Jun 02, 2026
Expression and Purification of Rice SUMOylation machinery
- Joana Marques1,
- Isabel A. Abreu1
- 1Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB NOVA)
- Joana Marques: Validated and wrote the Protocol
- Isabel A. Abreu: Reviewed and approved the final version of the protocol
Protocol Citation: Joana Marques, Isabel A. Abreu 2026. Expression and Purification of Rice SUMOylation machinery. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoe6edl4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 23, 2026
Last Modified: June 02, 2026
Protocol Integer ID: 315616
Keywords: SUMOylation, SEC, ULP1, ÄKTA Pure, SUMO, E2, SCE, E1, SAE, Protein Purification, purification of rice sumoylation machinery sumoylation, purification of the rice sumoylation machinery, rice sumoylation machinery sumoylation, rice sumoylation machinery, purification, eukaryote
Funders Acknowledgements:
Fundação para a Ciência e a Tecnologia (Portugal)
Grant ID: 2020.06917.BD
Fundação para a Ciência e a Tecnologia (Portugal)
Grant ID: FilliGRAIN-PROTECT: https://doi.org/10.54499/PTDC/ASP-PLA/1920/2021
Fundação para a Ciência e a Tecnologia (Portugal)
Grant ID: GREEN-it ‘Bioresources4sustainability’: UID/04551/2025, DOI: 10.54499/UID/04551/2025; UID/PRR/04551/2025, DOI: 10.54499/UID/PRR/04551/2025
Abstract
SUMOylation is a post-translational modification that occurs in Eukaryotes. This protocol describes the production and purification of the Rice SUMOylation machinery.
Guidelines
Columns should be cleaned according to the manufacturer’s instructions and stored in 20% ethanol when not in use. All buffers should be filtered (0.22 µm) and degassed before use.
All chromatography steps were carried out at 4 °C to preserve protein stability.
Protease inhibitors and DTT should be added to the buffers immediately before use.
Materials
E.coli Strain:
Rosetta (DE3) pLysS (Invitrogen)
Media:
LB media (For 1 L: 10 g Tryptone, 5 g Yeast Extract, and 10 g NaCl)
Antibiotics:
Ampicillin: 100 mg/ml, sterilize by filtration and store in 1 ml aliquots at −20°C.
Kanamycin: 50 mg/ml, sterilize by filtration and store in 1 ml aliquots at −20°C.
Chloramphenicol: 30 mg/mL, sterilize by filtration and store in 1 ml aliquots at −20°C.
Other reagents:
Isopropyl-β-D-thiogalactoside (IPTG): 1 M, filter-sterilize and store in aliquots at −20°C.
Dithiothreitol (DTT): 1 M, stored in aliquots at −20°C.
MgCl2: 1 M, autoclave before use.
Buffers:
SAE1/SAE2 complex:
- Cell lysis: 20 mM KPi, pH 7.5, 500 mM NaCl, 20 mM imidazole, 2mM PMSF, 5 mM MgCl2, and DNAse I (5–10 µg/mL of lysate)
- Buffer A: 20 mM KPi, pH 7.5, 500 mM NaCl, and 20 mM imidazole
- Buffer B: 20 mM KPi, pH 7.5, 500 mM NaCl, and 500 mM imidazole
- Hiload Superdex Superdex 200 pg preparative SEC column buffer: 20 mM Tris-HCl (pH 7.5), 50 mM NaCl
- Storage Buffer: 20 mM Tris-HCl, pH 7.5, 50 mM NaCl + 20% glycerol
SCE1:
- Cell lysis: 20 mM NaPi, pH 7.5, 500 mM NaCl, 20 mM imidazole, 2 mM PMSF, 5 mM MgCl2, and DNAse I (5–10 µg/mL of lysate)
- Buffer A: 20 mM NaPi, pH 7.5, 500 mM NaCl, and 20 mM imidazole
- Buffer B: 20 mM NaPi, pH 7.5, 500 mM NaCl, and 500 mM imidazole
- Hiload Superdex Superdex 200 pg preparative SEC column buffer:
- Storage Buffer: 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 0.7 mM DTT + 20% glycerol
SUMO1:
- Cell lysis: 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM PMSF, 5 mM MgCl2, and DNAse I (5–10 µg/mL of lysate)
- Buffer A: 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 20 mM imidazole
- Buffer B: 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 500 mM imidazole
