Chapter 11
Affinity Precipitation of Avidin by Using Ligand-Modified Surfactants 1
Downloaded by CORNELL UNIV on August 5, 2016 | http://pubs.acs.org Publication Date: January 24, 1990 | doi: 10.1021/bk-1990-0419.ch011
Roberto Z. Guzman , Peter K. Kilpatrick, and Ruben G. Carbonell Department of Chemical Engineering, North Carolina State University, Raleigh, NC 27695-7905
The use of ligand-modified double-tailed phospholipid surfactants for selectively precipitating the tetrameric protein avidin from model and crude solutions is described. Dimyristoylphosphatidylethanolamine (DMPE) was derivatized by covalently attaching biotin, the specific ligand for the egg white protein avidin. The biotinylated surfactant (DMPE-B) was solubilized in aqueous buffer solution by the ethoxylated alcohol octaethyleneglycol mono-n-dodecylether (C12E8) at concentrations above the critical micelle concentration of the nonionic surfactant. The mixed surfactant solution of DMPE-biotin and C12E8 was then combined with protein solutions containing avidin which resulted in dilution of the nonionic surfactant below its critical micelle concentration. Upon binding of avidin to DMPE-B, the hydrocarbon tail groups of the phospholipid apparently aggregated with other DMPE-B molecules complexed to other avidin molecules. Because each avidin can bind four DMPE-B molecules, the result is a three-dimensional network of modified phospholipidavidin complexes. This large aggregate grew until it precipitated from solution, as evidenced by gross turbidity which was monitored spectrophotometrically. The avidin-surfactant aggregates were then separated from solution by centrifugation and decantation of the supernatant The avidin complex was resolubilized in a high concentration of (10 M) C12E8 in buffer solution, denatured by guanidinium chloride addition to debind the DMPE-biotin, and renatured after removal of the phospholipid by dilution and ultrafiltration. The technique was demonstrated with avidin solutions using lysozyme, bovine serum albumin, and myoglobin as model impurities. Greater than 85% recovery of avidin was achieved with no measureable coprecipitation of the model impurities. Avidin was also recovered from partially purified egg white solutions, which had been pretreated by ion exchange chromatography to remove hydrophobic and negatively charged protein impurities. Hence, greater than 90% of the avidin in the partially purified egg white fraction was recovered in pure active form, as evidenced by spectrophotometric assay and sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). -3
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Current address: Department of Chemical Engineering, University of Arizona, Tucson, A Z 85721 0097-6156/90/0419-0212$07.25/0 © 1990 American Chemical Society Hamel et al.; Downstream Processing and Bioseparation ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
Downloaded by CORNELL UNIV on August 5, 2016 | http://pubs.acs.org Publication Date: January 24, 1990 | doi: 10.1021/bk-1990-0419.ch011
11.
GUZMAN ET AL.
Affinity Precipitation of Avidin
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P r e c i p i t a t i o n is a commonly used purification step i n protocols for isolating and recovering proteins from crude biological mixtures (1). The precipitation of proteins is commonly effected by a d d i n g a component w h i c h decreases the solubility of the desired biomacromolecule. The biomolecules i n the resulting super-saturated protein solution r a p i d l y aggregate to form seed particles for growth of larger precipitates. The differential solubility agents added to produce precipitation include, but are not l i m i t e d to, electrolytes, organic solvents, and p H modifiers. These additives act to attenuate repulsive electrostatic interactions between the protein molecules. The resulting increased importance of hydrophobic attractions between the biomolecules leads to agglomeration. Pure protein precipitates are rarely obtained, partly because simple addition of differential solubility agents leads to regions of h i g h local super-saturation. The resulting rapid aggregation of protein molecules tends to be non-specific. The result is simultaneous agglomeration and entrainment of different biomacromolecules and an impure precipitate. Fisher et aL (2) attempted to control the local concentration of precipitating agent i n protein solutions by using a dialytic membrane to control the rate of addition. The aim was to aggregate pure, dense crystals of the desired protein and ultimately produce a purer product. However, i n a very complex mixture, containing several proteins w i t h similar physical properties, it may be difficult to find a set of conditions for precipitation that w i l l make the process very selective. A n alternative approach that can be used to impart greater selectivity to protein precipitation is to attach a ligand, w h i c h possesses specific affinity for the desired biomolecule, to a polymer w h i c h has functional groups w h i c h make it easy to precipitate. Schneider et aL (3) have successfully exploited this so-called affinity precipitation technique i n the purification of the proteolytic enzyme trypsin from bovine pancreas. In their scheme, a competitive reversible inhibitor of trypsin, m-aminobenzamidine, was reacted w i t h acryloyl chloride to form N-acryloyl-m-aminobenzamidine, one of the m o n o m e r i c components of the p o l y m e r used i n the precipitation. One of the other monomeric units was N - a c r y l o y l - p aminobenzoic acid, which has a p K i n aqueous solution of about 5.5. These two monomers were p o l y m e r i z e d w i t h acrylamide to synthesize a substituted Polyacrylamide. The m-aminobenzamidine group served as a specific ligand for trypsin while the benzoic acid substituent acted as a precipitation aid. A t pH>6.0, the polymer was fully ionized and soluble while at pH
