Affinity precipitation of proteins

Lali A, Balan S, John R, D Souza F (1998), Carboxymethyl cellulose as a new heterobifunctional ligand carrier for affinity precipitation of proteins, Bioseparation 7 195-205. 

Smart particles capable of reversible transition between aggregated and dispersed states were used for affinity precipitation of proteins. Thermosensitive (44) or pH-sensitive latices (45) or salt-sensitive liposomes that have polsrmerized membranes (35) are examples of such systems. 

Galaev, I. Y., Kumar, A., Mattiasson, B. (1999). Metal-copolymer complexes of IV-isopropylaerylamide for affinity precipitation of proteins. Journal of Macromolecular Science—Pure and Applied Chemistry, 36, 1093-1105. 

Dissing U, Mattiasson B. Polyelectrolyte complexes as vehicles for affinity precipitation of proteins. J Biotechnol 1996 52 1-10. 

Morris, JE, Hoffman AS, Fisher RR. Affinity precipitation of proteins by polyligands. Biotechnol Bioeng 1993 41 991-997. 

Mattiasson B, Kumar A, Galaev lY. Affinity precipitation of proteins Design criteria for an efficient polymer. J Molec Recogn 1998 11 211-216. 

Water-soluble polymers and polyelectrolytes (e.g., polyethylene glycol, polyethylene imine polyacrylic acid) have been used success-hilly in protein precipitations, and there has been some success in affinity precipitations wherein appropriate ligands attached to polymers can couple with the target proteins to enhance their aggregation. Protein precipitation can also be achieved using pH adjustment, since proteins generally exhibit their lowest solubility at their isoelectric point. Temperature variations at constant salt concentration allow for frac tional precipitation of proteins. 

Affinity Precipitation of Avidin from Partially Purified Egg Whites. Following the ion exchange fractionation of egg white solution, described in the experimental section above, fractions of eluted protein solution (obtained from the elution described in Figure 6) were mixed with... 

In secondary effect affinity precipitation, which will be the variant considered exclusively from here onward, the redissolution of the affinity complex after precipitation is usually straightforward. The target molecule may be recovered either directly from the precipitate, which is clearly preferable from a process-engineering point of view, or after redissolution of the affinity complex. Authors who investigated the aspect of AML-recycling usually encountered no problems in reusing the affinity reagent several times. Below one recently published case study is detailed as a demonstration of the potential of secondary effect affinity precipitation for protein recovery. 

Kaul R, Guoqiang D, LaJi A et al. Affinity isolation of proteins based on precipitation of Eudragit-ligand-protein complexes. In Pyle DL, ed. Separations for Biotechnology 3. Cambridge The Royal Society of Chemistry, 1994 242-248. 

Protein isolation with affinity precipitation has been discussed in detail by Mattiasson and co-workers (see, e.g. Galaev and Mattiassion, 1997) and they have provided an exhaustive tabulation. Polymers varied from alginate.s/chitosan to dextran to NIPAM. Examples of the used proteins are from trypsin, p-glucosidase, xylanase, alkaline protease, etc. It is remarkable that affinity precipitation can sometimes give results comparable to affinity chromatography. 

Figure 6. Affinity chromatography of EGD from Clostridium thermocellum. Nucleic acid preparation, heat treatment and ammonium sulfate precipitation (0-70%, 70-100%) were carried out as described (10). The final precipitate ( 50 mg protein), dissolved in 50 mM sodium acetate, pH 5.0, was applied (after centrifugation) on the affinity column (2 x 25 cm) (4 -aminobenzyl l-thio-/ -cellobioside coupled to Sepharose 4B) (11). Protein was monitored at 280 nm and the activity of the fractions (2 ml) determined using 2 -chloro-4 -nitrophenyl / -cellobioside (pH 6.5, 25°C) as described in the text. Elution with 10 mM G2 was started as indicated.

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