Mini protein system

The experimental procedures used for the cloning, expression, and purification of selected proteins (either from naïve or homology sequence screening) in the Ek/LIC 46 vector and E. coli strain BL21 (DE3) were performed as described previously (Alcaide et al., 2015 (link)). The primers used for amplification are listed in Supplementary Material. All proteins studied here were N-terminally His₆-tagged, and the soluble His-tagged proteins were produced and purified at room temperature after binding to a nickel–nitrilotriacetic acid (Ni–NTA) His-Bind resin (from Merck Life Science S.L.U., Madrid, Spain) as described previously (Giunta et al., 2020 (link)), with slight modifications (the expression culture was scaled up to 1 L using 50 ml pre-inoculum). The purity was assessed as >98% using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE; Supplementary Figure 1) in a Bio-Rad Mini Protein system (Laemmli, 1970 (link)). Protein concentrations were determined according to the Bradford method with bovine serum albumin as the standard (Bradford, 1976 (link)). A total of approximately 0.8–37 mg of purified recombinant proteins was obtained from each 1 L culture on average, as follows: Est_A1 (6.4 mg/L), Est_A2 (25 mg/L), Est_A3 (13 mg/L), Est_A4 (37 mg/L), Est_A5 (41 mg/L), Est_A6 (7 mg/L), Est_A7 (0.8 mg/L), Est_A8 (19 mg/L), Est_B1 (1.0 mg/L), and Est_B2 (32 mg/L).

To analyze the activity of MMP-2, cells were seeded in 24-well plates and grown in GM. Upon reaching confluence, cells were treated with IL-17 and/or Dox and cultivated for an additional 24 h in a serum-free medium. Afterward, the conditioned medium was collected and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using a mini protein system (Bio-Rad, Richmond, CA, USA), as previously shown [20 (link)]. SDS-PAGE was performed in 8% polyacrylamide gels containing 0.1% gelatin under non-reducing conditions. The gel was then washed in 2% Triton X-100 for 30 min and incubated in 100 mM Tris-HCl, pH 8.5, with 10 mM CaCl2. After 24 h, gels were stained with Coomassie Brilliant blue R-250 for 30 min, causing the appearance of transparent bands inside the colored gel, which corresponds to the activity of MMP-2. Following the staining, the gel was scanned using the ChemiDoc Imaging System (Bio-Rad), and the activity of MMP-2 was quantified using NIH-Image J software V 1.8.0.

The total protein and membrane protein of cells were prepared with protein extraction reagent (Tiangen, Beijing, China) and a Membrane and Cytosol Protein Extraction Kit (Beyotime, Shanghai, China), respectively. The protein levels were assayed by the BCA protein kit (Tiangen, Beijing, China). Equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) filters membrane using a Mini-Protean 3 System (Bio-Rad, Hercules, CA, USA). The membrane was blocked in blocking buffer with 5% skim milk for 1 h and then incubated with specific primary polyclonal/monoclonal antibodies overnight at 4 °C. After incubation with horseradish peroxidase conjugated secondary antibody for 2 h, immunoreactive proteins were visualized with the ECL Plus Western Blotting Detection Reagents (Fude-bio, Hangzhou, China) and detected using a Mini-Protein System (Bio-Rad). Protein bands were quantitated with NIH ImageJ software and normalized by GAPDH bands for analysis.

The native PAGE experiment was performed with Mini PROTEIN®System (BIO-RAD, USA), under the following conditions: 15% (w/v) separating gel with a 4% (w/v) stacking gel (non-denaturing polyacrylamide gel) in Tris–glycine (pH 7.2), and 6-µL samples were loaded. The proteins were stained with Coomassie Blue G250.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was conducted by using a Mini-PROTEIN® System (Bio-Rad, USA) and a 12% gel according to the manufacturer’s protocol. Proteins were transferred to a nitrocellulose blotting membrane and were probed with primary antibodies, followed by an HRP-conjugated secondary antibody. Immunolabeled proteins were detected by incubation with an enhanced chemiluminescence (ECL) substrate, followed by the exposure of the membrane to autoradiography film.