- Storage Buffer: 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 1 mM DTT + 20% glycerol
Purification equipment:
Equipment
ÄKTA pure
NAME
Protein purification system
TYPE
Cytiva
BRAND
29046665
SKU
LINK
Columns:
Equipment
HisTrap HP His tag protein purification columns
NAME
protein purification columns
TYPE
Cytiva
BRAND
17524801
SKU
LINK
5mL
SPECIFICATIONS
Equipment
HiLoad Superdex 200 pg preparative SEC column
NAME
Protein Purification column
TYPE
Cytiva
BRAND
28989336
SKU
LINK
16/60
SPECIFICATIONS
Concentrator:
Equipment
Ultra Centrifugal Filter, 50 kDa MWCO
NAME
Centrifugal concentrator
TYPE
Amicon®
BRAND
UFC9050
SKU
LINK
Protocol materials
SUMO ProteaseInvitrogen - Thermo FisherCatalog #12588018
Safety warnings
It's important to set the pressure alarm for each column before use.
For optimal protein production we recommend using either vector pet28a/b or pet28g (https://dx.doi.org/10.17504/protocols.io.j8nlk9e75v5r/v1).
Ethics statement
Not applicable.
Before start
SUMOylation is a post-translational modification (PTM). This PTM is an ATP-dependent cascade that results in the covalent attachment of a mature Small Ubiquitin-like Modifier (SUMO) peptide to a target protein (Figure A). The cascade starts with the activation of SUMO by the E1 complex, followed by transfer of SUMO to the SUMO-conjugating enzyme, and ends with the covalent attachment of the SUMO peptide to the target protein.
Scheme 1. Schematic representation of the SUMOylation pathway. Before activation, the Small Ubiquitin-like Modifier (SUMO) peptide is processed by a SUMO-specific protease to expose its C-terminal diglycine motif. SUMO is then activated by the heterodimeric SUMO-activating enzyme (SAE) in an ATP-dependent reaction. Following activation, SUMO is transferred from the catalytic cysteine of SAE2 to the active-site cysteine of the SUMO-conjugating enzyme (SCE). SCEs can conjugate SUMO directly to a target protein or facilitate the reaction with the assistance of a SUMO E3 ligase. SUMOylation is reversible, as SUMO proteases can cleave SUMO from modified substrates, restoring the unmodified protein.
In vitro SUMOylation assays are essential for demonstrating the occurrence and specificity of this modification. To perform this assay, E1 complex, SUMO peptide, and SUMO conjugating enzyme are sufficient for SUMO conjugation to occur (Werner, 2009). Here, we present detailed protocols for the expression and purification of the Rice SUMOylation machinery: heterodimeric SUMO-activating enzyme (SAE), SUMO-conjugating enzymes (SCEs), and SUMO peptides.
OsSAE1/2: Cloning
The E1 complex subunits were cloned separately into the pET200 expression vector, generating the constructs pET200-OsSAE1 and pET200-OsSAE2.
Note
The E1 complex can be produced either by co-expressing both subunits in the same E. coli strain or by expressing each subunit independently. In this protocol, independent expression followed by in vitro reconstitution was selected, as this approach yields higher amounts of recombinant protein (Werner, 2009).
OsSAE1/2: Protein production
Bacterial cell growth: E. coli Rosetta (DE3) pLysS cells transformed with either pET200-OsSAE1 or pET200-OsSAE2 were grown separately at 37 °C and 180 rpm in LB medium (per 1 L: 10 g tryptone, 5 g yeast extract, and 10 g NaCl) supplemented with 50 μg/mL kanamycin (Kan) and 30 μg/mL chloramphenicol (Cam). Cultures were incubated until the OD₆₀₀ reached 0.8. For each construct, cells were grown in 5-L Erlenmeyer flasks containing 2 L of medium.
