*4.2. Protein Expression and Purification*

Expression of LrtA was carried out in BL21(DE3) or C41 [41] strains with a final ampicillin concentration of 100 mg/mL at 37 ◦C. The cells were cultured in 1 L flasks. Protein expression was induced with a final concentration of 1.0 mM IPTG when the absorbance of the cell culture at 600 nm was 0.4–0.9, and the cells were grown for 15–16 h at 37 ◦C. Cells were harvested at 8000 rpm in a JA-10 rotor (Beckman Coulter, Miami, FL, USA) for 15 min. The pellet from 5 L of culture was re-suspended in 50 mL of buffer A (500 mM NaCl, 5 mM imidazole, 20 mM Tris buffer (pH 8), 0.1% Triton X-100 and 1 mM β-ME), supplemented with a tablet of Sigma Protease Cocktail EDTA-free and 2 mg of DNase (per 5 L of culture). After being incubated with gentle agitation at 4 ◦C for 10 min, cells were disrupted by sonication (Branson sonicator, 750 W, Richmond, VA, USA), with 10 cycles of 45 s at 55% of maximal power output and an interval of 15 s between the cycles. All the sonication steps and the interval waits were carried out in ice. The lysate was clarified by centrifugation at 18,000 rpm for 40 min at 4 ◦C in a Beckman JSI30 centrifuge with a JA-20 rotor (Beckman Coulter, Miami, FL, USA).

The clarified lysate from such first centrifugation did not contain a large amount of LrtA, and thus, we suspected that most of the protein was present in the cell debris precipitate. Therefore, the precipitate was treated with buffer A supplemented with 8 M urea and a tablet of Sigma Protease Cocktail EDTA-free and 2 mg of DNase (per 5 L of culture). The re-suspended sample was treated with another 10 cycles of sonication in ice, and the sample was clarified by centrifugation at 20,000 rpm for 30 min at 4 ◦C. LrtA was in the supernatant and was purified by immobilized affinity chromatography (IMAC). The supernatant was added to 5 mL of Ni-resin previously equilibrated in buffer A supplemented with 8 M urea. The mixture was incubated for 20 min at 4 ◦C, and afterwards, the lysate was separated from the resin by gravity. On-column refolding was carried out during the washing step with 20 mL of buffer B (20 mM Tris buffer (pH 8.0), 500 mM NaCl, 1 mM β-ME, and 20 mM imidazole); the protein was eluted by gravity from the column with buffer C (20 mM Tris buffer (pH 8.0), 500 mM NaCl, 1 mM β-ME, and 500 mM imidazole). The eluted LrtA was extensively dialyzed against buffer D (100 mM sodium phosphate buffer (pH 8.0) with 500 mM NaCl). Precipitate in the dialysis tubing after five dialysis steps in buffer D was removed by centrifugation at 20,000 rpm for 30 min at 4 ◦C. The final yield of protein was 4.5–6.5 mg/L of culture (with both cellular strains assayed), and the protein was 90–95% pure as judged by SDS gels (Figure S14). This purity percentage takes into account the possible contamination due to the presence of deoxyribonucleotides, as judged by their absorbance at 260 nm (see below).

We attempted to re-purify the protein recovered from IMAC by using gel filtration chromatography in a Superdex 16/600, 75 pg column (GE Healthcare, Barcelona, Spain) connected to an AKTA FPLC system (GE Healthcare) by monitoring the absorbance at 280 nm; nevertheless, the protein was bound to the column and did not come out within its bed volume. Binding to the column has been also observed during purification of the recombinant HPF from *Staphylococcus aureus* [5], another member of the long subfamily of HPF.

The eluted protein from IMAC showed absorbance at 260 nm, suggesting that it was probably contaminated with di- or tri-deoxyribonucleotides (from the cleavage of the DNase used during purification and even though sample was dialyzed against 500 mM of NaCl). Presence of deoxyribonucleotides has been also observed in the recombinant HPF from *S. aureus* after its purification [5]. We tried to remove the deoxyribonucleotides by using different concentrations of polyethylenimine (PEI), ranging from 0.2 to 1% (*v*/*v*) [42], but most of LrtA co-precipitated with the oligonucleotides. The total protein concentration, *P*<sup>c</sup> (in mg/mL) was determined by using the expression [43]: *P*<sup>c</sup> = 1.55 A280 − 0.75 A260, where A280 and A260 are the absorbance of the dialyzed protein solution at 280 and 260 nm, respectively. However, it is important to note that the presence of deoxyribonucleotides did not affect the spectroscopic signals either of both fluorescence and far-UV CD, because DNA is spectroscopically silent in fluorescence, and in the far-UV region of CD spectra between 210–240 nm, deoxyribonucleotides do not absorb [44,45]. It is also interesting to note that deoxyoligonucleotides in aqueous solution or intact DNA show a small or slightly positive ellipticity around 222 nm [45–49] where we have carried out the study of the CD biophysical properties of the protein (see Results section).
