ABSTRACT
The development of higher-throughput, potentially lower-cost means to isolate proteins, for a variety of end uses, is of continuing emphasis. Polypropylene (PP) capillary-channeled polymer (C-CP) fiber columns are modified with the biotin-binding protein streptavidin (SAV) to capture biotinylated proteins. The loading characteristics of SAV on fiber supports were determined using breakthrough curves and frontal analysis. Based on adsorption data, a 3-min on-column loading at a flow rate of 0.5 mL min-1 (295.2 cm h-1) with a SAV feed concentration of 0.5 mg mL-1 produces a SAV loading capacity of 1.4 mg g-1 fiber. SAV has an incredibly high affinity for the small-molecule biotin (10-14 M), such that this binding relationship can be exploited by labeling a target protein with biotin via an Avi-tag. To evaluate the capture of the biotinylated proteins on the modified PP surface, the biotinylated versions of bovine serum albumin (b-BSA) and green fluorescent protein (b-GFP) were utilized as probe species. The loading buffer composition and flow rate were optimized towards protein capture. The non-ionic detergent Tween-20 was added to the deposition solutions to minimize non-specific binding. Values of 0.25-0.50% (v/v) Tween-20 in PBS exhibited better capture efficiency, while minimizing the non-specific binding for b-BSA and b-GFP, respectively. The C-CP fiber platform has the potential to provide a fast and low-cost method to capture targeted proteins for applications including protein purification or pull-down assays.
