SUMO1 Human His

What is SUMO1 and what are its key structural features?

SUMO1, also known as Sentrin, UBL1, and SMT3C, is synthesized as a 101 amino acid propeptide with a predicted molecular weight of 11.5 kDa. It belongs to the SUMO protein family, which functions similarly to ubiquitin in being covalently attached to target proteins, but instead of triggering degradation, SUMO1 modification regulates various cellular processes including nuclear transport, transcriptional regulation, apoptosis, and protein stability . All SUMO proteins share a conserved Ubiquitin domain and a C-terminal diglycine cleavage/attachment site. During SUMOylation, the C-terminal glycine residue of mature SUMO1 (after cleavage of a four amino acid C-terminal prosegment) is enzymatically attached to specific lysine residues on target proteins via isopeptide bonds .

Human SUMO1 is the most unique among the four SUMO proteins, sharing only 44%, 47%, and 41% amino acid sequence identity with SUMO2, SUMO3, and SUMO4, respectively. Interestingly, human SUMO1 shares 100% amino acid sequence identity with the mouse ortholog, suggesting strong evolutionary conservation .

What is the difference between His6-SUMO1 and His6-Pro-SUMO1 recombinant proteins?

His6-SUMO1 and His6-Pro-SUMO1 differ primarily in their processing state:

• His6-SUMO1: This is the mature, processed form of SUMO1 with an N-terminal 6-His tag, ready for direct conjugation to substrate proteins. The C-terminal has been processed to expose the diglycine motif necessary for conjugation to target proteins .

• His6-Pro-SUMO1: This is the precursor form that still contains the C-terminal tetrapeptide extension (prosegment) that must be cleaved to expose the diglycine motif before SUMO1 can be conjugated to substrate proteins .

The choice between these forms depends on your experimental design. For direct SUMOylation assays, the mature His6-SUMO1 is preferable, while His6-Pro-SUMO1 may be useful for studying SUMO processing enzymes or as a negative control in conjugation studies.

How should I select between carrier-free and BSA-containing SUMO1 His preparations?

The selection depends on your specific application requirements:

Carrier-free SUMO1 His recombinant protein is typically formulated in a buffer containing HEPES pH 8.0, NaCl, and DTT without any carrier protein like BSA . This format is particularly useful for applications where the presence of BSA could interfere with experimental results . In contrast, BSA-containing preparations are recommended when protein stability and extended shelf-life are primary concerns .

How can I set up a SUMOylation assay using recombinant SUMO1 His protein?

A standard in vitro SUMOylation assay requires the following components:

• Recombinant His6-SUMO1 protein (mature form with exposed diglycine motif)

• SUMO-activating enzyme (E1): SAE1/UBA2 complex

• SUMO-conjugating enzyme (E2): UBE2I/Ubc9

• Target substrate protein

• ATP and Mg²⁺ as cofactors

• Buffer system (typically HEPES pH 7.5-8.0)

• Optional: SUMO ligase (E3) to enhance conjugation efficiency for specific substrates

Methodology:

• Prepare reaction buffer containing 50 mM HEPES pH 8.0, 100 mM NaCl, 5 mM MgCl₂, 1 mM DTT, and 5 mM ATP

• Add 1-2 μg SUMO E1 enzyme (SAE1/UBA2), 1 μg SUMO E2 enzyme (UBE2I/Ubc9)

• Add 1-5 μg target substrate protein

• Add 1-10 μg recombinant His6-SUMO1

• Incubate at 30°C for 1-3 hours

• Analyze results by SDS-PAGE followed by western blotting using anti-SUMO1 and anti-substrate antibodies

The reaction can alternatively be performed using HeLa Fraction II, which contains the necessary enzymatic machinery for SUMOylation . Successful SUMOylation is typically evidenced by the appearance of higher molecular weight bands representing SUMO1-conjugated substrate proteins.

What are the optimal storage conditions for maintaining SUMO1 His protein activity?

To maintain optimal activity of SUMO1 Human His recombinant proteins, follow these storage guidelines:

• For short-term storage (2-4 weeks): Store at 4°C if the entire vial will be used within this period .

• For long-term storage: Store frozen at -20°C in a manual defrost freezer .

• Avoid repeated freeze-thaw cycles as these significantly reduce protein activity .

• For enhanced stability during long-term storage, consider adding a carrier protein (0.1% HSA or BSA) if using a carrier-free preparation .

• Upon receipt of shipped product (typically on dry ice), immediately store at the recommended temperature .

When handling the protein, minimize exposure to room temperature and work quickly on ice to prevent degradation. For aliquoting, prepare single-use volumes to avoid repeated freeze-thaw cycles of the stock solution.

