Recombinant Zea mays Unknown protein from spot 168 of 2D-PAGE of etiolated coleoptile

What is the Unknown protein from spot 168 of 2D-PAGE of etiolated coleoptile?

The Unknown protein from spot 168 of 2D-PAGE of etiolated coleoptile refers to a specific protein isolated from maize (Zea mays) seedlings grown in darkness (etiolated). This protein was initially identified through two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), appearing as spot 168 on the gel . While its precise function remains uncharacterized, it was isolated from the coleoptile, which is the protective sheath covering the emerging shoot in grass seedlings. Based on patterns observed in other Zea mays proteins, it may potentially belong to the germin-like protein (GLP) family, which plays important roles in plant development and stress responses .

What antibodies are available for studying this protein?

Polyclonal antibodies raised in rabbits against the recombinant form of this protein are commercially available. These antibodies have been affinity-purified and are suitable for various applications including ELISA and Western Blot. The antibodies are typically stored in a buffer containing 50% glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative . When designing experiments with these antibodies, researchers should be aware that they are specifically reactive to Zea mays and should be stored at -20°C or -80°C to maintain efficacy, avoiding repeated freeze-thaw cycles.

How should I properly store and handle antibodies against this protein?

For optimal preservation of antibody function, store the antibody at -20°C or -80°C immediately upon receipt. Avoid repeated freeze-thaw cycles as these can significantly diminish antibody activity and specificity . When working with the antibody, aliquot the stock solution into smaller volumes based on your experimental needs to minimize freeze-thaw events. Before use, thaw the antibody aliquot slowly at 4°C or on ice rather than at room temperature. During experiments, keep the antibody on ice and return unused portions to -20°C promptly. Document the number of freeze-thaw cycles and discard antibodies that have undergone multiple cycles to ensure experimental reproducibility.

What are the typical applications for studying this unknown protein?

The primary applications for studying this unknown protein include Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) techniques, which are effective for detection and quantification . For Western blot analysis, separating proteins via SDS-PAGE followed by transfer to a membrane allows for specific detection using the anti-spot 168 antibody. ELISA provides quantitative analysis of the protein in solution. Additional applications may include immunohistochemistry to localize the protein within plant tissues, immunoprecipitation to study protein-protein interactions, and mass spectrometry for detailed structural characterization. These methods collectively enable comprehensive analysis of the protein's expression patterns, cellular localization, and potential functional partners.

What is known about the protein's subcellular localization?

While the specific subcellular localization of the unknown protein from spot 168 has not been definitively characterized in the provided search results, we can make educated inferences based on patterns observed in Zea mays proteins with similar properties. If this protein shares characteristics with germin-like proteins (GLPs) in Zea mays, it may be localized either in the cytoplasm or in extracellular regions . Computational prediction tools such as CELLO and PSORT are commonly used to predict protein localization based on sequence features. Most ZmGLPs are either expressed in the cytoplasm or extracellular regions, with a few showing chloroplast-specific (ZmGLP4-11 and ZmGLP10-1) or periplasm-specific (ZmGLP4-10 and ZmGLP4-16) expression patterns . Experimental verification using subcellular fractionation techniques combined with Western blot analysis or immunofluorescence microscopy would be necessary to conclusively determine this protein's localization.

How can I characterize the enzymatic activity of this unknown protein?

Characterizing the enzymatic activity of the unknown protein requires a multifaceted approach. Begin with bioinformatic analysis using tools like InterPro, Pfam, and BLAST to identify conserved domains that might suggest enzymatic function. If the protein shares similarities with germin-like proteins, consider testing for oxalate oxidase, superoxide dismutase, or ADP glucose pyrophosphatase/phosphodiesterase activities .

Design targeted enzyme assays based on predicted activities. For oxalate oxidase activity, measure hydrogen peroxide production in the presence of oxalic acid. For superoxide dismutase activity, use the nitro blue tetrazolium reduction assay. If no clear enzymatic function is predicted, conduct systematic screening with various substrates, monitoring changes in substrate concentration or product formation using spectrophotometric, fluorometric, or chromatographic methods.