Protein expression induction: Recombinant protein expression was induced by adding 200 µM IPTG, followed by incubation for 4 h at 30 °C with shaking at 180 rpm, prior to cell harvesting.
Cell harvesting: Cells were harvested by centrifugation at 16,000 × g for 1 h at 4 °C.
OsSAE1/2: Cell lysis
Cell resuspension: Cell pellets corresponding to 7.7 g (wet weight) of OsSAE1 and 9.8 g (wet weight) of OsSAE2 were each resuspended in 100 mL of Buffer A (20 mM KPi, pH 7.5; 500 mM NaCl; 20 mM imidazole) supplemented with 2 mM PMSF, 5 mM MgCl₂, and DNase I (5–10 µg/mL lysate).
Cell lysis: Cells were lysed using a French press with three passes at 15,000 psi. The lysate was subsequently centrifuged at 20,000 × g for 1 h 30 min at 4 °C. The clarified supernatant was collected, and the pellet was discarded.
Extract preparation: The clarified supernatant was filtered through a 0.22 µm cellulose nitrate syringe filter to obtain the protein extract for subsequent purification steps.
OsSAE1/2: Protein Purification - HisTrap Affinity Chromatography
All chromatography steps were performed at 4 °C. A HisTrap HP column (Cytiva) was first equilibrated, and the filtered protein extract was loaded onto the column using a peristaltic pump (see Materials) at a flow rate of 5 mL/min. All subsequent chromatographic steps were carried out using an ÄKTA Pure system (see Materials) at a flow rate of 3 mL/min, with a maximum pressure limit of 0.5 MPa.
Equipment
HisTrap HP His tag protein purification columns
NAME
protein purification columns
TYPE
Cytiva
BRAND
17524801
SKU
LINK
5mL
SPECIFICATIONS
Equipment
ÄKTA pure
NAME
Protein purification system
TYPE
Cytiva
BRAND
29046665
SKU
LINK
Column preparation: The column was washed with 5 column volumes (25 mL) of water, followed by 5 column volumes of binding buffer for equilibration.
Column preparation and washing: The column was washed with 5 column volumes (25 mL) of water, followed by 5 column volumes of Buffer A (20 mM KPi, pH 7.5; 500 mM NaCl; 20 mM imidazole) for equilibration. After sample loading, the column was connected to the ÄKTA Pure system, and non-bound proteins were removed by washing with Buffer A until the UV baseline stabilised.
Protein elution: Proteins were eluted using a 0–100% imidazole gradient generated by mixing Buffer A with Buffer B (20 mM KPi, pH 7.5; 500 mM NaCl; 500 mM imidazole) over 20 column volumes (100 mL). Eluted fractions were collected in 2 mL fractions.
Figure 1. Purification of 6×His-tagged OsSAE1/6×His-tagged OsSAE2 by HisTrap HP affinity chromatography.
Mixed protein extracts from E. coli Rosetta (DE3) pLysS cells expressing 6×His-tagged OsSAE1 and 6×His-tagged OsSAE2 were loaded onto a HisTrap HP column (Cytiva) and purified using an ÄKTA Pure system at a flow rate of 1 mL/min. The column was washed with Buffer A (20 mM KPi, pH 7.5; 500 mM NaCl; 20 mM imidazole), and bound proteins were eluted using a linear gradient of Buffer B (20 mM KPi, pH 7.5; 500 mM NaCl; 500 mM imidazole).
The injected sample, flow-through, and eluted fractions were analyzed by 10% SDS–PAGE.
Figure 2. SDS–PAGE analysis of eluted fractions containing 6×His-tagged OsSAE1 and 6×His-tagged OsSAE2.
