Pqe30 plasmid

DNA fragments coding for CpsC, CpsC-ΔCter, CpsD and ParB were obtained by PCR using chromosomal DNA from S. pneumoniae D39 as template. For the chimera CpsC/D, we used DNA from strain TIGR4. Oligonucleotides used are described in S2 Table. The chimera DNA fragment was constructed by fusion of fragments obtained using primer pairs I-III and IV-II. The obtained DNA fragment was cloned between the BamHI and HindIII cloning sites of the pQE30 plasmid (Qiagen). parB was cloned between the NdeI and PstI cloning sites of the pT7.7 plasmid [64 (link)]. To construct plasmids for yeast two-hybrid, the PCR DNA fragments were digested by EcoRI and PstI and ligated either into pGAD-C1 or pGBDU-C1 vectors [65 (link)]. The nucleotide sequences of all DNA fragments were checked to ensure error-free amplification. Plasmids and primers used in this study are listed in S1 and S2 Tables, respectively.

The amplified PCR product of 0.7 kb corresponding to BYK gene was extracted from the agarose gel and treated with Klenow fragment (New England Bio labs, USA) following the manufacturer’s protocol. The blunt-ended PCR product was cloned in the SmaI site of pQE-30 plasmid (QIAGEN Inc., USA) and transformed into E. coli DH5α, and the obtained clone named as BYK-1[7 (link),21 (link)].

The pQE30 plasmid (Qiagen) was used to construct vectors for the expression of fusion proteins comprising: four copies of the M2e peptide (two copies M2e consensus among human influenza viruses A – M2eh and two copies of M2e from A/California/07/09 H1N1pdm09 – M2es); HA2–2(76–130) of phylogenetic group 2 influenza viruses; and flagellin from Salmonella typhimurium. In the first hybrid protein (Flg-HA2–2-4M2ehs), the HA2–2 fragment was linked to the C-terminus of flagellin, followed by 4 copies of M2e peptide. In the second protein (FlgSh-HA2–2-4M2ehs) the hypervariable region of flagellin was replaced by the HA2–2(76–130) fragment, and 4 copies of the M2e peptide were attached to flagellin’s C-terminus. The creation of chimeric genes was accomplished via standard genetic engineering methods. Flagellin PCR product was obtained via amplification of Salmonella typhimurium genomic DNA and cloned. Nucleotide sequences encoding the HA2–2(76–130) consensus sequence and tandem copies of M2e were synthesized in vitro. In this manner, two recombinant protein expression vectors (pQE30_Flg_HA2–2_4M2e and pQE30_FlgSh_HA2–2_4M2e) were created.

The pQE30 plasmid was obtained from Qiagen (Chatsworth, CA), and the Phusion site-directed mutagenesis system from Thermo Scientific (Waltham, MA). The Ni²⁺-chelating Sepharose column and HiLoad 16/600 Superdex 200 pg column were from GE Healthcare (Kowloon, HK). Triton X-114 and protease inhibitor cocktails were purchased from Sigma-Aldrich (St. Louis, MO). The ultrafiltration membrane (30 kDa cut off) was from Millipore (Bedford, MA). Cell culture medium RPMI was purchased from Hyclone (Cambridge, MA), while FBS, penicillin, and streptomycin were from Life Technologies (Gaithersburg, MD). Quantikine ELISA assay kits for human IL-8 and recombinant human TLR4 protein were from R&D Systems (Minneapolis, MN). Rabbit antibodies to HpGroES were produced in our laboratory24 (link). The mouse mAbs against human TLR4 (HTA125) were acquired from BioLegend (San Diego, CA). TRITC-conjugated anti-rabbit and FITC-conjugated anti-mouse IgG antibodies were sourced from Millipore (Bedford, MA).

Recombinant TRIP-1 protein was expressed in bacteria using the pQE-30 plasmid (Qiagen) system. Briefly, 973-bp fragment corresponding to the coding region of TRIP-1 was cloned into EcoRI/XhoI restriction sites in pQE-30 vector. This plasmid was transformed into E. coli bacteria BL21-Gold (DE3). The protein expression was induced by the addition of 1 mM IPTG. The expressed protein was purified using Ni-NTA agarose (Qiagen) column under native conditions. The manufacturer’s protocol was followed for protein elution. Specifically, the buffer containing 250 mM imidazole, pH 8.0 was used to elute TRIP-1 from the column.

The HspB8 gene cloned in pGEX‐6P‐1 vector (GE Healthcare LifeSciences, Little Chalfont, England) was kindly provided by Serena Carra (University of Modena‐Reggio Emilia, Italy) and the protein was expressed in Escherichia coli BL21‐CodonPlus(DE3)‐RIL strain (argU (AGA, AGG), ileY (AUA), leuW (CUA)) as N‐terminal glutathione S‐transferase (GST) fusion protein. BAG3 gene sequence (NCBI code number NM_004281.3) was synthetized and cloned in pQE‐30 plasmid (Qiagen, Hilden, Germany) between BamHI and HindIII sites. His‐tagged protein was expressed in E. coli M15 (pREP4) strain (F⁻, Φ80ΔlacM15, thi, lac⁻, mtl⁻, recA⁺, KmR). The Josephin domain (JD, 182 aa) is the structured part of the ataxin‐3 proteins that does not contain the poly Q stretch. The JD‐encoding gene was cloned in pET21‐a vector and the protein was expressed in E. coli BL21 Tuner (DE) pLacI (E. coli B F⁻ ompT hsdSB (rB⁻ mB⁻) gal dcm lacY1 (DE3) pLacI (CamR); Novagen, Germany) and purified as previously described (Bonanomi et al., 2015 (link)).