Test activity in the presence of different metal ions (Mn²⁺, Cu²⁺, Fe²⁺, Zn²⁺) and other potential cofactors, as many plant proteins require specific cofactors for activity. Determine optimal conditions by assaying activity across different pH values (4-9) and temperatures (10-50°C). Use specific enzyme inhibitors to help classify the enzyme family. For detailed mechanistic insights, determine the protein's three-dimensional structure through X-ray crystallography or cryo-EM.

What bioinformatic approaches can help predict the function of this uncharacterized protein?

Multiple bioinformatic strategies can help predict the function of this uncharacterized protein. Begin with sequence homology analysis using BLAST, FASTA, or HHpred to identify homologous proteins with known functions. Extend the search beyond Zea mays to other plant species, as functional homologs may exist in distantly related organisms.

Identify conserved domains and motifs using tools like InterPro, SMART, and Pfam. If the protein contains a cupin domain like other Zea mays GLPs, it may suggest functional similarities . Predict the protein's three-dimensional structure using AlphaFold2, I-TASSER, or Phyre2, as structural similarities often indicate functional similarities even when sequence homology is low.

Examine genes adjacent to the one encoding your protein of interest, as functionally related genes are often clustered in plant genomes. Identify genes that show similar expression patterns across various conditions through co-expression network analysis, as co-expressed genes often participate in common biological processes. Construct phylogenetic trees to understand the evolutionary relationships with other proteins, potentially revealing functional clusters.

Predict potential interaction partners using tools like STRING, suggesting participation in specific cellular pathways. Assign probable GO terms based on sequence features using algorithms like PANNZER2 and DeepGOPlus. Predict subcellular localization using tools like CELLO, PSORT, and TargetP to understand where the protein functions within the cell . Combining multiple prediction methods using meta-servers like COFACTOR or COACH improves prediction accuracy more than any single method alone.

How does this protein compare to other germin-like proteins (GLPs) in Zea mays?

Based on the available research on Zea mays germin-like proteins (ZmGLPs), we can draw potential comparisons with the unknown protein from spot 168. Most ZmGLPs are located on chromosome 4 (20 genes), with others on chromosomes 10 (5 genes) and 2 (1 gene) . Determining the chromosomal location of the gene encoding the unknown protein would help establish its relationship to known GLP clusters.

ZmGLPs show conserved features including a peptide signal (MASS) at the protein start, GER motif 1 (M1) approximately 25 amino acids from the N-terminus, GER motif 2 (M2) about 90-100 amino acids from the N-terminus, GER motif 3 (M3) roughly 155-165 amino acids from the N-terminus, and the KGD motif upstream of GER motif 3 .

If the unknown protein is a GLP, it likely contains a cupin domain and shares physicochemical properties with other ZmGLPs. The table below summarizes key properties of several ZmGLPs for comparison:

Most ZmGLPs are localized in the cytoplasm or extracellular regions, with a few showing chloroplast- or periplasm-specific expression . While many ZmGLPs remain functionally uncharacterized, known functions include roles in plant development, germination, and responses to biotic and abiotic stresses .

What expression patterns might be expected for this protein during plant development?

If the unknown protein from spot 168 shares characteristics with other Zea mays germin-like proteins (ZmGLPs), several expression patterns might be expected during plant development. Based on ZmGLP expression studies, this protein may show highest expression in root tissues (particularly root tips), crown roots, elongation and maturation zones, radicle, and cortex tissues .

Regarding developmental stage-specific expression, levels might peak during germination stages, where GLPs often play critical roles, maturity stages, as observed with many ZmGLPs , and early etiolation phases, given its isolation from etiolated coleoptiles .

Expression may be modulated by light conditions (especially given its isolation from etiolated tissue), biotic stresses (particularly fungal pathogens like Aspergillus flavus, Colletotrichum graminicola, Cercospora zeina, Fusarium verticillioides, and Fusarium virguliforme) , and potentially abiotic stresses, though ZmGLPs generally show limited expression changes under these conditions .

To experimentally verify these expression patterns, researchers should consider qRT-PCR analysis across different tissues and developmental stages, RNA-seq to capture global expression profiles, in situ hybridization to precisely localize expression within specific tissues, reporter gene constructs (e.g., protein-GFP fusions) to visualize expression in planta, and immunohistochemistry using the available antibody to detect protein accumulation patterns .

How can I determine if this protein is involved in stress responses in Zea mays?

