Utilization of Group Specific Ligands in the Downstream Processing of Proteins by Affinity Precipitation
In affinity precipitation, stimuli-responsive affinity macroligands are used to first capture and then reversibly coprecipitate and thereby isolate the target molecules. To create the affinity macroligand, an affinity mediator/ligand is linked to a stimuli-responsive polymer. In this paper three generic affinity ligands, namely chelated nickel (for capture of poly-histidin-tagged molecules), an anti-APP-antibody (for capture of APP-tagged fusion proteins) and Protein A (for antibody capture) are investigated for their applicability in affinity precipitation. Ligands were linked in terminal position to thermo-responsive poly-(N-isopropylacrylamide) oligomers (mass 2500 g mol−1). The Ni2+-activated affinity macroligands were able to capture the target molecule in a highly specific manner even from bacterial lysate. Unspecific coprecipitation was not observed. After recovery the target molecule could be quantitatively released from the affinity complex. In the case of the anti-peptide antibody, it was shown that such molecules could be linked to the stimuliresponsive polymer without changing the latter's overall solubility behaviour. The capture of a peptide-tagged fusion protein was demonstrated. The protein A-activated affinity macroligand was created in two ways; by direct chemical (carbodiimid) coupling and by combining an avidin-activated stimuli-responsive polymer and a biotinylated Protein A. The second affinity macroligand showed the expected binding strength to the target molecule and resulted in the capture of up to one mol of target molecules per mol of binding sites. The affinity macroligand produced by direct coupling, on the other hand, showed reduced binding, presumably due to steric hindrance of the binding site by the stimuli-responsive polymer.
Journal: Food and Bioproducts Processing - Volume 84, Issue 1, March 2006, Pages 28-36