Therefore, the present work should boost specific studies dedicated to LGG SpaCBA pili and the characterization of the interactions occurring with their protein partners at the molecular level. This simple protocol could be useful to numerous laboratories to purify pili from LGG easily. One can obtain ∼50 μg of purified pili, starting from 1 L culture at OD 600nm ≈ 1, in 2–3 working days. No other proteins were detectable by SDS-PAGE and the 260/280 nm ratio (∼0.6) of the UV spectrum confirmed the absence of any other co-purified macromolecules. Contrary to previously published methods, this purification protocol does not require specific antibodies nor complex laboratory equipment, including for the multimodal chromatography step, and provides high degree of protein purity. Here, we propose a chromatography-based protocol, mainly relying on multimodal chromatography (core bead technology using Capto Core 700 resin), to purify sortase-dependent SpaCBA pili from the probiotic strain Lacticaseibacillus rhamnosus GG (LGG). Purifying these high molecular weight proteins is challenging and has certainly slowed down their characterization. They are found both in Gram-negative and Gram-positive bacteria but differ in their structural organization. These filamentous proteins play a pivotal role in bacterial adhesion with the surrounding environment. Pili are polymeric proteins located at the cell surface of bacteria. 3Institut Universitaire de France, Parris, France.2Laboratoire de Chimie Physique et Microbiologie pour les Matériaux et l’Environnement (LCPME), UMR 7564, CNRS-Université de Lorraine, Nancy, France.1Laboratoire d’Ingénierie des Biomolécules, Université de Lorraine, Nancy, France.

Raphael Dos Santos Morais 1* Sofiane El-Kirat-Chatel 2 Jennifer Burgain 1 Blandine Simard 1 Sarah Barrau 1 Cédric Paris 1 Frédéric Borges 1 Claire Gaiani 1,3*

Capto core 400