Recombinant UPF0102 protein SAV_2633/SAV2633 (SAV_2633)

What is UPF0102 protein SAV_2633/SAV2633 and what organism does it originate from?

UPF0102 protein SAV_2633/SAV2633 (abbreviated as SAV_2633) is a protein belonging to the UPF0102 family with UniProt accession number Q82JX1. It originates from Streptomyces avermitilis strain ATCC 31267 / DSM 46492 / JCM 5070 / NBRC 14893 / NCIMB 12804 / NRRL 8165 / MA-4680, which is a gram-positive bacterium known for producing avermectins, a class of antiparasitic compounds . This protein is classified as an "uncharacterized protein family" (UPF), indicating that while its sequence is known, its specific biological function remains to be fully elucidated. The protein is composed of 148 amino acids and represents the full-length native sequence without any truncations .

What are the recommended storage conditions for preserving activity of recombinant UPF0102 protein SAV_2633/SAV2633?

Optimal storage conditions for recombinant UPF0102 protein SAV_2633/SAV2633 depend on the formulation and intended use duration. The shelf life is affected by multiple factors including buffer composition, storage temperature, and the intrinsic stability of the protein itself . For liquid formulations, the recommended storage is at -20°C/-80°C with an expected shelf life of approximately 6 months. Lyophilized formulations offer extended stability with a shelf life of up to 12 months when stored at -20°C/-80°C .

It is strongly advised to avoid repeated freeze-thaw cycles as these can significantly compromise protein integrity and activity. For short-term use (up to one week), working aliquots can be stored at 4°C . Researchers should consider adding glycerol (typically 5-50% final concentration, with 50% being standard) when reconstituting the protein to enhance stability during storage and prevent damage from freeze-thaw cycles.

Which expression systems provide optimal yields for UPF0102 protein SAV_2633/SAV2633 production?

Multiple expression systems have been validated for the production of recombinant UPF0102 protein SAV_2633/SAV2633, each with distinct advantages and limitations. Based on comparative studies, the highest yields and shortest production timelines are achieved using prokaryotic systems, particularly E. coli, followed by yeast expression systems . These systems are cost-effective and well-suited for basic structural and biochemical studies where post-translational modifications may not be critical.

For research requiring native-like post-translational modifications, insect cell expression using baculovirus vectors and mammalian cell systems provide better options . While commercially available SAV_2633 is often sourced from mammalian cell expression systems to ensure proper folding , expression efficiency tends to be lower compared to bacterial systems, resulting in reduced yields and increased production costs.

The table below summarizes the comparative performance of different expression systems for recombinant UPF0102 protein SAV_2633/SAV2633 production:

What purification strategy yields the highest purity for UPF0102 protein SAV_2633/SAV2633?

Achieving high purity (>85% by SDS-PAGE) for UPF0102 protein SAV_2633/SAV2633 typically involves a multi-step purification strategy tailored to the expression system and tag configuration . The commercially available recombinant protein is often produced with affinity tags, though the specific tag type is determined during the manufacturing process and may vary between production batches .

A typical purification workflow for tagged SAV_2633 includes:

• Initial capture using affinity chromatography (e.g., IMAC for His-tagged protein)

• Intermediate purification via ion exchange chromatography to remove host cell proteins

• Polishing steps such as size exclusion chromatography to achieve final purity

• Optional tag removal if required for downstream applications

What experimental approaches are recommended for determining the function of UPF0102 protein SAV_2633/SAV2633?

As a member of the UPF (Uncharacterized Protein Family) group, SAV_2633's biological function remains incompletely understood, making functional characterization a priority for researchers. A comprehensive functional analysis strategy should incorporate multiple complementary approaches:

• Bioinformatic Analysis: Sequence-based prediction tools can identify conserved domains and potential homologs with known functions. Structural modeling based on the amino acid sequence can provide insights into potential binding sites and catalytic regions.

• Expression Pattern Analysis: Investigating the conditions under which the native protein is expressed in Streptomyces avermitilis can provide contextual clues about its role. This includes analyzing expression during different growth phases and under various environmental stresses.

• Protein-Protein Interaction Studies: Pull-down assays, yeast two-hybrid screening, or proximity labeling approaches can identify binding partners, potentially revealing the biological pathways in which SAV_2633 participates. The recombinant protein with appropriate tags facilitates these interaction studies .

• Gene Knockout/Complementation: Creating knockout strains in the native organism followed by phenotypic characterization and complementation studies can reveal the consequences of SAV_2633 absence and confirm functional hypotheses.

• Biochemical Activity Assays: Testing for common enzymatic activities (e.g., hydrolase, transferase) using purified recombinant protein against various substrates may uncover catalytic functions. The high purity preparation (>85% by SDS-PAGE) is essential for reliable activity determination .

What structural features have been identified in UPF0102 protein SAV_2633/SAV2633?

The amino acid sequence reveals potential structural elements, including multiple arginine-rich regions that may be involved in DNA/RNA binding or protein-protein interactions . The sequence "SLAARRLTES GMTVLERNWR" (positions 41-60) contains a potential alpha-helical region based on secondary structure prediction algorithms.

For experimental structure determination, the availability of recombinant protein with >85% purity provides a starting point for crystallization trials . When designing structural biology experiments, researchers should consider:

• The full-length protein (148 amino acids) may present crystallization challenges due to potential flexible regions. Construct optimization through limited proteolysis followed by mass spectrometry could identify stable domains suitable for crystallization.

• NMR spectroscopy represents an alternative approach for structural characterization, particularly given the relatively small size of the protein (approximately 16-17 kDa based on sequence).

