Use of NaCl 5M for the extraction of Lentilactobacillus kefiri S-layer proteins: characterization of the extracts and immunomodulatory activity on murine macrophages

ASSANDRI MH; CAPOSTGANO C; TREJO FM; SERRADELL MA

Mendoza

Congreso; LVIII Annual Meeting of SAIB; 2022

SAIB

Self-assembled S-layer proteins (SLP) are non-covalently bound to the cell wall of several prokaryotic species, including potentially probiotic L. kefiri (Lk) strains. Recently, we have demonstrated that different SLP-Lk are able to enhance macrophage activation in vitro, and particularly, SLP-Lk8348 showed adjuvant capacity in vivo. Thus, regarding the potentiality of these proteins for their animal or human use, we aimed to replace LiCl by NaCl as a safer and cheaper extraction agent to obtain SLP from Lk strains of interest. To achieve this, cultures of Lk8348 and Lk83111 were grown in MRS broth at 37°C for 48 hs, and then the SLP were isolated using 5M NaCl for 10 or 30 minutes at room temperature under stirring. Usual extraction using 5M LiCl for 10 minutes was carried out as control. SLP extracts were characterized by SDS-PAGE and concentration of total proteins was determined by Bradford´s method. Effect on murine macrophage cell line RAW264.7 activation was determined evaluating both IL-6 secretion and CD86 surface expression as markers. Regarding the amount of protein isolated after incubation for 10 minutes, 5M NaCl showed lower efficiency of extraction than 5M LiCl for both SLP-Lk8348 and SLP-Lk83111. The SDS-PAGE revealed no apparent differences between 5M LiCl and 5M NaCl extracts obtained after 10 minutes of incubation. However, the incubation for 30 minutes with 5M NaCl showed evidence of protein degradation in both SLP-Lk8348 and SLP-Lk83111 extracts. Regarding the immunomodulatory properties, both NaCl and LiCl extracts of SLP-Lk83111 were able to induce secretion of IL-6 (P