All chemicals and media were from Sigma-Aldrich (ST.Louis, USA), unless indicated otherwise. Bicinchoninic acid (BCA) assay reagent was from Pierce (Rockford, IL, U.S.A.). DNAse I was from Roche Diagnostics (Mannheim, Germany) and chicken egg white lysozyme was obtained from Fluka (Buchs, Switzerland; 84,468 U/mg). Hiprep 26/10 desalting and Superdex Peptide 10/300 columns were purchased from GE Healthcare, (Uppsala, Sweden). PageRuler™ Prestained Protein Ladder was from (Fermentas, Vilnius, Lithuania). Phosphate buffer saline (PBS) was obtained from Braun (Melsungen AG, Germany).
Bacterial strains and plasmids
Escherichia coli BL21 (DE3) containing the T7 RNA polymerase under control of the lacUV5 promoter was purchased from Invitrogen (Breda, The Netherlands). BL21 (DE3) was transformed with pET-SUMO-SA2  and pSUPER-dtUD1 constructs (kindly donated by Prof. Patrick J. Loll)  separately according to the pET-SUMO supplier protocol (Invitrogen, Carlsbad, USA). Briefly, 6.5 ng of the plasmid DNA was added to 50 μl of chemically competent E.coli BL21 (DE3) in a Eppendorf tube and shaken gently. The tube was put on ice for 30 min after which the cells were placed in a water bath of 42°C for 30 sec. Next, the tube was placed on ice. For recovery of cells, 250 μl of SOC medium was added to the tube. To make a stock for the transformed E.coli bacteria, 100 μl of the bacterial suspension was transferred into a LB plate containing 50 μg/ml of kanamycin and incubated at 37°C overnight. A single colony was selected and grown in 5 ml LB overnight. The overnight grown bacteria were cooled on ice and glycerol was added up to 30% of final volume before storage at −80°C.
Media composition and protein expression
LB medium (peptone; 10 g/L, yeast extract; 5 g/L and 10 g/L of NaCl) was used for the pre-culture preparation and expression of SUMO-SA2 and SUMO protease (dtUD1). Auto induction medium (ZYM) was made according to the Studier method  and was used for SUMO-SA2 expression. In short, 1 L of ZYM medium that contained Tryptone (10 g/l), Yeast Extract (5 g/l), MgSO4 (1 mM), 20 mL of 50 × 5052 solution (glycerol 250 g/l, glucose 25 g/l, alpha lactose 100 g/l in RO water), kanamycin (100 mg/l) and 50 ml of 20× NPS solution ((NH4)2SO4 (66 g/l), KH2PO4 (136 g/l), Na2HPO4 (142 g/l). One liter of autoclaved ZYM or LB media was inoculated with 5 ml of overnight seed culture of the transformed E. coli strain BL21 (DE3). LB medium was incubated in a shaking incubator (Innova 4335, New Brunswick Scientifc,USA) at 37°C/250 rpm and induced with 1 mM IPTG when the culture reached OD600 = 0.6-0.8. Next, bacteria were harvested after 4 hrs by centrifugation at 5,000× g for 30 min at 4°C. Inoculated autoinduction medium was shaken at 37°C/250 rpm and bacteria were collected after 16 hrs at 5,000× g for 30 min at 4°C.
Purification of SUMO-SA2
For the isolation and purification of the SA2 peptide, bacterial pellets were suspended in the lysis buffer (3 ml for each gram of biomass) (20 mM Na2HPO4, 150 mM NaCl, 20 mM imidazole, 5 mM MgCl2 1.5% N-lauroylsarcosine, pH 8) supplemented with DNAse I 1 μg/ml and chicken egg white lysozyme 300 μg/ml. The resulted suspension was incubated on ice for 30 min. Subsequently, urea was added to the suspension to achieve 4 M final concentration. Lysis was accomplished using a Braun Labsonic tip-sonicator (Braun Biotech, Melsungen, Germany) for 5 min with 30 second stop between each 30 second pulse and passing two times through high pressure homogenizer. Next, the cell lysate was centrifuged (30 minutes, 40.000 g, 20°C) and supernatant was filtered through a 0.45 μm filter. SUMO-SA2 was purified by affinity chromatography using a 50 ml packed column of Ni-NTA Superflow (Qiagen, Chatsworth, CA) attached to an AKTA Purifier (GE Healthcare, Uppsala, Sweden). The column was washed with 5 column volumes of binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 40 mM imidazole, pH 8) after which the cleared lysate was loaded onto a 50 ml packed Ni2+-NTA column at 0.5 ml/min at room temperature. After loading the cleared lysate, the column was washed with the binding buffer until the A280 reached to the baseline. His-tagged proteins were eluted from the column with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 8).