How do buffer components affect SUMO1 His protein stability and activity?

Buffer components significantly impact SUMO1 His protein stability and enzymatic activity in SUMOylation reactions:

SUMO1 His recombinant proteins are typically formulated in buffers containing HEPES pH 8.0, NaCl, and DTT . The presence of DTT is particularly important as a reducing agent to maintain the activity of the SUMOylation machinery . For reaction buffers, the addition of ATP and Mg²⁺ is essential for the SUMO E1 enzyme to activate SUMO1 via adenylation .

How can I distinguish between specific and non-specific SUMO1 conjugation in my experiments?

Distinguishing between specific and non-specific SUMO1 conjugation requires careful experimental design and appropriate controls:

• Consensus Site Mutation Analysis:

  • Mutate the predicted SUMO1 consensus sites (Ψ-K-X-D/E) in your target protein

  • Compare SUMOylation patterns between wild-type and mutant proteins

  • Specific conjugation will be significantly reduced or eliminated in the mutant

• E2 Enzyme (UBE2I/Ubc9) Dependence:

  • Perform parallel reactions with and without the E2 enzyme

  • Specific SUMOylation is strictly dependent on E2 enzyme presence

  • Any modification occurring without E2 is likely non-specific

• SUMO Protease Treatment:

  • Treat SUMOylated samples with SUMO-specific proteases (SENPs)

  • Specific SUMO1 conjugates will be cleaved, reversing the modification

  • Persistent high-molecular-weight bands after SENP treatment indicate non-specific interactions

• Denaturing vs. Native Conditions:

  • Analyze samples under denaturing conditions (SDS-PAGE)

  • Specific SUMO1 conjugation involves covalent isopeptide bonds that resist denaturation

  • Non-covalent interactions will be disrupted under denaturing conditions

Although SUMOylation typically occurs at the consensus sequence Ψ-K-X-D/E, it has also been observed in cases where this consensus site is not conserved . Therefore, the absence of a consensus site does not definitively rule out specific SUMOylation.

What factors affect the efficiency of in vitro SUMOylation reactions using SUMO1 His?

Several factors can influence the efficiency of in vitro SUMOylation reactions:

To troubleshoot low conjugation efficiency:

• Verify the activity of all enzymatic components using positive control substrates

• Ensure the mature form of SUMO1 with exposed diglycine motif is being used

• Consider including an appropriate E3 ligase if known for your substrate

• Check for the presence of inhibitors (high salt, detergents, or chelating agents)

• Extend reaction time or increase enzyme concentrations

For specific substrates, addition of a suitable E3 ligase can dramatically enhance conjugation efficiency, though SUMOylation can occur without E3 ligases for some substrates where SUMO1 is transferred directly from UBE2I/Ubc9 .

How can SUMO1 His be used to study disease mechanisms, particularly in neurodegenerative disorders?

SUMO1 His recombinant proteins are valuable tools for investigating the role of SUMOylation in disease mechanisms, especially neurodegenerative disorders:

• Alzheimer's Disease Studies:
  SUMO1 has been shown to influence the generation of Amyloid-beta peptide by promoting the accumulation of BACE-1 . Researchers can use SUMO1 His in experimental models to:

  • Identify which BACE-1 lysine residues are specifically SUMOylated

  • Determine how SUMOylation affects BACE-1 enzyme activity, stability, and subcellular localization

  • Test whether inhibiting SUMO1 conjugation to BACE-1 reduces Amyloid-beta production

  • Investigate the potential therapeutic value of targeting the SUMOylation pathway

• Methodological Approaches:

  • In vitro SUMOylation assays of purified neuronal proteins using recombinant SUMO1 His

  • Mass spectrometry-based identification of SUMOylation sites on disease-relevant proteins

  • Cellular models expressing His-tagged SUMO1 to facilitate purification of SUMOylated proteins

  • Comparison of SUMOylation patterns between healthy and disease-state brain tissue

• Protein Aggregation Studies:

  • Investigate how SUMOylation affects the aggregation propensity of proteins involved in neurodegeneration

  • Examine cross-talk between SUMOylation and other post-translational modifications (phosphorylation, ubiquitination)

• Experimental Design Example - BACE-1 SUMOylation Analysis:

  a) Prepare wild-type and SUMO consensus site mutant BACE-1 proteins
  b) Perform in vitro SUMOylation with recombinant SUMO1 His
  c) Analyze by immunoblotting and mass spectrometry to identify modification sites
  d) Test the effect of SUMOylation on BACE-1 enzyme activity in vitro
  e) Validate findings in cellular models using overexpression or knockdown approaches

In Alzheimer's disease research, carrier-free SUMO1 His preparations are particularly valuable for mass spectrometry applications aiming to identify precise SUMOylation sites on disease-relevant proteins.

