Search

IntroductionThe S-layer proteins are a class of self-assembling proteins that form bi-dimensional lattices named S-Layer on the cell surface of bacteria and archaea. The protein SlpA, which is the major constituent of the Lactobacillus acidophilus S-layer, contains in its C-terminus region (SlpA284 − 444), a protein domain (named here as SLAPTAG) responsible for the association of SlpA to the bacterial surface. SLAPTAG was adapted for the development of a novel affinity chromatography method: the SLAPTAG-based affinity chromatography (SAC).MethodsProteins with different molecular weights or biochemical functions were fused in-frame to the SLAPTAG and efficiently purified by a Bacillus subtilis-derived affinity matrix (named Bio-Matrix or BM). Different binding and elution conditions were evaluated to establish an optimized protocol.ResultsThe binding equilibrium between SLAPTAG and BM was reached after a few minutes of incubation at 4°C, with an apparent dissociation constant (KD) of 4.3μM. A reporter protein (H6-GFP-SLAPTAG) was used to compare SAC protein purification efficiency against commercial immobilized metal affinity chromatography. No differences in protein purification performance were observed between the two methods. The stability and reusability of the BM were evaluated, and it was found that the matrix remained stable for more than a year. BM could be reused up to five times without a significant loss in performance. Additionally, the recovery of bound SLAP-tagged proteins was explored using proteolysis with a SLAP-tagged version of the HRV-3c protease (SLAPASE). This released the untagged GFP while the cut SLAPTAG and the SLAPASE were retained in the BM. As an alternative, iron nanoparticles were linked to the BM, resulting in BMmag. The BMmag was successfully adapted for a magnetic SAC, a technique with potential applications in high-throughput protein production and purification.DiscussionThe SAC protocol can be adapted as a universal tool for the purification of recombinant proteins. Furthermore, the SAC protocol utilizes simple and low-cost reagents, making it suitable for in-house protein purification systems in laboratories worldwide. This enables the production of pure recombinant proteins for research, diagnosis, and the food industry.

Brucella spp. are the etiological agent of animal and human brucellosis. We have reported previously that cyclophilins of Brucella (CypA and CypB) are upregulated within the intraphagosomal replicative niche and required for stress adaptation and host intracellular survival and virulence. Here, we characterize B. abortus cyclophilins, CypA, and CypB from a biochemical standpoint by studying their PPIase activity, chaperone activity, and oligomer formation. Even though CypA and CypB are very similar in sequence and share identical chaperone and PPIase activities, we were able to identify outstanding differential features between them. A series of differential peptide loops were predicted when comparing CypA and CypB, differences that might explain why specific antibodies (anti-CypA or anti-CypB) were able to discriminate between both cyclophilins without cross-reactivity. In addition, we identified the presence of critical amino acids in CypB, such as the Trp134 which is responsible for the cyclosporin A inhibition, and the Cys128 that leads to CypB homodimer formation by establishing a disulfide bond. Here, we demonstrated that CypB dimer formation was fully required for stress adaptation, survival within HeLa cells, and mouse infection in B. abortus. The presence of Trp134 and the Cys128 in CypB, which are not present in CypA, suggested that two different kinds of cyclophilins have evolved in Brucella, one with eukaryotic features (CypB), another (CypA) with similar features to Gram-negative cyclophilins.