Recombinant Zea mays Unknown protein from spot 206 of 2D-PAGE of etiolated coleoptile

What is the Unknown protein from spot 206 of 2D-PAGE of etiolated coleoptile?

This is a protein originally identified in maize (Zea mays) etiolated coleoptile tissue through two-dimensional gel electrophoresis (2D-PAGE). The protein appears as spot 206 on 2D gels and has been recombinantly produced for research purposes. This protein is part of the complex protein network involved in coleoptile development in dark-grown (etiolated) maize seedlings. The characterization of this protein contributes to our understanding of cellular and physiological activities during seedling growth in maize. The protein has been assigned the Uniprot accession number P80615 and is now commercially available as a recombinant product for research applications .

What is the amino acid sequence and basic properties of this protein?

The amino acid sequence of this protein is: ITEEVAAAAA VGAGGYVXXL GEAGHHHLFN HE. It is a full-length protein comprising 32 amino acids (expression region 1-32). In its recombinant form, it has a purity of >85% as determined by SDS-PAGE analysis. The protein is expressed in yeast systems to maintain proper folding and post-translational modifications similar to the native form . While the exact molecular weight is not specified in the provided information, based on its amino acid composition, it would have a relatively low molecular weight characteristic of small proteins or peptides identified through 2D-PAGE techniques.

How does this protein relate to other proteins in maize development?

This protein was identified in the context of studies examining protein changes associated with mesocotyl growth in maize. The mesocotyl connects the coleoptilar node and the basal part of the seminal root of maize seedlings and is crucial for pushing the shoot out of the soil during germination. Studies like the one conducted with the maize hybrid Zhengdan 958 have identified 88 differentially abundant proteins (DAPs) during different growth stages of the mesocotyl . While the specific function of the protein from spot 206 is not explicitly stated in the available information, it is likely part of the protein network involved in cellular and physiological activities during seedling growth, potentially related to processes such as cell wall synthesis, carbohydrate biogenesis, or cytoskeleton organization that are critical during this developmental stage.

What are the recommended storage and handling conditions for this recombinant protein?

For optimal stability and activity, the recombinant Zea mays unknown protein from spot 206 should be stored at -20°C, or for extended storage, at -20°C to -80°C. Repeated freezing and thawing is not recommended as it can lead to protein degradation and loss of activity. For working solutions, it is advisable to prepare aliquots and store them at 4°C for up to one week to minimize freeze-thaw cycles .

The protein should be briefly centrifuged prior to opening to bring the contents to the bottom of the vial. For reconstitution, it is recommended to use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL. The manufacturer recommends adding 5-50% glycerol (final concentration) for long-term storage, with 50% being the default glycerol concentration . The shelf life in liquid form is approximately 6 months at -20°C/-80°C, while the lyophilized form maintains stability for approximately 12 months at -20°C/-80°C.

What techniques were used to isolate and identify this protein from maize?

The isolation and identification of this protein involved a multi-step process characteristic of proteomic studies. In similar studies with maize, researchers extracted soluble proteins from etiolated mesocotyls grown in darkness for specific time periods corresponding to different growth stages (e.g., 48 h, 84 h, and 132 h) . The extracted protein mixture was then separated using two-dimensional gel electrophoresis (2D-PAGE), which separates proteins based on their isoelectric point in the first dimension and molecular weight in the second dimension.

After gel separation, protein spots of interest (such as spot 206) were excised and subjected to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF) analysis for identification . This technique involves enzymatic digestion of the protein (typically with trypsin), generating peptide fragments that are then analyzed by mass spectrometry. The resulting peptide mass fingerprint and MS/MS spectra are compared against protein databases to identify the protein. Following identification, the protein was recombinantly expressed in yeast to produce larger quantities for research purposes .

What methods can be used to validate protein purity and activity?