The soluble fraction from E. coli Rosetta (DE3) pLysS cells expressing OsSAE1 /OsSAE2 was loaded onto the ÄKTA Pure system (injected, Inj). Non-bound proteins were collected in the flow-through (FT). Protein eluted fractions 8 to 24 were loaded in a 10% SDS–PAGE and stained with Coomassie Blue (BlueSafe, NZYTech). The molecular weight marker (M) and corresponding molecular masses are indicated on the left.
Fractions 10 to 13 were pooled and concentrated to a final volume of 3 mL prior to injection onto a HiLoad Superdex 200 pg preparative SEC column (Cytiva). Concentration was performed using an Amicon Ultra-15 centrifugal filter unit (50 kDa molecular weight cutoff) by centrifugation at 4,000 × g and 4 °C in a swing-bucket benchtop centrifuge. Samples were centrifuged in 10-min intervals until the desired final volume was reached.
Note
No protein precipitation was observed during the concentration process.
OsSAE1/2: Protein Purification - HiLoad Superdex 200 Size-Exclusion Chromatography
All chromatography steps were performed at 4 °C using an ÄKTA Pure system (see Materials) at a flow rate of 1 mL/min and with a maximum pressure limit of 0.5 MPa. The HiLoad Superdex 200 pg preparative SEC column (Cytiva) was equilibrated prior to injection of the concentrated protein sample at a flow rate of 1 mL/min.
Equipment
HiLoad Superdex 200 pg preparative SEC column
NAME
Protein Purification column
TYPE
Cytiva
BRAND
28989336
SKU
LINK
16/60
SPECIFICATIONS
Equipment
ÄKTA pure
NAME
Protein purification system
TYPE
Cytiva
BRAND
29046665
SKU
LINK
Column preparation: The column was washed with 5 column volumes (620 mL) of water, followed by 5 column volumes of running buffer (20 mM Tris-HCl, pH 7.5; 50 mM NaCl) for equilibration.
Column loading: The concentrated pooled fractions (fractions 10 to 13; total volume of 3 mL) obtained from the HisTrap purification step were injected onto the column using the ÄKTA Pure system, using a 3 mL loop.
Note
Sample volume is critical for achieving optimal separation resolution in size-exclusion chromatography. For high-resolution separation, a loading volume corresponding to approximately 0.5–1% of the column volume (CV) is recommended. However, loading volumes of up to 1–2% of the CV are commonly used and generally still provide good separation resolution.
Protein elution: Proteins were eluted with running buffer (20 mM Tris-HCl, pH 7.5; 50 mM NaCl), and 1 mL fractions were collected throughout the separation.
Figure 3. Purification of the E1 heterodimeric complex (6×His-tagged OsSAE1/6×His-tagged OsSAE2) by preparative size-exclusion chromatography (SEC).
Pooled fractions obtained from the HisTrap purification step were concentrated and injected onto a HiLoad Superdex 200 pg preparative SEC column (Cytiva). Proteins were separated using arunning buffer consisting of 20 mM Tris-HCl, pH 7.5, and 50 mM NaCl. Eluted fractions were collected in 1 mL fractions.
The injected sample and the collected SEC fractions were analyzed by 10% SDS–PAGE.
Figure 4. SDS–PAGE analysis of 6×His-tagged OsSAE1/6×His-tagged OsSAE2 eluted from a preparative SEC column.
Pooled fractions obtained from the HisTrap purification step were concentrated and injected onto a HiLoad Superdex 200 pg preparative SEC column (Cytiva) (Inj). Eluted fractions 8 to 28 were analyzed by 10% SDS–PAGE and stained with Coomassie Blue (BlueSafe, NZYTech). The molecular weight marker (M) and corresponding molecular masses are indicated on the left. Fractions 20 to 24 were selected for further purification.
Note
For optimal E1 enzyme activity, fractions containing both subunits at an approximately 1:1 ratio should be selected.
Protein concentration: The pooled protein fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (30 kDa molecular weight cutoff) by centrifugation at 4,000 × g and 4 °C in a swing-bucket benchtop centrifuge. Samples were centrifuged in 10-min intervals until the desired final volume was reached.