To remove excess imidazole and NaCl, the elution buffer was exchanged with cleavage buffer (20 mM hepes, 150 mM NaCl, pH 8.0) by loading onto a Hiprep 26/10 desalting column.
As the molecular weight of SA2 peptide (1.142 KD) is 8% of the molecular weight of the SUMO-SA2 (14295 KD), the highest expected amount of SA2 that can be released after enzymatic cleavage can be calculated.
Purification of SUMO protease
The same protocol as described above was used for the purification of SUMO protease without addition of urea. Moreover, the elution buffer was replaced by the storage buffer (50 mM NaH2PO4, 300 mM NaCl, 1 mM DTT, pH 8.0) prior to protein quantification using the BCA assay which BSA used as a standard. Finally, 0.2 mg/ml dilutions were made by adding glycerol 50% (v/v) and stored at −80°C until required.
Purification of SA2 peptide
SUMO protease was added at a 1:500 molar ratio to the purified SUMO-SA2 solution supplemented with 1 mM DTT and the mixture was incubated under gentle shaking for 6 hrs at 30°C to allow SUMO cleavage from the SA2 peptide.
To separate SA2 peptide from cleaved SUMO, SUMO protease and uncleaved SUMO-SA2, selective precipitation by ethanol was performed.
The pH of protein solution after cleavage was adjusted to 11.5 then ethanol was added up to 50% of the total volume to precipitate all proteins except SA2. After centrifugation at 5000xg at 4°C for 15 min, supernatant was collected and pH of supernatant was adjusted to 2 by adding 1 M HCl to precipitate SA2. The precipitate was collected and suspended in 0.1 M HCl and centrifuged at 5000× g at 4°C. This procedure was repeated 3 times.
Subsequently, recovered peptide was confirmed by HPLC, and mass spectrometry. Finally the peptide pellet was lyophilized at −50°C and at 0.5 mbar in a Chris Alpha 1–2 freeze-drier (Osterode am Harz, Germany) for 12 hrs and stored at −20°C.
Characterization of produced peptide
The produced proteins were evaluated by SDS-PAGE. Samples were boiled in Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) for 5 min and loaded at 20 μl/well onto NuPAGE 10% Novex Bis-Tris gels (12 wells, 1.0-mm thickness; NuPAGE, Invitrogen, Carlsbad, CA, USA). Electrophoresis was performed at room temperature applying a constant voltage of 175 V for 50 min. The gel was stained with Page Blue™ Protein Staining Solution (Fermentas GMBH, St. Leon-Rot, Germany) and destained overnight by washing with RO water.
Size exclusion chromatography
Cleavage of SUMO-SA2 was followed by Size Exclusion Chromatography on a Superdex Peptide 10/300 GL column at a flow rate of 0.7 ml/min with phosphate buffered saline at pH = 7.4 or pH = 11.5 as the mobile phase. Prior to loading the samples a Gel Filtration LMW Calibration kit (GE Healthcare, Uppsala, Sweden) was used to validate column performance.
HPLC analysis and mass spectrometry
1 mg of Lyophilized SA2 peptide was dissolved in 1 ml of DMSO and 20 μl of the peptide solution was diluted 5 times in RO water. 50 μl of prepared sample was injected onto a Sunfire C18 column (waters Corporation, Milford, USA). A gradient was run at 1.0 ml/min flow rate from buffer A (5% actonitrile, 0.1% trifluoroacetic acid, 95% water) in 30 minutes to buffer B (100% acetonitrile, 0.1% trifluoroacetic acid). UV absorption was monitored at 220 nm, 280 nm and also fluorescent emission at 350 nm of tryptophan residue upon excitation at 295 nm was recorded.
Furthermore, Electrospray ionization (ESI) mass spectrometry was carried out using a Shimadzu LCMS QP-8000(Duisburg, Germany) single quadrupole bench top mass spectrometer (m/z range, 2000), coupled with a QP-8000 data system.