To investigate whether this unknown protein is involved in stress responses in Zea mays, implement a systematic experimental approach. Begin with stress-induced expression analysis by exposing maize plants to various stresses (drought, salinity, cold, heat, pathogen infection), collecting samples at multiple time points (0, 3, 6, 12, 24, 48, 72 hours post-treatment), quantifying protein expression using Western blot with the available antibody , and complementing with transcript analysis using qRT-PCR.

Perform comparative proteomics by conducting 2D-PAGE or LC-MS/MS proteomics on control and stressed tissues, comparing spot 168 intensity across different stress conditions, and analyzing post-translational modifications that might occur during stress responses.

Employ functional genomics approaches by generating transgenic maize lines overexpressing the protein, creating knockout/knockdown lines using CRISPR-Cas9 or RNAi, assessing stress tolerance phenotypes in these modified lines, and measuring physiological parameters (ROS levels, membrane integrity, photosynthetic efficiency).

Identify stress-related protein interactors using co-immunoprecipitation with the antibody , perform yeast two-hybrid or BiFC assays to confirm direct interactions, and analyze if these interactions change under stress conditions.

If the protein contains a cupin domain like other ZmGLPs , test for enzymatic activities that might be relevant to stress responses, such as superoxide dismutase activity (oxidative stress response), oxalate oxidase activity (pathogen response), or ADP glucose pyrophosphatase/phosphodiesterase activity (metabolic regulation).

What purification strategies are most effective for isolating this recombinant protein?

Purifying the recombinant unknown protein from spot 168 of 2D-PAGE requires a tailored purification strategy. For expression system selection, consider E. coli expression using pET or pGEX vectors with tags like 6xHis or GST for initial attempts, plant-based expression in Nicotiana benthamiana for proper post-translational modifications, or yeast expression in P. pastoris or S. cerevisiae for proteins requiring eukaryotic processing.

To optimize soluble expression, test multiple growth temperatures (16°C, 25°C, 30°C, 37°C), vary IPTG concentration (0.1-1.0 mM) for bacterial systems, add solubility enhancers like sorbitol or betaine to growth media, co-express with chaperones (GroEL/GroES, DnaK/DnaJ) if folding issues occur, and consider fusion partners beyond purification tags (MBP, SUMO, Trx) to enhance solubility.

Implement a multi-step purification strategy beginning with affinity chromatography using the tag (IMAC for His-tag or glutathione-agarose for GST), followed by ion exchange chromatography based on the protein's predicted pI, and a polishing step using size exclusion chromatography to achieve highest purity and remove aggregates.

For tag removal, incorporate a protease cleavage site (TEV, PreScission, or thrombin) between the tag and protein, optimize cleavage conditions (temperature, time, enzyme:protein ratio), and separate cleaved protein from tag and protease through reverse affinity chromatography.

Perform quality control assessments including SDS-PAGE with Coomassie or silver staining to verify purity, Western blot using the available antibody to confirm identity, mass spectrometry to verify molecular weight and sequence, dynamic light scattering to assess homogeneity and aggregation state, and circular dichroism to evaluate secondary structure integrity.

How can I develop a high-throughput screening assay to identify compounds that interact with this protein?

Developing a high-throughput screening (HTS) assay for compounds interacting with the unknown protein from spot 168 requires careful assay design and optimization. Begin by selecting an appropriate assay format based on readout mechanism. Consider thermal shift assay (TSA) to monitor protein stability changes upon ligand binding using fluorescent dyes like SYPRO Orange, which offers advantages including low protein consumption and minimal compound interference. Fluorescence polarization (FP) provides a mix-and-read format ideal for HTS if a fluorescent probe that binds the protein can be identified. Surface plasmon resonance (SPR) is useful for fragment screening and binding kinetics determination, though throughput is lower than other methods. AlphaScreen/AlphaLISA offers high sensitivity for detecting protein-ligand interactions without separation steps. If enzymatic function is identified, design activity-based assays monitoring substrate conversion or product formation.

For assay development and optimization, determine minimum protein concentration yielding reproducible signal, optimize buffer conditions (pH, ionic strength, additives) to maximize signal-to-noise ratio, evaluate DMSO tolerance (typically need ≥1% compatibility for compound libraries), establish positive controls (known binders if available) and negative controls, determine Z' factor (aim for ≥0.7) to ensure assay robustness, and assess day-to-day and plate-to-plate variability.

