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

The SLAP TAG is a novel molecular TAG derived from a protein domain present in the sequence of Lactobacillus acidophilus SlpA (SlpA 284–444 ). Proteins from different biological sources, with different molecular weights or biochemical functions, can be fused in frame to the SLAP TAG and 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 SLAP TAG -based affinity chromatography (SAC). The binding equilibrium between SLAP TAG and BM was reached after a few minutes at 4°C, being the apparent dissociation constant (K D ) 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 (H 6 -GFP-SLAP TAG ) to compare the efficiency of the SAC against a commercial system based on a Ni 2+ -charged agarose matrix, observing no differences in the H 6 -GFP-SLAP TAG purification 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 SLAP ASE (a SLAP-tagged version of the HRV-3c protease) that released a tag-less GFP (SLAP TAG -less). Additionally, iron nanoparticles were linked to the BM and the resulting BM mag was successfully adapted for a magnetic SAC, a technique that can be potentially applied for high-throughput-out protein production and purification.