Note
No protein precipitation was observed during the concentration process. Following concentration, glycerol was added to a final concentration of 20% (v/v) for long-term storage. Protein samples were aliquoted to the desired volumes, flash-frozen in liquid nitrogen, and stored at −70 °C.
OsSCE1a: Cloning
The OsSCE1a was cloned into the pDEST17 expression vector, generating the construct pDEST17-OsSCE1a.
Note
This expression vector yielded low amounts of protein. For future cloning, we would recommend using pET28a/b or pET28g (Luís & Abreu 2025).
OsSCE1a: Protein production
Bacterial cell growth: E. coli Rosetta (DE3) pLysS cells transformed with pDEST17-OsSCE1a were grown at 37 °C with shaking at 180 rpm in LB medium (per 1 L: 10 g tryptone, 5 g yeast extract, and 10 g NaCl) supplemented with 100 µg/mL ampicillin (Amp) and 30 µg/mL chloramphenicol (Cam). Cultures were incubated until reaching an OD₆₀₀ of 0.8–1.0. For each construct, cells were grown in 5-L Erlenmeyer flasks containing 2 L of medium.
Protein expression induction: Recombinant protein expression was induced by adding 200 µM IPTG, followed by incubation for 5 h at 30 °C with shaking at 180 rpm, prior to cell harvesting.
Cell harvesting: Cells were harvested by centrifugation at 16,000 × g for 1 h at 4 °C.
OsSCE1a: Cell lysis
Cell resuspension: A cell pellet corresponding to 6.4 g (wet weight) was resuspended in 30 mL of Buffer A (20 mM NaPi, pH 7.5; 500 mM NaCl; 20 mM imidazole) supplemented with 2 mM PMSF and DNase I (5–10 µg/mL lysate).
Cell lysis: Cells were lysed using a French press with three passes at 15,000 psi. The lysate was subsequently centrifuged at 20,000 × g for 1 h 30 min at 4 °C. The clarified supernatant was collected, and the pellet was discarded.
Extract preparation: The clarified supernatant was filtered through a 0.22 µm cellulose nitrate syringe filter to obtain the protein extract for subsequent purification steps.
OsSCE1a: Protein Purification - HisTrap
Chromatography setup: All chromatography steps were performed at 4 °C. The HisTrap HP column (Cytiva) was equilibrated, and the protein extract was loaded using a peristaltic pump (see Materials) at a flow rate of 5 mL/min. All subsequent chromatographic steps were carried out on an ÄKTA Pure system (see Materials) at 2 mL/min, with a maximum pressure limit of 0.5 MPa.
Equipment
HisTrap HP His tag protein purification columns
NAME
protein purification columns
TYPE
Cytiva
BRAND
17524801
SKU
LINK
5mL
SPECIFICATIONS
Equipment
ÄKTA pure
NAME
Protein purification system
TYPE
Cytiva
BRAND
29046665
SKU
LINK
Column preparation: The column was washed with 5 column volumes (25 mL) of water, followed by 5 column volumes of binding buffer for equilibration.
Column loading: The 30 mL protein extract was loaded onto the column by recirculating the sample 2–3 times to maximise binding of the His-tagged protein.
Following sample loading, the column was connected to the ÄKTA Pure system, and non-bound proteins were removed by washing with Buffer A (20 mM NaPi, pH 7.5; 500 mM NaCl; 20 mM imidazole) until the UV baseline stabilized.
Note
This step ensures the removal of contaminating endogenous E. coli proteins.
Protein elution: OsSCE1a protein was eluted using a 0–100% imidazole gradient generated by mixing Buffer A with Buffer B (20 mM NaPi, pH 7.5; 500 mM NaCl; 500 mM imidazole) over 20 column volumes (100 mL). Eluted fractions were collected in 2 mL fractions.
Figure 5. Purification of 6×His-tagged OsSCE1a by HisTrap HP affinity chromatography.