Validating the purity and activity of the recombinant protein is crucial for reliable experimental results. For purity assessment, SDS-PAGE analysis is commonly employed, with the recombinant protein showing >85% purity according to the manufacturer . Additional validation methods could include:

• Western blotting using specific antibodies against the protein or tag

• Mass spectrometry for accurate mass determination and sequence verification

• Size-exclusion chromatography to assess aggregation state and homogeneity

• Dynamic light scattering to evaluate size distribution and potential aggregation

For activity validation, appropriate functional assays should be developed based on the protein's presumed function. While the specific function of the protein from spot 206 is not explicitly stated, activity assays might include:

• Enzyme activity assays if the protein has enzymatic functions

• Protein-protein interaction studies using pull-down assays or co-immunoprecipitation

• Cell-based assays to assess biological activity in relevant systems

• Circular dichroism spectroscopy to confirm proper folding

How can this protein be used in proteomic studies of plant development?

This recombinant protein can serve as a valuable standard or reference in proteomic studies investigating maize development, particularly those focused on coleoptile and mesocotyl growth. Specific applications include:

• Comparative proteomics: The protein can be used as a standard to normalize gel-to-gel variations in 2D-PAGE experiments examining protein changes during different developmental stages or under various environmental conditions.

• Antibody development: The recombinant protein can be used to generate specific antibodies for immunodetection of the native protein in plant tissues, enabling more targeted studies of its expression and localization.

• Protein interaction studies: The recombinant protein can be employed in pull-down assays, yeast two-hybrid screens, or co-immunoprecipitation experiments to identify potential interaction partners, providing insights into the protein's function in cellular processes.

• Structural studies: The purified protein could be used for crystallography or NMR studies to determine its three-dimensional structure, which might provide clues about its function.

These applications can contribute to a better understanding of the molecular mechanisms underlying maize seedling development, particularly the etiolation process that occurs during growth in darkness .

What experimental design would be appropriate for studying this protein's function?

A comprehensive experimental design to elucidate the function of this unknown protein would involve multiple complementary approaches:

• Expression analysis: Quantitative RT-PCR and Western blot analysis to monitor the protein's expression levels across different tissues, developmental stages, and in response to various stimuli (light, hormones, stress conditions).

• Subcellular localization: Fusion with fluorescent proteins (e.g., GFP) followed by confocal microscopy to determine the protein's localization within the cell, providing clues about its potential function.

• Genetic approaches:

  • CRISPR/Cas9-mediated gene knockout or RNAi-mediated knockdown to observe loss-of-function phenotypes

  • Overexpression studies to assess gain-of-function effects

  • Complementation studies in knockout lines to confirm phenotype rescue

• Biochemical characterization:

  • In vitro activity assays based on predicted function

  • Protein-protein interaction studies (Y2H, BiFC, co-IP)

  • Post-translational modification analysis

• Comparative analysis: Cross-species comparison with homologs in other plants to infer potential functions based on evolutionary conservation.

For control experiments, it would be important to include appropriate wild-type controls, empty vector controls (for overexpression studies), and non-targeting controls (for knockdown/knockout studies) .

How might this protein relate to germin-like proteins in maize?

While the specific classification of the unknown protein from spot 206 is not explicitly stated in the provided information, it's worth considering its potential relationship with germin-like proteins (GLPs), which form an important family in maize and other plants. Zea mays contains 26 germin-like protein genes (ZmGLPs) primarily located on chromosomes 2, 4, and 10 .

The protein from spot 206 might share characteristics with GLPs if it contains features such as:

• The presence of a cupin domain, which is characteristic of GLPs

• Conservation of the GER box with the consensus sequence GxxxxHxHPxAxEh

• The presence of the KGD motif involved in protein-protein interactions

• Similar subcellular localization patterns (cytoplasmic or extracellular)

If sequence analysis reveals homology with GLPs, this would provide valuable insights into potential functions, as GLPs play roles in plant development and stress responses. For example, ZmGLPs show high expression during germination and at maturity levels, particularly in the root, root tips, crown root, elongation and maturation zones, radicle, and cortex .

A comparative analysis examining the protein sequence, structural features, and expression patterns would be necessary to establish any relationship with the GLP family. This could be accomplished through sequence alignment tools, structural prediction software, and gene expression analysis.

What challenges might researchers face when working with proteins identified through 2D-PAGE?

