Adaptation of the binding domain ofLactobacillus acidophilusS-layer protein as a molecular tag for affinity chromatography development

Abstract

AbstractThe SLAPTAGis a novel molecular TAG derived from a protein domain present in the sequence ofLactobacillus acidophilusSlpA (SlpA284–444). Proteins from different biological sources, with different molecular weights or biochemical functions, can be fused in frame to the SLAPTAGand efficiently purified by the specific binding to a bacterial-derived chromatographic matrix named here Bio-Matrix (BM). Different binding and elution conditions were evaluated to set an optimized protocol for the SLAPTAG-based affinity chromatography (SAC). The binding equilibrium between SLAPTAGand BM was reached after a few minutes at 4°C, being the apparent dissociation constant (KD) of 4.3 µM, a value which is similar to different Kd determined for other S-layer proteins and their respective bacterial cell walls. A reporter protein was generated (H6-GFP-SLAPTAG) to compare the efficiency of the SAC against a commercial system based on a Ni2+-charged agarose matrix, observing no differences in the H6-GFP-SLAPTAGpurification performance. The stability and reusability of the BM were evaluated, and it was determined that the matrix was stable for more than a year, being possible to reuse it five times without a significant loss in the efficiency for protein purification. Alternatively, we explored the recovery of bound SLAP-tagged proteins by proteolysis using the SLAPASE(a SLAP-tagged version of the HRV-3c protease) that released a tag-less GFP (SLAPTAG-less). Additionally, iron nanoparticles were linked to the BM and the resulting BMmagwas successfully adapted for a magnetic SAC, a technique that can be potentially applied for high-throughput-out protein production and purification.