Purification of avidin

Cuatrecasas, P., and Wilchek, M. (1968) Single-step purification of avidin from egg white by affinity chromatography of biocytin-Sepharose columns. Biochem. Biophys. Res. Comm. 33, 235-246. 

The use of a novel affinity precipitation scheme, applicable to multibinding site biomolecules, has been illustrated by the purification of avidin from model protein solutions and from partially purified egg whites. The... 

In another example, ligands can be biotinylated with a cleavable biotinylation reagent and then incubated with receptor molecules. The resulting complex can be isolated by affinity chromatography on immobilized (strept)avidin. Final purification of the ligand-receptor can be accomplished by cleaving the biotin modification sites while the complex is still bound to the support. The receptor complex thus can be eluted from the column without the usual harsh conditions required to break the avidin-biotin interaction. 

It is essential that the procedure described in Section 3.1.2. be performed approx 24 h before the procedure described in Section 3.1 3, because the deprotected thiolated avidin and the maleimide derivative of the protein are unstable. Purification of the maleimide-derivatized protein by size exclusion chromatography can be performed more rapidly than dialysis, however, the former leads to dilution of the protein and a decrease in the yield of the conjugate. 

The application of biotinylated receptor substrates is another approach, incubating the labeled substrate with the receptors prior to isolation on an avidin-coated support. In such cases, biotinylation with a cleavable biotinylation reagent such as Sulfo-NHS-SS-biotin or NHS-Iminobiotin would be essential for recovery of the isolated receptor. Alternatively, the receptor could be recovered by substrate competition. Perhaps one of the major drawbacks to the application of affinity techniques is the relative low molecular weight or small size of the receptor substrates, making them difficult ligands to immobilize. However, affinity procedures have been applied to the purification of a number of different receptors although relatively little work has been reported on those involved in the processing of neurotransmittors, neuropeptides, and hormones [1,2]. 

Figure 1. Structural formulas of the ethoxylated alcohol and phospholipid surfactants used in the affinity precipitation of avidin. Octaethyleneglycol mono-n-dodecylether (C12E8) was used as a solubilizing surfactant and dimyristoylphosphatidylethanolamine (DMPE) was used as the insoluble surfactant to which biotin was covalently attached. (The structural formula of the derivatized phospholipid and a discussion of the reaction and purification scheme have been previously described by Powers et (7)).

Hood E, Witcher DR, Maddock S, et al. Commercial production of avidin from transgenic maize characterization of transformant, production, processing, extraction, and purification. Mol. Breed., 1997 3 291-306. 

Figure 36. Biotin-avidin system for detection and purification of oligonucleotides. Modified from [277] Examples for direct ligand incorporation.

Campos S K, Parrott M B, Barry M A (2004). Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector. Molec. Ther. 9 942-954. 

A. Zocchi, A. M. Jobe, J.-M. Neuhaus, T. R. Ward, Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris, Prot. Expr. Purif., 2003, 32, 167-174. 

All aspects of the biochemical role of biotin have not yet been clarified. The vitamin has been implicated in the metabolism of carbohydrates, lipids, proteins, and nucleic acids. Available evidence indicates that biotin acts as a CO2 carrier in a number of carboxyla-tion and decarboxylation reactions connected with carbohydrate and fatty acid metabolism. A number of experimental procedures are used to establish the participation of biotin in a given biochemical reaction (1) the study of enzyme activity in biotin-deficient animals (2) the effect of avidin administered in vivo or added to the incubation mixture on the activity of the enzyme under study and (3) purification of the enzyme and demonstration of the existence of enzyme-bound biotin. Studies of this kind have established that biotin is required for the carboxylases of jS-methyl-crotonyl CoA, acetyl-CoA, propionyl CoA, and oxaloacetic transcarboxylase. Only some of the results are presented here [74-76]. 

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