Researchers working with proteins identified through 2D-PAGE, such as the unknown protein from spot 206, face several technical challenges:

• Sample complexity and reproducibility:

  • Biological variation between samples can affect spot patterns

  • Technical variations in 2D-PAGE methodology can lead to inconsistent spot detection

  • Overlapping protein spots can complicate accurate isolation of a single protein

• Protein identification limitations:

  • Low-abundance proteins may be difficult to detect and identify

  • Post-translational modifications can alter migration patterns and complicate identification

  • Hydrophobic or membrane proteins are often underrepresented in standard 2D-PAGE

• Recombinant protein production challenges:

  • Ensuring proper folding of the recombinant protein to match the native structure

  • Achieving appropriate post-translational modifications in heterologous expression systems

  • Maintaining stability and activity during purification processes

• Functional characterization:

  • Connecting a protein spot to its biological function can be difficult

  • Unknown proteins without clear homology to characterized proteins present special challenges

  • Validating in vitro findings in the context of the whole organism

To address these challenges, researchers should employ complementary techniques such as mass spectrometry-based proteomics, RNA-seq for transcriptome correlation, and various in vivo and in vitro functional assays .

How can post-translational modifications of this protein be analyzed?

Analysis of post-translational modifications (PTMs) is crucial for understanding protein function, as PTMs can significantly affect protein activity, localization, and interactions. For the unknown protein from spot 206, several complementary approaches can be employed:

• Mass spectrometry-based methods:

  • High-resolution MS/MS analysis of the purified protein to identify modified residues

  • Enrichment techniques for specific modifications (e.g., phosphopeptide enrichment using TiO₂ or IMAC for phosphorylation analysis)

  • Quantitative proteomics approaches (SILAC, iTRAQ, TMT) to compare modification levels under different conditions

• Site-specific techniques:

  • Site-directed mutagenesis of potential modification sites followed by functional assays

  • Phospho-specific or other modification-specific antibodies for immunodetection

  • Protein microarrays to profile PTMs in different contexts

• In silico prediction tools:

  • Computational algorithms to predict potential modification sites

  • Structural modeling to understand the impact of modifications on protein conformation

Based on information from search result , proteins in the Zea mays GLP family commonly undergo N-glycosylation and phosphorylation. Table 1 in the search results indicates that most ZmGLPs possess multiple N-glycosylation sites (ranging from 0-14) and phosphorylation sites (ranging from 0-2) . If the protein from spot 206 shares characteristics with these proteins, it may undergo similar modifications that could be critical for its function.

What are common issues when working with recombinant proteins and how can they be addressed?

Working with recombinant proteins like the Zea mays unknown protein from spot 206 can present several challenges. Here are common issues and strategies to address them:

• Protein instability and degradation:

  • Add protease inhibitors during extraction and purification

  • Optimize buffer conditions (pH, salt concentration, reducing agents)

  • Store protein with glycerol (5-50%) as recommended by the manufacturer

  • Prepare small aliquots to avoid repeated freeze-thaw cycles

• Low solubility or aggregation:

  • Adjust buffer composition (consider detergents for hydrophobic proteins)

  • Optimize protein concentration to prevent aggregation

  • Consider fusion tags that enhance solubility (e.g., MBP, SUMO)

  • Use gentle reconstitution methods, avoiding vigorous shaking

• Loss of activity:

  • Ensure proper folding by optimizing expression conditions

  • Include stabilizing agents such as glycerol or specific cofactors

  • Verify that the reconstitution process maintains the protein's native structure

  • Perform activity assays immediately after reconstitution when possible

• Inconsistent experimental results:

  • Standardize protein quantification methods

  • Include internal controls in all experiments

  • Ensure consistent storage and handling between experiments

  • Document lot-to-lot variations and their potential impact on results

For specific troubleshooting of the recombinant Zea mays unknown protein from spot 206, researchers should refer to the manufacturer's technical notes and consider reaching out to technical support for product-specific guidance .

What experimental controls should be included when studying this protein?

