Recombinant Human SUN domain-containing protein 5 (SUN5)

What is the structural composition of human SUN5 protein?

Human SUN domain-containing protein 5 (SUN5) is a 379-amino acid protein characterized by its distinctive M-sun domain. The full sequence includes: MPRSSRSPGDPGALLEDVAHNPRPRRIAQRGRNTSRMAEDTSPNMNDNILLPVRNNDQALGLTQCMLGCVSWFTCFACSLRTQAQQVLFNTCRCKLLCQKLMEKTGILLLCAFGFWMFSIHLPSKMKVWQDDSINGPLQSLRLYQEKVRHHSGEIQDLRGSMNQLIAKLQEMEAMSDEQKMAQKIMKMIHGDYIEKPDFALKSIGASIDFEHTSVTYNHEKAHSYWNWIQLWNYAQPPDVILEPNVTPGNCWAFEGDRGQVTIQLAQKVYLSNLTLQHIPKTISLSGSLDTAPKDFVIYGMEGSPKEEVFLGAFQFQPENIIQMFPLQNQPARAFSAVKVKISSNWGNPGFTCLYRVRVHGSVAPPREQPHQNPYPKRD . The protein contains transmembrane domains and the characteristic SUN domain that defines its functional properties.

What alternative names and identifiers are associated with SUN5?

SUN5 is known by several alternative names in scientific literature:

• SPAG4L (Sperm-associated antigen 4-like protein)

• TSARG4 (Testis and spermatogenesis-related gene 4 protein)

• Sad1 and UNC84 domain-containing protein 5

The protein is identified in UniProt with the accession number Q8TC36 .

What are the optimal storage conditions for recombinant SUN5 protein?

Recombinant SUN5 protein should be stored at -20°C to -80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles. Working aliquots can be stored at 4°C for up to one week. For long-term storage, it is recommended to add glycerol (5-50% final concentration, with 50% being the default) and store at -20°C/-80°C. The protein is typically provided in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 .

What is the recommended protocol for reconstitution of lyophilized SUN5 protein?

For optimal reconstitution of lyophilized SUN5 protein:

• Briefly centrifuge the vial prior to opening to bring the contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol (5-50% final concentration) for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles, which can compromise protein integrity

What expression systems are most effective for producing recombinant human SUN5?

Based on available data, E. coli has been successfully used as an expression system for producing recombinant full-length human SUN5 protein with N-terminal His tags. This bacterial expression system allows for efficient production of the 379-amino acid protein with greater than 90% purity as determined by SDS-PAGE . For experiments requiring post-translational modifications more similar to native human proteins, researchers might consider mammalian expression systems, though specific comparative data between expression systems is not provided in the search results.

What is the role of SUN5 in nuclear membrane dynamics?

SUN5, as a SUN-domain containing protein, functions as a crucial nuclear membrane protein involved in maintaining nuclear morphology. Like other SUN proteins, it forms part of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex that spans the nuclear envelope. While specific mechanistic details for SUN5 are not fully elaborated in the search results, SUN proteins generally connect the nuclear interior to the cytoskeleton, playing vital roles in nuclear positioning, migration, and cellular mechanics .

How does SUN5 contribute to reproductive biology and fertility?

SUN5 shows significant expression during pollen development and germination, suggesting an important role in plant reproductive processes. Studies have demonstrated that the promoter of SUN5 (specifically GbSUN5_At) remains active throughout the entire period of pollen development and germination, with strong expression observed in pollen grains and pollen tubes during germination . In human biology, SUN5's alternative name as Testis and spermatogenesis-related gene 4 protein (TSARG4) further suggests a role in reproductive processes .

What evidence supports SUN5's involvement in meiotic processes?

While the search results don't directly demonstrate SUN5's role in meiosis, they provide context for understanding how SUN domain-containing proteins function in these processes. SUN proteins interact with KASH domain proteins across the nuclear envelope, forming LINC complexes. Specifically, the KASH5 protein interacts with SUN1 in the perinuclear space, where SUN1 binds telomeres at chromosome ends, completing attachments between dynein and chromosomal cargo essential for meiosis progression and fertility . Given SUN5's classification in the same protein family, it likely plays related roles in nuclear-cytoskeletal connections during meiotic processes.

What methodologies are effective for studying SUN5 expression patterns in different tissues?

Based on research methodologies described in the search results, several effective approaches for studying SUN5 expression include:

• Promoter-reporter fusion systems:

  • Fusion of SUN5 promoter regions with reporter genes like GUS or GFP

  • Introduction into model organisms via Agrobacterium-mediated transformation

  • Visualization of expression patterns using fluorescence microscopy or histochemical staining

• Gene silencing approaches:

  • Virus-induced gene silencing (VIGS) using appropriate vector systems

  • Verification of silencing efficiency via qRT-PCR analysis

  • Assessment of phenotypic effects through specialized assays (e.g., pollen viability tests)

• CRISPR-Cas9 targeted editing:

  • Design of sgRNAs targeting the SUN domain coding region

  • Construction of expression vectors for transformation

  • Phenotypic analysis of edited plants/cells

What are the methodological challenges in designing CRISPR-Cas9 experiments targeting SUN5?

