Kits for dna isolation

Restriction endonucleases, T4 DNA ligase, Phusion DNA polymerase, molecular mass markers and kits for DNA isolation were from Thermo Fisher Scientific (Waltham, MA, USA). LB (Luria-Bertani) medium was from Amresco (Solon, OH, USA), Tryptic Soy Broth, Brain Heart Infusion and Yeast Extract were from Difco-Becton Dickinson and Co. (Franklin Lakes, NJ, USA), general laboratory reagents were from Sigma-Aldrich (Moscow, Russia), liquid chromatography media were from GE Healthcare (Moscow, Russia), reagents for Western blot were from Bio-Rad (Moscow, Russia). The commercially available reagent “Absorbed staphylococcal anatoxin” (Medgamal, Moscow, Russia) was used as a crude immunogen. This reagent represents cell-free broth culture of S. aureus O15 inactivated by 0.4% formalin, concentrated by sequential treatment with trichloro acetic acid at pH=3.5 and 70% ethanol and absorbed on aluminium hydroxide. The same preparation but without formalin treatment and aluminium hydroxide absorption was used in Western blot and protein electrophoresis as “staphylococcal anatoxin” (i.e. non-absorbed).

Restriction endonucleases, T4 DNA ligase, Phusion DNA polymerase, molecular mass markers, and kits for DNA isolation were obtained from Thermo Fisher Scientific (Moscow, Russia). The lysogeny broth Miller recipe (LB) medium was obtained from Amresco (Solon, OH, USA); liquid chromatography media from GE Healthcare (Moscow, Russia); reagents for agarose and polyacrylamide gel electrophoresis from Bio-Rad (Moscow, Russia); and general laboratory reagents from Merck (Moscow, Russia).
Gene cloning and recombinant protein production were carried out in Escherichia coli DH10B and Rosetta (DE3), respectively (Merck). Vectors for cloning and recombinant protein expression in E. coli included pMal-c5x (cloning with the NH₂-terminal maltose-binding protein (MBP), New England Biolabs, Ipswich, MA, USA) and pGEX4T-1 (cloning with the NH₂-terminal glutathione-S-transferase (GST), GE Healthcare). Anti-MBP antibody (#E8032S) was purchased from New England Biolabs, and anti-FLAG antibody (#14793) from Cell Signaling (Leiden, The Netherlands). The source of the green fluorescent protein coding sequence (eGFP) was the plasmid pEGFP-C, obtained from Clontech (Takara Bio Europe SAS, Saint-Germain-en-Laye, France).