptase (from avian or murine RNA tumour viruses) [9068-38-6] [EC 2.7.7.49]. Purified by solubilising the virus with non-ionic detergent. Lysed virions were adsorbed on DEAE-cellulose or DEAE-Sephadex columns and the enzyme eluted with a salt gradient, then chromatographed on a phosphocellulose column and enzyme activity eluted in a salt gradient. Purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 7977 Smith Methods Enzymol 65 560 1980 see commercial catalogues for other transcriptases.]... 

Purified by (NH4)2S04 fractionation, followed by PC cellulose chromatography and affinity chromatography (using Sepharose 4B to which (G)n was covalently bonded). [Schmukler et al. J Biol Chem 250 2206 7975.]... 

Fraction A was examinated to purify by affinity chromatography on ConA -cellulose. Some impurities were removed but the separation of... 

The affinity chromatography on ConA - cellulose indicated the presence of small N-glycosylation of all forms of exopolygalacturonases present in carrot roots (unpublished results). This method was usefull for purification of these enzymes from other protein inpurities but was completely uneffective by separation of individual forms (Fig. 4). 

This method was used, for example, for the solid-phase immunoassay of thyroxine (affinity chromatography). Various activation methods (CDI, periodate, and cyanogen bromide procedures) were compared with each other for coupling antibodies to magnetizable cellulose/iron oxide solid-phase particles. 211]... 

The mRNA is then isolated from this total cellular extract by affinity chromatography using oligo-dT-cellulose or poly(U)-sepharose. 

In an attempt to separate the domains from the cores, we used limited degradation with several proteases. CBH I (65 kda) and CBH II (58 kda) under native conditions could only be cleaved successfully with papain (15). The cores (56 and 45 kda) and terminal peptides (11 and 13 kda) were isolated by affinity chromatography (15,16) and the scission points were determined unequivocally. The effect on the activity of these enzymes was quite remarkable (Fig. 7). The cores remained perfectly active towards soluble substrates such as those described above. They exhibited, however, a considerably decreased activity towards native (microcrystalline) cellulose. These effects could be attributed to the loss of the terminal peptides, which were recognized as binding domains, whose role is to raise the relative concentration of the intact enzymes on the cellulose surface. This aspect is discussed further below. The tertiary structures of the intact CBH I and its core in solution were examined by small angle X-ray scattering (SAXS) analysis (17,18). The molecular parameters derived for the core (Rj = 2.09 mm, Dmax = 6.5 nm) and for the intact CBH I (R = 4.27 nm, Dmax = 18 nm) indicated very different shapes for both enzymes. Models constructed on the basis of these SAXS measurements showed a tadpole structure for the intact enzyme and an isotropic ellipsoid for the core (Fig. 8). The extended, flexible tail part of the tadpole should thus be identified with the C-terminal peptide of CBH I. 

Cellulose is another example of polysaccharides which is used as support in affinity chromatography. Cellulose has a historical significance. Phospo- and DNA-cellulose are used especially for DNA related separations [14]. Antibody and enz)nne purifications have also been carried out. However its fibrous and non-rmiform character limits its use since cellulose detains macromolecules [11]. 

Membranes have been used for affinity chromatography in various formats, such as stacked sheets, in rolled geometries, or as hollow fibers. Materials that are commonly used for these membranes are cellulose, polysulfone, and polyamide. Because of their lack of diffusion pores, the surface area in these materials is as low as it is in nonporous beads. However, the flat geometry and shallow bed depth of membranes keep the pressure drop across them to a minimum degree. This means that high flow rates can be used, which makes these membranes especially well-suited for capturing proteins from dilute feed streams. 

Ap tamers can be selected in various ways. The most frequently used approaches are affinity chromatography [21] and modified cellulose filtration [4,22]. The choice of method depends on the properties of the target (for example, its capability to be immobilized on a matrix or to be bound to modified cellulose filters) and the aim of selection. If the desired aptamers should, for example, bind molecules on the surface of intact cells, the selection scheme should employ these cells adhering to the surfaces of tissue culture flasks [23]. 

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