Construction of pEXP-htrA plasmid
The htrA (> CP009792.1:4,438,735–4,440,180) gene was obtained by PCR amplification using the pCHTRA7 plasmid as a template (unpublished results). This plasmid is derived from pCR2.1TOPO (Invitrogen, USA) which have cloned the htrA gene, 457 base pairs (bp) upstream and 412 bp downstream of the Y. pseudotuberculosis YPIII htrA gene. The primers used for the amplification were: htrATG_mut_del 5′ATG AAA AAA ACC ACG TTAG and htrA_CTG_rev 5′CTG CAT CAA TAA ATA GAG TG 3′, using Taq Platinum from Invitrogen®. The 1 444 pb PCR product was cloned into the pEXP5-CT/TOPO (Invitrogen, USA) vector. Random selected clones were digested with BglII and EcoRI, and clones with the restriction pattern expected were sequenced.
The oppA gene (> CP009792.1:2,946,314–2,947,951) is cloned into the pEXP5-CT expression vector, pEXP-oppA (unpublished results), the construction was used as a DNA template for inverse PCR. In order to obtain mutant R41A:D42A, two mutagenic primers were designed following the instructions from the QuickChange II (Agilent, USA) method. Primers BsgI_mut02_del 5′CCTGAATCCAATATTTCACGTCCACTACTTGAAGGGCTGGTG and BsgI_mut02_rev 5′CACCAGCCCTTCAAGTAGTGCAGCTGAAATATTGGATTCAGG (in bold, the nucleotides changed to generate the amino acid substitution), two recognition sites for restriction enzymes BsgI and PvuII (underlined) were also included. PvuII was used to select the clones that had the desired mutation.
To obtain the OppAD419G:Y420G and OppAR41A:D42A/D419G:Y420G mutants, an inverse PCR was performed using the plasmid pEXP-OppA as a template for mutant OppAD419G:Y420G and plasmid pEXP-OppAR41A:D42A for mutant OppAR41A:D42A:D419G:Y420G. For this inverse PCR we used two primers designed back to back and phosphorylated at the 5′end, in this system only one primer is mutagenic. The primers used were: 01__del / 5Phos / GC TTG GTG TGC AGGTGG CAA TGA GCC ATC CTC CTT CCT AAA TAT G (nucleotides changed to replace amino acids D419G / Y420G are in bold) and 01_rev / 5Phos / GG CCC GGG CAA CAT CAT AAG TCC CCT GGT GAC GGG TAT CC (the cut site for the SmaI enzyme is underlined). The PCR product was digested by the enzyme DpnI, after which it was self-circularized by ligation reaction using the enzyme T4 DNA Ligase Thermo Scientific® (Thermo Fisher Scientific, USA). It was subsequently transformed into chemically competent TOP10 cells and plated on LB agar supplemented with Amp 100 µg/mL, the selection was the same as that used for the OppAR41A:D42A mutant. Random clones were selected and its plasmids were digested with SmaI, the clones with the expected restriction pattern were selected for sequencing.
Purification of proteins
Plasmids pEXP-OppA, pEXP-OppAR41A:D42A, pEXP-OppAD419G:Y420G, pEXP-OppAR41A:D42A/D419G:Y420G, and pEXP-htrA were transformed into E. coli BL21 (pLys). Cells harboring these plasmids were grown in LB medium supplemented with Amp 100 µg/mL and incubated at 37 °C with constant stirring. At an O.D.600 of 0.5, IPTG (Invitrogen, USA) was added to the cultures at a final concentration of 0.15 mM. After three hours of induction, cells were harvested, washed with PBS buffer, and frozen at − 70 °C overnight. The pellet was resuspended in lysis buffer (20 mM KH2PO4, 250 mM KCl, 20 mM imidazole, 1 mg/mL of lysozyme pH 7.0) and incubated on ice for 30 min. Then, N-lauroyl-sarcosine was added to a final concentration of 0.1% and the mix was incubated on ice for 30 min more. The suspension was then sonicated and centrifuged at 5000 rpm for 15 min, the supernatant was dialyzed in 20 mM KH2PO4, 250 mM KCl, pH 7 buffer (buffer A). After that, the bacterial extracts were loaded onto a Sephadex G-50 column equilibrated in 20 mM KH2PO4, 250 mM KCl pH 7 buffer, and eluted in the same buffer. The fractions were analyzed by 10% SDS-polyacrylamide gel to determine which fractions contain proteins, these fractions were allowed to interact with Ni-NTA resin for 2 h at 4 °C with constant stirring. After that, protein purification was performed following the manufacturer’s protocol for protein purification in native conditions. In order to eliminate imidazole, proteins were equilibrated in 20 mM KH2PO4, 250 mM KCl buffer pH 7 by gel filtration chromatography on a Sephadex G-50 column. Fractions were quantified by Bradford assay and immediately used for the chaperone activity assays.
