Pet28a vector

Recombinant N-terminal hexahistidine tag (His-Tag) containing cytolysins: Streptococcus pneumoniae pneumolysin (PLY), Gardnerella spp. vaginolysin (VLY), Streptococcus intermedius intermedilysin (ILY), Clostridium perfringens perfringolysin O (PFO), Listeria monocytogenes listeriolysin O (LLO), Streptococcus pyogenes streptolysin O (SLO) were expressed and purified as previously described in [27 (link)]. Production of Lactobacillus iner inerolysin (INY) was described in [32 (link)]. Briefly, the corresponding cytolysin-coding DNA lacking a putative signal sequence, except for PLY, was amplified from the respective bacterial isolates. The obtained PCR products were sequenced, cloned into pET28a(+) vector (Thermo Fisher Scientific, Waltham, MA, USA), and transformed into E. coli BL21 (DE3) strain (Merck KGaA, Darmstadt, Germany). Recombinant cytolysins were purified using Ni-chelate affinity chromatography columns (HisTrap HP, 17-5247-01, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) according to the manufacturer’s recommendations.

The plasmids containing a DNA sequence
encoding eGFP-NT2RepCT (flanked with a 6xHisTag at N terminal) protein
and RFP (mCherry) were obtained from a commercial supplier (Genscript).
DNA sequences of all truncation variants were derived from the DNA
sequence of eGFP-NT2RepCT by PCR (Thermo Fisher Scientific). All DNA
sequences were cloned into a pET-28a vector (which was genetically
modified to have two BsaI restriction enzyme recognition
sites in its multiple cloning sites) using golden gate cloning (Thermo
Fisher Scientific) for protein expression in E. coli or into a yeast-E. coli shuttle vector pRS413-Gal,
pRS416-Gal, or pRS413-GPD using XhoI and XbaI restriction enzymes (Thermo Fisher Scientific) for
protein expression in Saccharomyces cerevisiae. All
plasmids were transformed into the chemically competent E.
coli top 10 cells for plasmid amplification and long-term
storage. All plasmids were verified by DNA Sanger sequencing (Eurofins).

The construction of the pET28_bglhi plasmid containing the Bglhi has been previously described [15 ], and random mutagenesis of the bglhi coding sequence was performed by error-prone PCR (epPCR) using the pET28_bglhi plasmid as template. The reaction mixture (100 μL) contained 7 mM MgCl₂, 200 μM dNTPs, 100 pmol of each universal DNA primers (T7promoter and T7terminator, complementary to the 5´ and 3´ regions flanking the bglhi coding sequence in the pET-28a(+) vector), 200 ng template, 100 μM MnCl₂ and 5 units (U) of Taq polymerase (Thermo Scientific, Wilmington, USA) and its respective reaction buffer. Thermocyling involved a single initial heating to 95°C for 2 min, followed by 30 cycles of 94°C for 1 min, 45°C for 1 min and 72°C for 3 min and a final heating to 72°C for 10 min. The PCR products were separated by agarose gel electrophoresis and purified by Wizard® SV Gel Extraction kit (Promega, Madison, USA). The extracted DNA fragments were digested with restriction enzymes NheI and BamHI (Thermo Scientific, Waltham, MA, USA), and ligated into the pET-28a(+) vector (Novagen, Madison, WI, USA) linearized with the same enzymes. The random mutant library (denominated pET28_mut) was used to transform E. coli BL21 (DE3) by electroporation. The recombinant plasmid pET28_bglhi was also used to transform E. coli BL21 (DE3) as a control for all random mutant library screening steps.