Protein extract from E. coli Rosetta (DE3) pLysS cells expressing 6×His-tagged OsSCE1a was loaded onto a HisTrap HP column (Cytiva) and purified using an ÄKTA Pure system at a flow rate of 1 mL/min. The column was washed with Buffer A (20 mM NaPi, pH 7.5; 500 mM NaCl; 20 mM imidazole), and bound proteins were eluted using a linear gradient of Buffer B (20 mM NaPi, pH 7.5; 500 mM NaCl; 500 mM imidazole).
The injected sample, flow-through, and eluted fractions were analyzed by 10% SDS–PAGE.
Figure 6. SDS–PAGE analysis of eluted fractions containing 6×His-tagged OsSCE1a.
The soluble fraction from E. coli Rosetta (DE3) pLysS cells expressing OsSCE1a was loaded onto the ÄKTA Pure system (injected, Inj). Non-bound proteins were collected in the flow-through (FT). Protein eluted fractions 2 to 15 were loaded in a 10% SDS–PAGE and stained with Coomassie Blue (BlueSafe, NZYTech). The molecular weight marker (M) and corresponding molecular masses are indicated on the left.
OsSCE1a: Protein Dialysis and Concentration
Selected fractions 7 to 1 were pooled (approximately 40 mL) and transferred into SnakeSkin dialysis tubing. The tubing was tightly sealed at both ends and additionally secured using a clothespin.
Dialysis: The SnakeSkin dialysis tubing was placed in a beaker containing 4 L of dialysis buffer (10 mM Tris-HCl, pH 7.5; 50 mM NaCl; 0.7 mM DTT) and a magnetic stir bar. Dialysis was carried out overnight at 4 °C with continuous stirring.
Protein concentration: The dialyzed protein sample was concentrated using an Amicon Ultra-15 centrifugal filter unit (10 kDa molecular weight cutoff) by centrifugation at 4,000 × g and 4 °C in a swing-bucket benchtop centrifuge. Samples were centrifuged in 10-min intervals until the desired final volume was reached.
Note
No protein precipitation was observed during the concentration process. Following concentration, glycerol was added to a final concentration of 20% (v/v) for long-term storage. Protein samples were aliquoted to the desired volumes, flash-frozen in liquid nitrogen, and stored at −70 °C.
OsSUMO1: Cloning
The OsSUMO1 was cloned into the pET28a expression vector, generating the construct pET28a-OsSUMO1.
Note
SUMO1 protein may be produced in two different ways: precursor or mature forms (see Scheme 2). This should be defined during the cloning step by specifying where to terminate protein translation. If the SUMO protein is produced in its precursor form, the C-terminal diglycine motif must be exposed before performing in vitro SUMOylation assays. Producing precursor SUMO1 requires additional purification steps (see step 37 and section OsSUMO1: Maturation).
Scheme 2. SUMO1 maturation. In vivo, SUMO1 is produced in its precursor form. SUMO1 precursor is processed by a cysteine SUMO protease. This process exposes the C-terminal diglycine motif, which is required for SUMO1 conjugation to a target protein.
Note
If SUMO is produced in its precursor form, the SUMO peptide must be processed. Proteolytic processing of SUMO precursors can be performed in vitro using the commercially available yeast cysteine protease ScULP1t (Li & Hochstrasser 2003) (SUMO ProteaseInvitrogen - Thermo FisherCatalog #12588018 ). This means that SUMO1 and ScULP1t are incubated in an appropriate buffer (e.g., 10 mM Tris-HCl, pH 7.5; 50 mM NaCl; 1 mM DTT) (see section OsSUMO1: Maturation). After incubation, the mature SUMO peptide must be separated from the SUMO protease and the SUMO1 precursor. Therefore, carefully choosing the placement of the His-tag is essential, especially if the ScULP1t is also tagged with a His-TAG.
- Option 1, C-terminal His-tag: Since the SUMO protease will cleave off the C-terminal tail, which in this case includes the His-Tag, the mature SUMO can be purified by either a His-Trap or an SEC-based chromatography. This means this option is more versatile.
