Table_1_Adaptation of the binding domain of Lactobacillus acidophilus S-layer protein as a molecular tag for affinity chromatography development.PDF

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.

引言
S层蛋白是一类自组装蛋白，它们在细菌和古菌的细胞表面形成二维晶格，称为S层。蛋白质SlpA，为嗜酸乳杆菌S层的主体成分，在其C端区域（SlpA284 − 444）含有一个负责SlpA与细菌表面结合的蛋白结构域（以下简称SLAPTAG）。SLAPTAG被用于开发一种新型的亲和层析方法：基于SLAPTAG的亲和层析（SAC）。
方法
将不同分子量或生化功能的蛋白质与SLAPTAG在框内融合，并通过来自枯草芽孢杆菌的亲和基质（命名为Bio-Matrix或BM）高效纯化。评估了不同的结合和洗脱条件，以建立优化的协议。
结果
在4°C孵育数分钟后，SLAPTAG与BM之间的结合平衡得以达到，其表观解离常数（KD）为4.3μM。使用报告蛋白（H6-GFP-SLAPTAG）来比较SAC蛋白纯化效率与商业固定化金属亲和层析。两种方法在蛋白纯化性能上未观察到差异。评估了BM的稳定性和可重复使用性，发现该基质在超过一年的时间内保持稳定。BM可重复使用至五次，而性能无明显下降。此外，还探索了使用带有SLAP标签的HRV-3c蛋白酶（SLAPASE）进行蛋白质水解来回收结合的SLAP标记蛋白。这释放了未标记的GFP，而切割的SLAPTAG和SLAPASE则保留在BM中。作为替代方案，将铁纳米颗粒连接到BM上，得到BMmag。BMmag成功地适应了磁性SAC，这是一种在高通量蛋白生产和纯化中具有潜在应用的技术。
讨论
SAC协议可被调整为一种通用的蛋白纯化工具。此外，SAC协议利用简单且低成本的试剂，使其适合全球实验室的内部蛋白纯化系统。这为研究、诊断和食品工业生产纯重组蛋白提供了可能。