What approaches can be used to study the role of SUMO1 in tumor suppression mechanisms?

SUMO1 Human His recombinant proteins can be instrumental in dissecting the role of SUMOylation in tumor suppression, particularly through modification of key tumor suppressors like PTEN:

• PTEN SUMOylation Studies:
  SUMO1 modification of PTEN has been shown to regulate tumorigenesis by retaining PTEN at the plasma membrane, suppressing PI 3-Kinase/Akt-dependent tumor growth . Researchers can use SUMO1 His to:

  • Map the specific SUMOylation sites on PTEN

  • Determine how SUMOylation affects PTEN phosphatase activity

  • Investigate the mechanism by which SUMOylation alters PTEN subcellular localization

  • Examine the interplay between SUMOylation and other PTEN modifications

• Comprehensive Experimental Approach:

  Experimental Stage	Methodology	Expected Outcome
  SUMOylation Site Mapping	In vitro SUMOylation with recombinant SUMO1 His followed by mass spectrometry	Identification of specific lysine residues modified by SUMO1
  Functional Impact Assessment	Enzyme activity assays comparing wild-type and SUMOylation-deficient mutants	Quantification of how SUMOylation affects PTEN catalytic activity
  Subcellular Localization	Immunofluorescence and subcellular fractionation of cells expressing wild-type or SUMOylation-deficient PTEN	Determination of how SUMOylation affects PTEN membrane retention
  Signaling Pathway Analysis	Western blotting for phospho-Akt and downstream targets	Evaluation of how PTEN SUMOylation impacts PI3K/Akt signaling
  Tumor Growth Studies	Xenograft models with wild-type vs. SUMOylation-deficient PTEN	Assessment of SUMO1's role in tumor suppression in vivo

• Technical Considerations:

  • Use carrier-free SUMO1 His preparations for mass spectrometry applications

  • Include appropriate controls (SUMOylation-deficient mutants, catalytically inactive E2 enzyme)

  • Consider the potential role of specific E3 ligases in enhancing PTEN SUMOylation

  • Account for potential cross-talk with other post-translational modifications

This methodological framework can be adapted for studying other tumor suppressors potentially regulated by SUMO1 modification, providing insights into novel therapeutic strategies targeting the SUMOylation pathway in cancer.

How can I quantitatively analyze the kinetics of SUMO1 conjugation to different substrates?

Quantitative analysis of SUMO1 conjugation kinetics requires precise methodologies and careful data interpretation:

• Time-Course Analysis Protocol:
  a) Set up standard SUMOylation reactions with SUMO1 His and your substrate
  b) Sample the reaction at multiple time points (0, 5, 15, 30, 60, 120 minutes)
  c) Quench reactions immediately by adding SDS-PAGE loading buffer and boiling
  d) Analyze by SDS-PAGE and western blotting or fluorescence detection
  e) Quantify the ratio of SUMOylated to non-SUMOylated substrate at each time point

• Kinetic Parameter Determination:

  • Plot the percentage of SUMOylated substrate versus time

  • For initial rate measurements, use only early time points where the reaction is linear

  • Perform reactions with varying substrate concentrations to determine Km and Vmax

  • Analyze data using appropriate enzyme kinetics software or Michaelis-Menten equations

• Comparison Framework for Multiple Substrates:

  Parameter	Calculation Method	Interpretation
  Initial Rate (V₀)	Slope of linear portion of progress curve	Higher values indicate faster conjugation
  Time to 50% Conjugation (t₅₀)	Time at which 50% of substrate is SUMOylated	Lower values indicate more efficient conjugation
  Maximum Conjugation Level	Plateau level of SUMOylation at saturation	Higher values indicate greater proportion of substrate can be modified
  Km	Substrate concentration at half-maximal rate	Lower values indicate higher enzyme-substrate affinity
  kcat	Maximum number of substrate molecules modified per enzyme per time	Higher values indicate more efficient catalysis
  kcat/Km	Catalytic efficiency	Higher values indicate better substrates

• Methodological Considerations:

  • Ensure enzyme concentrations are substoichiometric to substrate for proper kinetic analysis

  • Maintain consistent temperature throughout experiments (typically 30°C)

  • Include internal standards for normalization between experiments

  • Consider the potential impact of E3 ligases on reaction kinetics

When comparing different substrates, presentation of data in a comprehensive table format allows for clear visualization of kinetic differences, highlighting which proteins are preferential SUMO1 targets and potentially identifying structural features that enhance SUMOylation efficiency.