When designing experiments to study the unknown protein from spot 206, including appropriate controls is essential for generating reliable and interpretable data:

• Positive and negative controls for protein detection:

  • Purified recombinant protein as a positive control for antibody specificity

  • Samples known to lack the protein (e.g., knockout mutants) as negative controls

  • Isotype controls for immunodetection experiments

• Controls for functional assays:

  • Heat-inactivated protein to control for non-specific effects

  • Related proteins with known functions as benchmarks

  • Vehicle-only controls that undergo identical processing steps

• Biological controls:

  • Wild-type plants grown under identical conditions

  • Tissues where the protein is not expressed (as determined by previous studies)

  • Time course samples to account for developmental variations

• Technical controls:

  • Loading controls for Western blots (housekeeping proteins)

  • Normalization controls for quantitative RT-PCR

  • Mock treatments to control for handling effects

In studies examining protein changes during maize mesocotyl growth, researchers used samples from different time points (48 h, 84 h, and 132 h) corresponding to different growth stages . Similar temporal controls would be valuable when studying the unknown protein from spot 206 to understand its expression dynamics and potential roles in development.

How can researchers evaluate whether this protein is suitable for their specific research questions?

Evaluating the suitability of the recombinant Zea mays unknown protein from spot 206 for specific research questions requires careful consideration of several factors:

• Relevance to the biological process of interest:

  • Review literature on protein expression patterns in relation to developmental stages, tissues, or stress responses

  • Consider the protein's potential functions based on sequence homology, structural features, or previously characterized related proteins

  • Evaluate preliminary data on the protein's involvement in the specific process being studied

• Technical compatibility with planned experiments:

  • Verify that the protein's properties (stability, solubility, etc.) are compatible with planned experimental conditions

  • Ensure that available detection methods (antibodies, activity assays) are sufficiently sensitive and specific

  • Confirm that the recombinant form adequately represents the native protein in terms of structure and modifications

• Pilot experiments to assess feasibility:

  • Conduct small-scale preliminary experiments to validate methods

  • Test protein behavior under experimental conditions

  • Evaluate background signals and potential interference factors

• Available alternatives and complementary approaches:

  • Consider whether alternative proteins, genetic approaches, or systems might better address the research question

  • Evaluate the need for complementary approaches to provide a comprehensive understanding

Researchers should also consider the known context of this protein, which was identified in studies of maize mesocotyl growth. If the research question relates to processes such as cellular growth, cell wall synthesis, or responses to dark conditions (etiolation), this protein may be particularly relevant based on the proteomic studies in which it was identified .

What bioinformatic tools are most useful for analyzing this protein?

Several bioinformatic tools can be valuable for analyzing the unknown protein from spot 206 and placing it in a broader biological context:

• Sequence analysis tools:

  • BLAST (Basic Local Alignment Search Tool) for identifying homologous proteins

  • Multiple sequence alignment tools (Clustal Omega, MUSCLE, T-Coffee) to compare with related proteins

  • PFAM, InterPro, or SMART for domain identification and functional prediction

• Structural prediction tools:

  • AlphaFold or RoseTTAFold for 3D structure prediction

  • PSIPRED for secondary structure prediction

  • DisProt or IUPred for disorder prediction

• Functional annotation tools:

  • Gene Ontology (GO) analysis for functional categorization

  • KEGG pathway analysis to identify potential metabolic pathways

  • STRING database for protein-protein interaction network analysis

• Subcellular localization prediction:

  • CELLO and PSORT, which have been used for predicting the localization of Zea mays proteins

  • TargetP for targeting peptide prediction

  • Plant-PLoc for plant-specific localization prediction

• Post-translational modification prediction:

  • NetPhos or PhosphoSitePlus for phosphorylation site prediction

  • NetNGlyc for N-glycosylation site prediction

  • NetOGlyc for O-glycosylation site prediction

These tools can be particularly useful for characterizing this unknown protein given the limited direct information available. For instance, from Table 1 in search result , we can see that similar analyses of ZmGLP proteins included prediction of molecular weight, isoelectric point, positive and negative residues, extinction coefficient, instability index, aliphatic index, GRAVY (Grand Average of Hydropathicity), domain identification, and subcellular localization .

How can researchers integrate data from different experimental approaches?

