Ngc medium pressure chromatography system

The pPAL7 vector system (Bio-Rad Laboratories, Hercules, CA, USA) was used for the induction of recombinant PIMTs in E. coli BL21(DE3). Vectors with PvPimt genes were artificially synthesized by GenScript Biotech (Piscataway, NJ, USA). The cells were lysed using the “BugBuster” protein extraction reagent (Merck Millipore, Burlington, MA, USA), centrifuged at 16,000× g for 20 min at 4 °C, and 1 or 5 mL of supernatant were used for fast protein liquid chromatography (FPLC) on NGC™ Medium-Pressure Chromatography Systems (Bio-Rad) with 1 or 5 mL of eXact column (Bio-Rad). The purified proteins were stored at −20 °C in a phosphate buffer with 10% glycerol until use. SDS PAGE was used for electrophoretic separation using a “sample buffer” with a composition of ×2 0.1 M Tris HCl, pH 6.8, 4% SDS, 12% β-mercaptoethanol, 20% glycerol, ≈0.0001% BPB, and a “running buffer”, with a composition of 12% Tris, 13.3% aspartic acid, 1% SDS, pH 6.0, in polyacrylamide gel with a composition of 0.3M Tris HCl, pH 7.2, 9% acrylamide, 0.1% SDS, 0.1% APS, and 0.1% TEMED, with a protein amount of 1 µg.

To obtain mutated HP0377 proteins for biochemical experiments, a set of recombinant plasmids were constructed. pUWM544 [21 (link)], which carries the hp0377 gene (amino acids 25–221) without its promoter and signal sequence, was used as a starting plasmid for all constructs. Point mutations were generated using the Quick Change Site-Directed Mutagenesis Kit (Qiagen) according to the manufacturer’s instructions, starting with pUWM544 as a template. Correct construction of the plasmids was verified by sequencing. Next, the hp0377-modified nucleotide sequences from pUWM544-derived recombinant plasmids were inserted into pET28a. All plasmids carried the HP0377-His₆ translation fusion. HP0377 and its mutated forms were overexpressed by autoinduction from an E. coli Rosetta strain and purified using NGC Medium-Pressure Chromatography Systems by Bio-Rad as previously described [21 (link)]. HP1227 was overexpressed by IPTG induction and purified from an E. coli Rosetta strain as previously described [21 (link)]. EcDsbC E. coli protein was overexpressed from E.coli BL21harboring pET28a/EcDsbC using autoinduction [30 (link)].

The recombinant phage-borne endolysin was prepared according to the method described previously by Maciejewska et al. (2017) (link). Briefly, the coding sequence of Klebsiella phage KP27 endopeptidase was amplified using Pfu polymerase (Thermo Fisher Scientific, Waltham, MA, United States) and cloned into the commercially available pEXP-5-CT/TOPO^® TA expression vector (Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer recommendations. The expression was conducted for 18 h at 20°C using E. coli BL21 (DE3) pLysS (Agilent Technologies, Santa Clara, CA, United States) and isopropyl-β-D-1-thiogalactopyranoside (IPTG; the final concentration of 0.5 mmol/L) as an inductor of the expression. The recombinant protein was purified from the filtered supernatant by affinity chromatography using NGC medium pressure chromatography systems (Bio-Rad, Hercules, CA, United States) combined with 5-ml nickel columns using Bio-Scale Mini Profinity IMAC Cartridges (Bio-Rad, Hercules, CA, United States) and dialyzed against a PBS buffer. The concentration of purified recombinant enzyme was then determined fluorimetrically (Qubit^® Protein Assay Kit, Molecular Probes, Thermo Fisher Scientific, Waltham, MA, United States).

HP0377 expression vector was constructed by amplifying the region encoding the mature HP0377 protein (without the signal sequence, amino acids 1–24) from the chromosome of H. pylori 26695, with primers hp377exI and hp377exII. The insert was cloned into pET28a with NcoI and XhoI restriction enzymes, to yield plasmid pUWM518. The cytoplasm-located HP0377 was overexpressed from pUWM518 by autoinduction [32 (link)], and then it was purified by affinity chromatography, dialyzed against Phosphate Buffered Saline (Sigma) and later used for rabbit immunization (Animal Facility, Faculty of Biology, University of Warsaw). The anti-HP0377 rabbit serum was specific and recognized native HP0377, as verified by Western blot analysis.
The HP0377 C89S and C92S expression vectors were constructed by cutting out hp0377-changed sequences from pUWM2045 and pUWM2064, respectively, and cloning the inserts into pET28a as described above, resulting in formation of pUWM2046 and pUWM2065. All plasmids carried the HP0377-His₆ translation fusion.
For biochemical experiments, the protein was expressed and purified from E. coli Rosetta harboring pUWM518, pUWM2046 and pUWM2065. The proteins were overexpressed by autoinduction and then purified by affinity chromatography using NGC Medium-Pressure Chromatography Systems by Bio-Rad as described above.