- Option 2, N-terminal His-tag: This option requires SEC-based chromatography as the step following cleavage.
Scheme 3. SUMO1 His-tag placement cloning options.
OsSUMO1: Protein production
Bacterial cell growth: E. coli Rosetta (DE3) pLysS cells transformed with pET28a-OsSUMO1 were grown at 37 °C with shaking at 180 rpm in LB medium (per 1 L: 10 g tryptone, 5 g yeast extract, and 10 g NaCl) supplemented with 50 µg/mL kanamycin (Kan) and 30 µg/mL chloramphenicol (Cam) until reaching an OD₆₀₀ of 0.8–1.0. Cultures were grown in 5-L Erlenmeyer flasks containing 2 L of medium.
Protein expression induction: Recombinant protein expression was induced by adding 400 µM IPTG, followed by incubation for 5 h at 30 °C with shaking at 180 rpm, prior to cell harvesting.
Cell harvesting: Cells were harvested by centrifugation at 16,000 × g for 1 h at 4 °C.
OsSUMO1: Cell lysis
Cell resuspension: A cell pellet corresponding to 9.88 g (wet weight) was resuspended in 50 mL of Buffer A supplemented with 1 mM PMSF and DNase I (5–10 µg/mL lysate).
Cell lysis: Cells were lysed using a French press with three passes at 15,000 psi. The lysate was subsequently centrifuged at 20,000 × g for 1 h 30 min at 4 °C. The clarified supernatant was collected, and the pellet was discarded.
Extract preparation: The clarified supernatant was filtered through a 0.45 µm cellulose nitrate syringe filter to obtain the protein extract for subsequent purification steps.
OsSUMO1: Protein Purification - HisTrap
All chromatography steps were performed at 4 °C. The HisTrap HP column (Cytiva) was equilibrated, and the filtered protein extract was loaded onto the column using a peristaltic pump (see Materials) at a flow rate of 5 mL/min. All subsequent chromatographic steps were carried out using an ÄKTA Pure system (see Materials) at a flow rate of 2 mL/min, with a maximum pressure limit of 0.5 MPa.
Equipment
HisTrap HP His tag protein purification columns
NAME
protein purification columns
TYPE
Cytiva
BRAND
17524801
SKU
LINK
5mL
SPECIFICATIONS
Equipment
ÄKTA pure
NAME
Protein purification system
TYPE
Cytiva
BRAND
29046665
SKU
LINK
Column preparation: The column was washed with 5 column volumes (25 mL) of water, followed by 5 column volumes of binding buffer for equilibration.
Column loading: The 50 mL protein extract was loaded onto the column by recirculating the sample 2–3 times to maximize binding of the His-tagged protein.
Following sample loading, the column was connected to the ÄKTA Pure system, and non-bound proteins were removed by washing with Buffer A (20 mM KPi, pH 7.5; 500 mM NaCl; 20 mM imidazole) until the UV baseline stabilized.
Protein elution: Proteins were eluted using an imidazole gradient generated by mixing Buffer A and Buffer B from 0% to 30% Buffer B. From fraction 11 onward, elution was performed using stepwise increases to 30%, 50%, and 100% Buffer B over a total of 20 column volumes (100 mL). Eluted fractions were collected in 2 mL fractions.
Figure 7. Purification of 6×His-OsSUMO1 by HisTrap HP affinity chromatography.
Protein extract from E. coli Rosetta (DE3) pLysS cells expressing 6×His-OsSUMO1 was loaded onto a HisTrap HP column (Cytiva) and purified using an ÄKTA Pure system at a flow rate of 1 mL/min. The column was washed with Buffer A (20 mM Tris-HCl, pH 7.5; 500 mM NaCl; 20 mM imidazole), and bound proteins were eluted using a linear gradient of Buffer B (20 mM Tris-HCl, pH 7.5; 500 mM NaCl; 500 mM imidazole).