• Cryo-EM may not be optimal for this protein due to its small size, unless it forms larger complexes with binding partners.

How can site-directed mutagenesis be applied to study UPF0102 protein SAV_2633/SAV2633 structure-function relationships?

Site-directed mutagenesis represents a powerful approach for investigating structure-function relationships in UPF0102 protein SAV_2633/SAV2633. When designing a mutagenesis strategy, researchers should consider the following methodological aspects:

What are the considerations for isotope labeling of UPF0102 protein SAV_2633/SAV2633 for NMR studies?

Isotope labeling of UPF0102 protein SAV_2633/SAV2633 for NMR studies requires careful planning to ensure high incorporation rates while maintaining protein folding and stability. The following methodological considerations are critical:

• Expression System Selection: While E. coli provides the most cost-effective platform for isotope labeling, researchers should verify that the protein expressed in minimal media maintains proper folding . If structural integrity is compromised, consider cell-free protein synthesis systems that offer rapid production with high incorporation efficiency.

• Labeling Strategy:

  • Uniform ^15^N labeling using ^15^NH₄Cl as the sole nitrogen source represents the entry point for initial NMR characterization

  • Dual ^13^C/^15^N labeling enables more comprehensive structural determination but substantially increases costs

  • Selective amino acid labeling can be employed to resolve assignment ambiguities in arginine-rich regions (positions 24-33, 79-88)

  • Deuteration may be necessary for optimal spectral quality given the presence of multiple glycine-rich regions that could lead to spectral crowding

• Media Optimization:

  • For minimal media growth, supplement with micronutrients essential for Streptomyces proteins

  • Consider using algal hydrolysates as cost-effective partially labeled nitrogen and carbon sources

  • Implement glucose feeding strategies to enhance biomass prior to induction

• Purification Considerations:

  • Maintain reducing conditions throughout purification to prevent disulfide formation

  • Include protease inhibitors to prevent degradation of isotopically labeled protein

  • Optimize buffer conditions for NMR (typically low-salt, deuterated buffers with appropriate pH for optimal spectral quality)

• Sample Preparation:

  • Target 0.3-0.5 mM protein concentration in NMR buffer

  • Verify monodispersity through dynamic light scattering prior to NMR data collection

  • Test sample stability at measurement temperature (typically 25°C) over the expected data collection period

What strategies can address low solubility issues when expressing UPF0102 protein SAV_2633/SAV2633?

Low solubility during expression represents a common challenge for recombinant proteins, including UPF0102 protein SAV_2633/SAV2633. Researchers encountering solubility issues can implement these methodological solutions:

• Expression Condition Optimization:

  • Reduce induction temperature to 16-20°C to slow protein production and promote proper folding

  • Decrease inducer concentration to reduce expression rate

  • Explore auto-induction media which provides gradual protein expression

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) to assist folding

• Construct Engineering:

  • Utilize solubility-enhancing fusion partners such as SUMO, MBP, or TrxA

  • Consider expressing individual domains if bioinformatic analysis identifies distinct structural regions

  • Introduce surface mutations that increase hydrophilicity without affecting core structure

• Buffer Optimization During Extraction and Purification:

  • Increase ionic strength (300-500 mM NaCl) to shield electrostatic interactions

  • Add solubility enhancers such as glycerol (10-20%), non-detergent sulfobetaines, or arginine (50-100 mM)

  • Optimize pH based on theoretical isoelectric point of SAV_2633

  • Include reducing agents (DTT or TCEP) to prevent non-native disulfide formation

• Alternative Expression Systems:

  • If E. coli expression consistently yields insoluble protein despite optimization, consider yeast or insect cell expression systems which may provide enhanced folding environments

  • For mammalian cell expression, optimize codon usage and signal peptides for improved soluble expression

• Refolding Approaches:

  • If inclusion body formation persists, develop a refolding protocol using gradual dialysis or on-column refolding

  • Screen multiple refolding conditions varying buffers, pH, additives, and protein concentration

How can researchers optimize reconstitution of lyophilized UPF0102 protein SAV_2633/SAV2633 to maintain activity?

Proper reconstitution of lyophilized UPF0102 protein SAV_2633/SAV2633 is critical for maintaining structural integrity and biological activity. The following methodological approach is recommended:

• Pre-Reconstitution Preparation:

  • Bring the vial to room temperature before opening to prevent condensation

  • Centrifuge briefly to collect all lyophilized material at the bottom of the vial

  • Prepare all buffers using high-quality, deionized sterile water that is freshly degassed

• Reconstitution Procedure:

  • Add deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL for optimal solubility

  • Add solution gently to the sides of the vial rather than directly onto the protein cake

  • Allow the protein to solubilize without aggressive mixing (gentle rotation instead of vortexing)

  • For complete solubilization, allow 10-15 minutes at room temperature with occasional gentle swirling

• Stabilization Strategy:

  • Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) to prevent freeze-thaw damage during storage

  • Consider supplementing with reducing agents if the protein contains cysteine residues

  • For long-term stability, prepare small single-use aliquots to avoid repeated freeze-thaw cycles

• Quality Control Assessment:

  • Verify protein concentration using UV absorbance at 280 nm with the theoretical extinction coefficient

  • Confirm protein integrity through SDS-PAGE analysis

  • If applicable, perform activity assays to ensure functional preservation

  • Check for aggregation using dynamic light scattering or size exclusion chromatography

• Storage Recommendations:

  • Store reconstituted protein in small aliquots at -20°C/-80°C for up to 6 months

  • For working aliquots needed within a week, storage at 4°C is acceptable

  • Document all freeze-thaw cycles and monitor for potential activity loss