Sumo protease

The strains used for antimicrobial activity determination, E. coli ATCC 25922, E. coli ATCC 078, E. coli UB 1005, Salmonella Typhimurium (S. Typhimurium) C 7731, S. Typhimurium ATCC 14028, P. aeruginosa ATCC 27853, Staphylococcus aureus (S. aureus) ATCC 29213, S. aureus ATCC 25923, Staphylococcus epidermidis (S. epidermidis) ATCC 12228, and Streptococcus faecalis (S. faecalis) ATCC 29212, were all preserved by the Institute of Animal Nutrition, Northeast Agricultural University. The vector pPICZaA was purchased from Liuhe Huada Gene Technology Co., Ltd. (Beijing, China). Restriction endonucleases were obtained from Thermo Fisher Co., Ltd. (Waltham, USA), and SUMO protease was purchased from Gene Copoeia (Guangzhou, China). A plasmid extraction kit was obtained from Genstar (Beijing, China). The Ni–NTA Sefinose (TM) resin kit was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The purity of other chemical reagents used was of analytical grade.

The SUMO–C–L protein was cleaved by SUMO protease (Genecopoeia, Rockville, MD, USA) at 30 °C for 3 h. The recombinant C–L protein was purified with Ni-NTA Sepharose column. The enzymatic solution and dialyzed fractions were analyzed by tricine-SDS-PAGE.

rLectin (Lectin domain of CLSP2) was amplified by RT-PCR from cDNA with specific primers (S6 Table). The PCR product was subcloned into PSFM (a kind gift from Dr. Haobo Jiang, Oklahoma State University), a vector with a Sumo at the N-terminal, which increases the solubility of the fusion protein and can be removed by SUMO protease afterwards. The N-terminal FLAG and the C-terminal Myc are short sequences for detection of the expression of fusion protein and its cleavage products using commercially available monoclonal antibodies against these two tags, respectively. SUMO-rLectin was first purified on a Ni-NTA (nickel-nitrilotriacetic acid, Qiagen) agarose column. Then, SUMO-rLectin was cleaved using SUMO protease, as per the manufacturer’s protocol (GeneCopoeia), and re-purified on the Ni-NTA agarose column. Monoclonal antibodies were prepared against KLH-peptide from CLSP2 (Beijing Protein Innovation). Polyclonal antibodies were prepared against recombinant CLSP2 and recombinant PPO3 (Beijing Protein Innovation). Specificity tests of these antibodies are presented in S1 Fig.