A Quantitative Analysis and Chemical Approach for the Reduction of Nonspecific Binding Proteins on Affinity Resins

Bioconjugate Chemistry

Volumn 14, Issue 6, 2003, Pages 1222-1230

  Tamura, Tsuruki ^a   Terada, Tomohiro ^a   Tanaka, Akito ^a  

^a Reverse Proteomics Research Institute Co   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

AFFINITY CHROMATOGRAPHY; BIOCHEMISTRY; ETHYLENE; ETHYLENE GLYCOL; HYDROPHOBICITY; POLYETHYLENE GLYCOLS; POLYOLS; PROTEINS; RESINS;

% REDUCTIONS; AFFINITY CHROMATOGRAPHY RESIN; AFFINITY RESINS; BINDING PROTEINS; CELLULAR TARGETS; HYDROPHILICS; NON-SPECIFIC BINDING; POLYMER SUPPORTS; PROTEIN-BINDING; SMALL MOLECULES;

LIGANDS;

ACTIN; BINDING PROTEIN; LIGAND; MACROGOL; POLYMER; RESIN; TACROLIMUS; TUBULIN;

AFFINITY CHROMATOGRAPHY; ALGORITHM; ARTICLE; CORRELATION ANALYSIS; HYDROPHOBICITY; LIGAND BINDING; PROTEIN BINDING; QUANTITATIVE ANALYSIS;

EID: 0345357014     PISSN: 10431802     EISSN: None     Source Type: Journal    
DOI: 10.1021/bc034099l     Document Type: Article

References (28)

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6. Information on one of methacrylate derivatives, "Toyopearl", can be found at the web site of TOSOH Corporation (http:// www.tosoh.com).
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14. The synthesis of mono TBS-protected FK506 was carried out by a manner similar to that of ascomycin described in the following literature: Hyun, O., Szumiloski, J. L., Beattie, T. R., Goulet, M. T., Staruch, M. J. et al. (1997). C32-Amino derivatives of the immunosuppressant ascomycin. Bioorg. Med. Chem. Lett. 7, 2199-2204.
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16. As described in the text, AffiGel is stable in aqueous solution and usually used in aqueous solutions. However, it is impossible to use an excess amount of ligand for immobilization since we usually synthesize the compound with linker moiety via more than several synthetic steps, and only small amounts are available. Thus, the immobilization reaction, such as amide formation, needed to be carried out in organic solvent to avoid the presence of water. But AffiGel is sometimes irreversibly denatured in organic solvent and sometimes gives false-positive targets; for example, we found a novel nonspecific binding protein when we synthesized affinity material bearing FK506 in acetonitrile while it was not detected when prepared in an aqueous solution.
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21. Liu, J., Farmer, J. D., Lane, W. S., Friedman, J., Weissman, I., and Schreiber, S. L. (1991) Calcineurin is a Common Target of Cyclophilin-Cyclosporin A and FKBP-FK506 Complexes. Cell 66, 807-815.
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23. TentaGel is used as basal matrix in peptide library screening. We thought that proteins only bind to the very outside surface of TentaGel, so it works well only when hydrophilic ligands such as peptides are immobilized on it, and it does not work when hydrophobic ligands such as FK506 are on it because they tend to bind to the hydrophobic basal resin, which consists of polystyrene. It is usually impossible because the length of spacer moiety of the usual affinity resins is not enough while the length of TentaGel is very long; the average number of PEG polymer is ca. 66.
24. Dean, P. M. (1987) Molecular foundation of drug-receptor interaction. Cambridge University Press, England.
25. It was known that enzyme activities were still observed after absorption on n-octyl-agarose (11). These results indicated that the conformation of proteins was not unique; that is, some of them were completely denatured and others were partially denatured; moreover, native proteins can also bind on the surface that are occupied by various conformational states of the enzymes.
26. There are many surface-active agents that include PEG in their structures as a hydrophilic moiety; for examples, polyoxyethylene(23) lauryl ether ("Brij 35") and other "Brij" series, polyoxyethylene branched monocyclohexyl ether ("Triton") and other "Triton" series, and polyoxyethylene(20) sorbitan monolaurate ("Tween20") and others in the "Tween" series.
27. For example, the average repetition number of oxyethylene unit of TentaGel's PEG moiety is 68, and the number is not unique.
28. Dextran is used as hydrophilic spacer in the Biacore chips. Dexatran plays as a spacer between gold foil and ligands in the Biacore system. We have observed nonspecific binding proteins after treatment of the gold foil bearing a ligand via the Dextran spacer with acetonitrile (data not shown).