What controls are essential when interpreting SUMO1 conjugation experiments?

Proper experimental controls are critical for accurate interpretation of SUMO1 conjugation data:

• Essential Negative Controls:

  Control Type	Implementation	Purpose
  No ATP	Omit ATP from reaction	Confirms ATP-dependence of conjugation (hallmark of enzymatic SUMOylation)
  No E1 Enzyme	Omit SAE1/UBA2 from reaction	Verifies E1-dependence of conjugation process
  No E2 Enzyme	Omit UBE2I/Ubc9 from reaction	Confirms requirement for E2 in the conjugation cascade
  Substrate-free	Reaction without target protein	Detects potential self-conjugation of SUMO1 or modification of enzymes
  SUMOylation-deficient Substrate	K→R mutations at consensus sites	Verifies specificity of conjugation to predicted sites
  Non-conjugatable SUMO1	Mutant lacking C-terminal Gly-Gly motif	Confirms requirement for SUMO1 C-terminus in conjugation

• Essential Positive Controls:

  Control Type	Implementation	Purpose
  Known SUMO1 Substrate	Include well-characterized substrate	Validates functionality of the SUMOylation machinery
  Complete Reaction	All components with optimal conditions	Establishes maximum expected conjugation efficiency
  E3 Ligase Enhancement	Addition of appropriate E3 for substrate	Demonstrates specificity and enhancement of conjugation
  SENP Treatment	Post-reaction treatment with SUMO protease	Confirms reversibility (hallmark of true SUMOylation)

• Antibody Controls for Western Blot Analysis:

  • Primary antibody only (no secondary)

  • Secondary antibody only (no primary)

  • Non-specific IgG instead of specific antibody

  • Pre-adsorption of antibody with recombinant SUMO1

• Addressing Common Misinterpretations:

  Observation	Possible Misinterpretation	Control to Resolve
  Multiple high MW bands	Non-specific binding	SENP treatment to confirm SUMOylation
  Unexpected MW shift	Multiple SUMO1 attachments	Mass spectrometry analysis of modified protein
  No visible modification	Failed reaction	Include positive control substrate
  Modification in absence of E3	Non-specific reaction	Compare reaction rates with/without E3

When displaying research data, include representative images of control experiments alongside test conditions and quantify the degree of SUMOylation relative to controls. This approach ensures that observed modifications represent genuine SUMO1 conjugation events rather than experimental artifacts.

How can I differentiate between the effects of SUMO1, SUMO2, and SUMO3 in my research?

Differentiating between the effects of different SUMO paralogs requires strategic experimental design and careful analysis:

• Comparative SUMOylation Analysis:

  Approach	Methodology	Expected Outcome
  Paralog-Specific Conjugation	Parallel in vitro reactions with His-tagged SUMO1, SUMO2, and SUMO3	Identifies which SUMO paralog(s) preferentially modify your target protein
  Site Mapping	Mass spectrometry after paralog-specific modifications	Determines whether different paralogs target the same or different lysine residues
  Kinetic Analysis	Time-course experiments with each paralog	Reveals differences in conjugation efficiency and rates
  E3 Dependence	SUMOylation with/without E3 ligases for each paralog	Identifies paralog-specific requirements for E3 enhancement

• Structural and Functional Impact Assessment:

  • Compare the effects of each SUMO paralog on:

    • Substrate protein activity (enzymatic assays)

    • Protein-protein interactions (co-immunoprecipitation, FRET)

    • Subcellular localization (immunofluorescence)

    • Protein stability (cycloheximide chase)

• Key Distinguishing Features to Consider:

  • SUMO1 shares only 44%, 47%, and 41% amino acid sequence identity with SUMO2, SUMO3, and SUMO4, respectively

  • SUMO2/3 contain internal SUMOylation sites allowing poly-SUMO chain formation, while SUMO1 typically acts as a chain terminator

  • Different SUMO paralogs may interact with distinct sets of SIM (SUMO-Interaction Motif)-containing proteins

• Paralog-Specific Tools and Approaches:

  • Use paralog-specific antibodies for western blotting

  • Employ tagged versions with different epitopes (His-SUMO1, FLAG-SUMO2, Myc-SUMO3)

  • Utilize RNAi or CRISPR approaches to selectively deplete individual SUMO paralogs

  • Use paralog-specific SENP proteases (different SENPs show paralog preferences)

When presenting research data comparing SUMO paralogs, side-by-side visualization of results in comprehensive tables enables clear identification of paralog-specific effects. This approach allows researchers to determine whether a given cellular process is regulated specifically by SUMO1 or more broadly by multiple SUMO family members, providing deeper insights into the complexity of SUMOylation-dependent regulation.