When designing CRISPR-Cas9 experiments targeting SUN5, researchers should consider several methodological challenges:

• Domain-specific targeting: The search results mention designing sgRNAs specifically in the coding region of the SUN domain. This approach requires precise targeting of functionally important regions while avoiding off-target effects. Researchers should carefully analyze the conserved regions within the SUN domain to design effective sgRNAs .

• Transformation efficiency: Agrobacterium-mediated transformation protocols need to be optimized for the specific experimental system. The search results mention using Agrobacterium tumefaciens LBA4404 as host cells and cotton cultivar Gh cv. HM-1 as the transformation receptor .

• Phenotypic analysis: Developing appropriate assays to detect phenotypic changes resulting from SUN5 modification is critical. Since SUN5 is involved in reproductive processes, specialized assays focusing on pollen development, germination, and fertility would be most informative .

How can researchers effectively analyze the interaction between SUN5 and other nuclear envelope proteins?

To effectively analyze interactions between SUN5 and other nuclear envelope proteins, researchers can employ several complementary approaches:

• Co-immunoprecipitation (Co-IP): This technique can identify protein-protein interactions by precipitating protein complexes using specific antibodies. For transmembrane proteins like SUN5, careful buffer selection is crucial, as demonstrated in studies with KASH5 where deletion of the transmembrane domain increased solubility for biochemical analysis .

• Size-exclusion chromatography (SEC): This method separates proteins based on size and can verify direct protein-protein interactions. SEC can identify co-elution of protein complexes, distinguishable from individual proteins alone .

• Pull-down assays: Using GST-fusion or His-tagged proteins to pull down potential interaction partners can demonstrate direct binding. This approach has been effectively used with related proteins such as KASH5 and dynein light intermediate chain (LIC) .

• Fluorescent protein tagging and co-localization: Expression of fluorescently tagged proteins can reveal co-localization at the nuclear envelope, providing evidence for potential interactions in vivo .

What technical considerations are important when studying SUN5's role in specialized cellular contexts?

When investigating SUN5's role in specialized cellular contexts such as reproductive tissues, researchers should consider several technical aspects:

• Tissue-specific expression systems: Utilizing promoters that drive expression in specific tissues where SUN5 functions naturally. The search results describe using a 3-kb upstream fragment from GbSUN5_At to study expression during pollen development and germination .

• Temporal expression analysis: SUN5 expression varies temporally during developmental processes. For instance, GFP signal strength changed at different time points after pollen germination (4, 6, and 10 hours), indicating dynamic expression patterns that should be captured through time-course experiments .

• Functional redundancy: Consider potential compensatory mechanisms from other SUN domain proteins when designing knockout or knockdown experiments. The search results mention multiple SUN family members (SUN1, SUN3, SUN5) with distinct domains but potentially overlapping functions .

• Appropriate control systems: When using gene silencing approaches like VIGS, appropriate controls (such as GhCHLI-silenced plants showing yellow bleaching phenotype) should be included to validate experimental outcomes .

What are the potential limitations when interpreting results from SUN5 knockdown experiments?

When interpreting results from SUN5 knockdown experiments, researchers should consider several potential limitations:

• Incomplete silencing: The search results describe VIGS experiments that reduced GhSUN5 transcription levels to about 65% of normal. This incomplete silencing may not be sufficient to observe clear phenotypes if residual expression maintains function .

• Functional redundancy: Other SUN domain proteins might compensate for reduced SUN5 function. The search results suggest that despite reduced GhSUN5 expression, pollen viability remained similar to control plants, possibly due to compensatory mechanisms .

• Tissue-specific effects: SUN5 functions may be more critical in specific tissues or developmental stages. Knockdown experiments should examine multiple tissues and developmental timepoints to capture potential phenotypes.

• Technical variability: The search results mention that only 22 of 50 VIGS plants showed reduced transcription levels, highlighting the variability in gene silencing efficiency that must be considered when interpreting results .

How should researchers approach contradictory findings regarding SUN5 function across different experimental systems?

When faced with contradictory findings regarding SUN5 function across different experimental systems, researchers should:

• Evaluate methodological differences: Different expression systems (E. coli vs. mammalian cells), protein constructs (full-length vs. domain deletions), or buffer conditions can significantly impact results. The search results highlight how deletion of the transmembrane domain in KASH5 greatly increased soluble protein levels, allowing for biochemical analysis that wouldn't be possible with the full-length protein .

• Consider species-specific differences: The search results reveal evolutionary divergence between plant and animal SUN proteins. When comparing findings across species, researchers should account for structural and functional adaptations that may have occurred through evolution .

• Examine context-dependent functions: SUN5 may perform different functions depending on cellular context, developmental stage, or tissue type. The promoter activity of SUN5 during pollen development and germination demonstrates how its function may be specialized to specific biological processes .

• Design integrative experiments: To resolve contradictions, develop experimental approaches that integrate multiple techniques (structural analysis, interaction studies, functional assays) to build a more comprehensive understanding of SUN5 function.