Histidine tagged protein purification kit

A schematic of the LPH structure was showed in Fig. 2a. The Sangon Biotech (Shanghai, China) was entrusted to synthesize the following gene sequences: LPH without signal sequence (51-351 aa); D316A mutation of LPH (LPH-AS1); D329A mutation of LPH (LPH-AS2); P295A, G297A and T298A mutations of LPH (LPH-AS3); LPH-3D (51-267 aa of LPH); SagA without signal sequence (51-530 aa), LRP without signal sequence (51-357 aa) and LPP without signal sequence (51-339 aa). All these sequences contained restriction enzyme sites EcoR I at the 5’ end and Xho I at the 3’ end. These synthetic constructs were ligated into EcoR I- and Xho I digested pET-28a respectively, and transformed into competent E. coli BL21(DE3). During inducing protein expression, kanamycin was used at 50 μg/mL, and isopropyl-β-d-thiogalactoside (Sigma-Aldrich) was used at 0.5 mM for LPH, LPH-AS1, LPH-AS2, LPH-AS3, LPH-3D, LRP, and LPP, and at 1 mM for SagA. After induction of protein expression for 6 h, bacteria were pelleted and lysed ultrasonically. His-tagged proteins were purified using Histidine-Tagged Protein Purification Kit (CWBIO, China) and desalted using phosphate buffered saline (PBS) containing 20% glycerin, followed by a clearing of potential endotoxin by passing through a Detoxi-Gel Endotoxin Removing Gel (Thermo Fisher Scientific).

Briefly, LGG genomic DNA was isolated and used as a template for PCR. Considering the transmembrane structure at the N-terminal of HM0539, primers aimed at 51–357 amino sequences were designed for amplification of extracellular protein fragment:
F: 5′-GTAGAATTCGTTAACGCGGCAACGAAAG
R: 5′-CGGGCCTCGAGTTAGTTGATCACTTCAA.
PCR products were purified using a GenElute^TM PCR Clean-Up Kit (Sigma-Aldrich), and then digested with EcoR I and Xho I, followed by ligating to the same restriction enzyme cutting sites of pET-31a(+) (Novagen). The recombinant plasmid was transformed into E. coli DH5α (Tiangen Biotech Co., Ltd., Beijing, China). After incubation, the kanamycin resistant bacterial colony was lysed for PCR assay. The expression plasmid producing the HM0539 was confirmed by DNA sequencing and transformed into E. coli BL21(DE3) (Tiangen Biotech Co., Ltd., Beijing, China). Recombinant HM0539 protein with a histidine tag was overexpressed via 0.5 mM isopropyl-β-D-thiogalactoside induction at 37°C and purified using Histidine Tagged Protein Purification Kit (Soluble Protein, CW0894, CWBioTech, Beijing, China), according to the manufacturer’s instructions. The potential endotoxin of HM0539 was removed using resin according to the supplier’s instructions (EndotoxinOUT Resin; Sangon Biotech, Shanghai, China).