Integrating data from multiple experimental approaches is essential for developing a comprehensive understanding of the unknown protein from spot 206. Here's a methodological framework for effective data integration:

• Data normalization and standardization:

  • Ensure comparable scales and units across different data types

  • Apply appropriate statistical transformations where necessary

  • Establish clear criteria for data quality and inclusion

• Correlation analysis:

  • Examine relationships between expression levels, protein abundance, and functional outcomes

  • Use statistical approaches (Pearson/Spearman correlation, regression analysis) to quantify relationships

  • Identify patterns across multiple experimental conditions or time points

• Multi-omics integration:

  • Combine proteomic data with transcriptomic, metabolomic, or phenotypic data

  • Use specialized tools for multi-omics data integration (e.g., mixOmics, DIABLO)

  • Develop integrated network models incorporating multiple data types

• Pathway and network analysis:

  • Map experimental findings to known biological pathways

  • Construct protein-protein interaction networks

  • Identify regulatory relationships through causal network analysis

• Visualization strategies:

  • Use heat maps, network diagrams, or other visualizations to represent complex relationships

  • Develop interactive visualizations for exploring multi-dimensional data

  • Present temporal data using time series visualizations

In the context of maize mesocotyl studies, researchers integrated proteomic data with physiological measurements (IAA levels, cellulose content, and POD activity) and verified protein accumulation using complementary techniques like immunoblotting and RT-qPCR . This multi-level approach provides stronger evidence for the biological significance of observed protein changes.

What are promising research directions for further characterizing this protein?

Several promising research directions could advance our understanding of the unknown protein from spot 206:

• Comprehensive functional characterization:

  • CRISPR/Cas9-mediated gene editing to generate knockout mutants

  • Phenotypic analysis of mutants under various conditions, particularly focusing on coleoptile and mesocotyl development

  • Complementation studies with the recombinant protein to confirm functional rescue

• Spatial and temporal expression profiling:

  • High-resolution analysis of protein expression during seedling development

  • Tissue-specific and subcellular localization studies

  • Response to environmental stimuli (light, hormones, stress)

• Interactome mapping:

  • Identification of protein interaction partners using techniques such as BioID, proximity labeling, or tandem affinity purification

  • Investigation of protein complexes formed under different conditions

  • Validation of interactions through in vivo imaging techniques

• Structural biology approaches:

  • X-ray crystallography or cryo-EM to determine three-dimensional structure

  • Structure-function relationship studies through targeted mutagenesis

  • Molecular dynamics simulations to understand protein behavior

• Comparative studies across varieties and species:

  • Analysis of orthologs in different maize varieties and related species

  • Evolutionary conservation studies to identify critical functional domains

  • Investigation of how protein function may have adapted to different environmental niches

These approaches would build upon existing knowledge about protein changes during maize mesocotyl growth and could potentially connect this unknown protein to characterized protein families such as the germin-like proteins in Zea mays .

How might this protein contribute to our understanding of plant development?

The study of the unknown protein from spot 206 has potential to enhance our understanding of plant development in several key areas:

• Etiolation and light-responsive growth:

  • Being identified in etiolated coleoptile tissue, this protein may play a role in the development of seedlings grown in darkness

  • It could be involved in the complex signaling networks that regulate the transition from skotomorphogenesis to photomorphogenesis

  • Understanding its function might provide insights into how plants respond to light availability during early development

• Cell wall dynamics and cell expansion:

  • Proteins involved in mesocotyl growth often participate in cell wall synthesis and modification

  • At 132 h of growth, the most striking differentially abundant proteins in maize mesocotyls were those involved in cell wall synthesis and modification

  • This protein might be part of the machinery that regulates cell expansion during rapid growth phases

• Hormone signaling pathways:

  • The mesocotyl growth exhibited significant changes in the levels of indole-3-acetic acid (IAA)

  • If this protein interacts with or is regulated by hormones, it could provide new insights into hormone-mediated development

  • It might function in integrating multiple hormonal signals to coordinate growth responses

• Stress responses during early development:

  • Many proteins involved in early seedling development also function in stress responses

  • If this protein shares characteristics with germin-like proteins, it might have roles in biotic or abiotic stress responses, as ZmGLPs show expression against various stresses

  • Understanding dual functions in development and stress response could illuminate how plants balance growth and defense