E coli bl21 de3 plyss

Site-directed mutagenesis was carried out as specified in the QuikChange II Site-Directed Mutagenesis Kit (Stratagene). The pET21-ferB plasmid served as a PCR template; the primers are listed in Table S1. After the mutagenesis protocol, the sequences of the new ferB constructs were verified by sequence analysis. The mutated plasmids were purified using the Plasmid Mini DNA Purification Kit (Qiagen) and transformed into the host expression strain E. coli BL21(DE3) pLysS (Invitrogen). Expression of the recombinant protein mutants and the purification procedure by Ni-IDA (GE Healthcare) chromatography were performed as described for the wild-recombinant type enzyme [13] (link). Correct folding of all mutant enzymes was confirmed by circular dichroism spectroscopy.

L. lactis M4 was a locally isolated strain from fresh milk. E. coli BL21(DE3)pLysS, E. coli TOP10, pCR-BluntII-TOPO vector, and pRSET A expression vector were purchased from Invitrogen (Invitrogen, USA). L. lactis M4 was grown at 30°C in M17 medium supplemented with 0.5% glucose. E. coli BL21(DE3)pLysS harboring and E. coli TOP10 harboring pCR/SOD were cultured aerobically at 37°C in Luria Bertani (LB) medium supplemented with 35 μg/mL chloramphenicol and 50 μg/mL ampicillin or 10 μg/mL kanamycin, respectively.

For heterologous expression of the recombinant enzymes, E. coli BL21 (DE3) pLysS (Invitrogen) were transformed with the expression constructs or the empty vector as a control. Expression, extraction and purification were done as described previously^{26 (link)}. The elution fractions from Strep Tactin sepharose resin (IBA, Göttingen, Germany) were analyzed via SDS-PAGE and the fractions with the highest amounts of purified recombinant proteins (or the corresponding fractions from vector control samples) were pooled and used in enzyme assays. Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Primers matching the 38 target sequences were designed and, appended to these primers, were sequences identical to the DNA sequence of the expression vector pET29a immediately upstream and downstream of its multiple cloning site. The pET29a sequences appended to the forward primers were chosen to facilitate the insertion of the amplified genes with the initiator methionine at the translation start site in the expression vector. The reverse primers were designed to remove the endogenous stop codon and allow read through of the hexa‐histidine tag encoded by the expression vector. Gibson assembly (HiFi assembly NEB) was used to include the PCR products of the SDR genes directly into pET29a plasmids. Amplifications of genes from the metagenome and cloning into the vector were successful for 37 out of 38 target sequences. Primers and adapters for Gibson cloning are given in in the SI Table S4. The recombinant vectors were then transformed into a chemically competent cloning strain E. coli Top10 (Invitrogen) and a chemically competent expression strain E. coli BL21*(DE3)pLysS (Invitrogen) and stored as glycerol stocks.

cDNA of the CPV-2 NS1 endonuclease gene was synthesized after codon optimization (Genscript, USA) based on the N-terminal amino acid sequence (amino acids 1–277 of the DNA-binding region) of CPV-2 NS1 endonuclease (GenBank Accession No. AAV36764.1) (Fig. S2). The CPV-2 NS1 endonuclease gene was imbedded into the pET-28a (+) vector (Novagen, Germany) with hexahistidine tags at the N- and C-termini. The plasmids were transformed into E. coli BL21 (De3) pLysS (Invitrogen, USA) for expression. The transformed cells were cultured in 1 L of LB containing 30 μg/ml kanamycin at 37°C until the OD₆₀₀ reached 0.6. Next, protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 24°C for 20 h. Cells were harvested by centrifugation at 12,000 ×g for 25 min and 4°C. The pellet was resuspended in 50 ml 50 mM Tris-HCl buffer (pH 7.0) containing 300 mM NaCl and disrupted by sonication. After centrifugation at 12,000 ×g for 30 min and 4°C, the supernatant was applied to a Ni-NTA agarose column (Qiagen, Germany) and washed with 50 mM Tris-HCl buffer (pH 8.0) containing 300 mM NaCl and 20 mM imidazole. CPV-2 NS1 endonuclease was eluted with 50 mM Tris-HCl buffer (pH 8.0) containing 300 mM NaCl, and 250 mM imidazole, and dialyzed against 50 mM Tris-HCl (pH 7.0) containing 300 mM NaCl at 4°C overnight. The purified protein was stored at −20°C until required.

Recombinant 6XHis-tag GreA or 6XHis-tag GreB were produced by cloning greA and greB genes into NdeI and BamHI sites of the pET14B vector (Novagen) using greA F and greA R or greB F and greB R primers, respectively (Tables b and c in S1 Text). All constructs were confirmed by sequencing. Plasmids were expressed in E. coli BL21 (DE3) pLysS (Invitrogen). Cells grown in LB broth at 37°C to an OD₆₀₀ of 0.5 were treated with 1 mM isopropyl β-D-1-thiogalactopyranoside. After 3 h, the cells were harvested, disrupted by sonication, and centrifuged to obtain cell-free supernatants. 6XHis-tag fusion proteins were purified using Ni-NTA affinity chromatography (Qiagen) as per manufacturer’s instructions. DksA protein was purified as described previously [56 (link)]. A GST-DksA fusion protein was purified using Glutathione-Sepharose 4B (bioWORLD, Dublin, Ohio, USA) according to manufacturer’s protocols. To remove the GST tag, PreScission protease was added to recombinant GST-DksA protein in phosphate-buffered saline (PBS) containing 10 mM DTT. After overnight incubation at 4°C, proteins were eluted and further purified by size-exclusion chromatography on Superdex 75 (GE Healthcare Life Sciences). Purified DksA proteins were aliquoted inside a BACTRON anaerobic chamber (Shel Lab, Cornelius, Oregon, USA). The purity and mass of the recombinant proteins were assessed by SDS/PAGE.