Chaperone-like activity assays
α-glucosidase renaturation assay
Urea in a concentration of 4 m at 20 °C was the means of denaturing 8.4 µg of α-glucosidase (α-Glucosidase from Saccharomyces cerevisiae, Sigma-Aldrich, USA), during 20 min, at 25 °C. After that, 100 µl of 50 mM KH2PO4, 200 mM KCl, pH 7 buffer was added, then 39 µg of bovine serum albumin (BSA), 26.7 µg of HtrA, 34 µg of OppA proteins were added in each treatment, finally were added 50 µl of protease inhibitor 10X and 50 mM KH2PO4, 200 mM KCl, pH 7 untill 500 µl of final volume mix. The treatments were: (i) the negative control was BSA in a 1150 nM concentration, (ii) target OppA was prepared in a concentration of 1150 nM, and (iii) the positive control HtrA was prepared in a 1000 nM concentration (1 µM). The SIGMAFAST™ (Sigma, USA. Cat. S8830) Protease Inhibitor reactant was added according to the protocol of the commercial kit.
The molar α-glucosidase: OppA molar ratio was 1:4, while the urea concentration during the refolding process was held at 80 mM, because at this concentration the activity of the enzyme α-glucosidase is not affected which was empirically determined.
In the following step, all the treatments were incubated at room temperature during one and a half hours and subsequently 2 µg maltose sugar was added and the mixture was stirred to obtain a solution. Finally, after another hour and a half the glucose concentration was quantified by standard glucose oxidase/peroxidase protocol from BioSystems® (Ref. 11,504). The tests were performed in triplicate for statistical proof.
Lactate dehydrogenase renaturation assay
1.25 µg of lactate dehydrogenase from rabbit muscle (Sigma-Aldrich®, Sigma, USA) was denatured with 1 molar guanidine hydrochloride (GdnHCl) during 10 min at 25 °C. After that, 100 mM of NaH2PO4 pH 7 buffer was added, the final volume mix was 500 µl. The mix was incubated at 25 °C for 90 min with 287 nM chaperones: BSA (negative control), OppAWT, OppA mutants and HtrA (as positive control). Again, The SIGMAFAST™ (Sigma, USA) Protease Inhibitor was added according to the protocol of the commercial kit. Lactate dehydrogenase (71.43 nM) activity assays were performed at 25 °C according to Sigma-Aldrich® (Sigma, USA) protocol. β-NADH was measured by spectrophotometry at 340 nm every 10 s for 5 min. LDH units in each assay were calculated according to LDH activity assay protocol from Sigma-Aldrich® (Sigma, USA).
The molar ratio beween LDH and chaperone-like proteins (OppA, BSA and HtrA) was set to the same proportion as before, 1 to 4, while GdnHCl was applied at a 20 mM concentration because at this concentration level the LDH activity remains unaltered, all of which had been empirically determined prior to our bioassays.
All the measurements were carried out in triplicate and results were expressed in terms of mean values ± standard deviation whereas the corresponding percentage of enzymatic activity values were calculated using statistical software. (Sigma Plot for Windows versión 10.0. Systat software, Inc., Germany). All tests were performed in triplicate for statistical proof applying One way ANOVA.