DEVELOPMENT OF A NEW AFFINITY CHROMATOGRAPHY SYSTEM BASED ON THE Lactobacillus S-LAYER PROTEIN

ROSET MS; BRIONES G; MURUAGA EJ

Virtual

Congreso; CONGRESO CONJUNTO SAIB-SAMIGE 2021; 2021

Affinity chromatography is a technique based on the reversible interaction of a ligand coupled to an inert matrix, and a protein or enzyme with the ability to bio-associate with the ligand. This technique allows purification of proteins with high selectivity, resolution, and capacity, achieving purifications of several orders of magnitude in a single step. Here we propose a novel protein purification method based on Lactobacillus S-layer protein. Lactobacillus acidophilus is a gram-positive lactic bacterium that carries a lattice of surface proteins linked to its cell wall called S-layer (surface layer). The predominant protein that forms the S-Layer is named SlpA. SlpA has a SLAP domain, encoded in the last ~ 159 amino acids, which is responsible for SlpA membrane association on the Lactobacillus membrane. SLAP is able to bio-associate with teichoic and lipoteichoic acids, among other ligands. In this project we propose the development of a new affinity chromatography system adapting the SLAP domain of Lactobacillus acidophilus SlpA protein as a molecular tag. Thus, SLAP-tagged recombinant proteins were able to bio-associate to Lactobacillus-derived affinity matrix allowing their purification with high efficiency. Also, a Bacillus subtilis natto derived matrix was tested with equivalent performance as the Lactobacillus derived matrix. Interestingly, B. subtilis is more convenient since it is able to grow faster than Lactobacillus in a less-expensive culture medium. Moreover, since B. subtilis has no S-layer, there is no need for S-layer removal, a step which is required when Lactobacillus is used. To optimize the protocol for protein affinity purification different binding and elution conditions were studied. As a result of this optimization a defined protocol was established showing that the optimal binding conditions were observed at 5 min of incubation at 0 ºC, in 50mM Tris-HCl buffer (pH 7,6 - 200mM NaCl). Optimal elution conditions were determined as 5 min at 24 ºC in Carbonate buffer (pH 10, 200mM NaCl). As a reporter, a fusion protein was constructed (GFP-SLAP) and used to optimize the purification system. Furthermore, a series of recombinant proteins from diverse organisms and variated molecular weight were probed to purify with this chromatographic system. In all the cases, proteins were successfully purified. Finally, the optimized chromatographic system was compared with the commercial, largely used but expensive nickel/poly-histidine chromatography. Both systems showed similar results for the GFP-SLAP fusion protein purification suggesting that our purification system was efficient and robust.