The injected sample, flow-through, and collected fractions were analyzed by 10% SDS–PAGE.
Figure 8. SDS–PAGE analysis of eluted fractions containing 6×His-OsSUMO1.
The soluble fraction from E. coli Rosetta (DE3) pLysS cells expressing OsSUMO1 was injected onto the ÄKTA Pure system (Inj). Non-bound proteins were collected in the flow-through (FT). Eluted protein fractions 1 to 17 were analysed by 10% SDS–PAGE and stained with Coomassie Blue (BlueSafe, NZYTech). The molecular weight marker (M) and corresponding molecular masses are indicated on the left.
OsSUMO1: Protein Dialysis, Concentration and Storage
Fractions 8 to 17 were pooled (approximately 25 mL) and transferred into SnakeSkin dialysis tubing for dialysis against buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 1 mM DTT. The tubing was tightly sealed at both ends and additionally secured using a clothespin.
Dialysis: The SnakeSkin dialysis tubing was placed in a beaker containing 4 L of dialysis buffer (10 mM Tris-HCl, pH 7.5; 50 mM NaCl; 1 mM DTT) and a magnetic stir bar. Dialysis was carried out overnight at 4 °C with continuous stirring.
Protein concentration: The dialyzed protein sample was concentrated using an Amicon Ultra-15 centrifugal filter unit (3 kDa molecular weight cutoff) by centrifugation at 4,000 × g and 4 °C in a swing-bucket benchtop centrifuge. Samples were centrifuged in 10-min intervals until the desired final volume was reached.
Note
No protein precipitation was observed during the concentration process. Following concentration, glycerol was added to a final concentration of 20% (v/v) for long-term storage. Protein samples were aliquoted to the desired volumes, flash-frozen in liquid nitrogen, and stored at −70 °C.
OsSUMO1: Maturation (optional)
Proteolytic processing of SUMO precursors can be performed in vitro using the commercially available yeast cysteine protease ScULP1t (Li & Hochstrasser 2003) (SUMO ProteaseInvitrogen - Thermo FisherCatalog #12588018 ).
Proteolytic processing: Approximately 25 mL of pooled OsSUMO1 fractions were incubated with 7 µg of ScULP1t overnight at 4 °C with continuous stirring.
Protocol references
Werner, A., Moutty, M.-C., Möller, U., & Melchior, F. (2009). Performing in vitro Sumoylation reactions using recombinant enzymes. In H. D. Ulrich (Ed.), Methods in Molecular Biology: SUMO protocols (Vol. 497, pp. 187–199). Humana Press. https://doi.org/10.1007/978-1-59745-566-4
Luís, I. M., & Abreu, I. A. (2025) Preparation of level 0 modules for Golden Gate assembly in pET28g, a
new tool to prepare pET-based vectors for protein expression in Escherichia coli. (dx.doi.org/10.17504/protocols.io.j8nlk9e75v5r/v1)
Li, S. J., & Hochstrasser, M. (2003). The Ulp1 SUMO isopeptidase: Distinct domains required for viability, nuclear envelope localization, and substrate specificity. Journal of Cell Biology, 160(7), 1069–1081. https://doi.org/10.1083/jcb.200212052
Acknowledgements
We thank and acknowledge Cristina Timóteo, Rita Pacheco, Teresa Baptista da Silva, and the Protein Purification facility at ITQB-NOVA for bacterial cell disruption and affinity purification.
We acknowledge the Portuguese Fundação para a Ciência e a Tecnologia (FCT) for a fellowship for JM (PD/BD/06917/2020, 10.54499/2020.06917.BD). This work was supported by FCT - Fundação para a Ciência e a
Tecnologia, I.P., through Green-it Bioresources for Sustainability R&D Unit (UID/04551/2025, DOI: 10.54499/UID/04551/2025; UID/PRR/04551/2025, DOI: 10.54499/UID/PRR/04551/2025); and FilliGRAIN-PROTECT (https://doi.org/10.54499/PTDC/ASP-PLA/1920/2021).