For compound library considerations, select appropriate libraries (natural products, FDA-approved drugs, diversity-oriented collections), determine appropriate screening concentration (typically 10-20 μM for primary screening), and include controls for compound interference and aggregation.

Implement a screening workflow consisting of single-point primary screening at one concentration, dose-response confirmation for hits (8-12 concentrations), orthogonal assays to confirm specific binding, and evaluation of structural novelty and chemical tractability of confirmed hits.

What approaches can be used to investigate protein-protein interactions involving this unknown protein?

Investigating protein-protein interactions (PPIs) involving the unknown protein from spot 168 requires a multi-technique approach. Begin with affinity-based isolation techniques such as co-immunoprecipitation (Co-IP) using the available antibody to pull down the protein along with its interaction partners from plant extracts, tandem affinity purification (TAP) by expressing the protein with dual affinity tags in maize for sequential purification steps, or proximity-dependent biotin identification (BioID) by fusing the protein to a biotin ligase to biotinylate nearby proteins.

Employ binary interaction detection methods including yeast two-hybrid (Y2H) screening against cDNA libraries from maize tissues where the protein is expressed, split-ubiquitin system (particularly useful if the protein is membrane-associated), bimolecular fluorescence complementation (BiFC) to visualize interactions in planta, Förster resonance energy transfer (FRET) to detect direct protein interactions at nanometer resolution in living cells, or protein fragment complementation assays (PCA) using split reporter proteins that regain activity upon interaction.

Utilize in vitro binding assays such as surface plasmon resonance (SPR) to determine binding kinetics and affinities between purified proteins, isothermal titration calorimetry (ITC) to measure thermodynamic parameters of interactions, microscale thermophoresis (MST) to detect interactions based on changes in thermophoretic mobility, or AlphaScreen/AlphaLISA bead-based proximity assays.

Apply crosslinking strategies like chemical crosslinking coupled with mass spectrometry (XL-MS) using crosslinkers of defined length to capture transient interactions, or photo-crosslinking by incorporating photo-activatable amino acids to capture interactions upon UV exposure.

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

Characterizing post-translational modifications (PTMs) of the unknown protein from spot 168 requires a comprehensive analytical strategy. Begin with predictive analysis using bioinformatic tools to predict potential modification sites like NetPhos and PhosphoSitePlus for phosphorylation, NetNGlyc and NetOGlyc for glycosylation, SUMOplot and GPS-SUMO for SUMOylation, and UbPred for ubiquitination. Search for conserved motifs associated with specific PTMs. From search results, we know ZmGLPs may contain N-glycosylation and phosphorylation sites , suggesting similar modifications might be present on this protein.

Implement mass spectrometry-based approaches with sample preparation strategies that enrich for phosphopeptides using TiO₂, IMAC, or phospho-antibodies, enrich for glycopeptides using lectin affinity, hydrazide chemistry, or HILIC, and use specific enrichment methods for other PTMs. Use MS analysis workflows including bottom-up proteomics (enzymatic digestion followed by LC-MS/MS), top-down proteomics (analysis of intact protein to preserve PTM combinations), and middle-down approach (limited proteolysis to generate larger peptides). Apply fragmentation techniques including ETD/ECD for labile modifications like phosphorylation and glycosylation, CID/HCD for routine peptide sequencing and PTM localization, and stepped collision energy for glycan structure elucidation.

Employ site-specific analytical methods such as Edman degradation for N-terminal modifications and sequencing, site-directed mutagenesis to confirm PTM sites by mutating predicted residues, specific antibodies for modification detection, and enzymatic demodification treatments to confirm modification type.

What are the challenges and solutions in crystallizing this protein for structural studies?

Crystallizing the unknown protein from spot 168 presents several challenges along with strategic solutions. Protein purity and homogeneity challenges arise because contaminating proteins or variably modified protein forms can impede crystallization. Solutions include implementing multi-step purification including affinity, ion exchange, and size exclusion chromatography, using dynamic light scattering (DLS) to assess sample monodispersity, considering limited proteolysis to remove flexible regions, and treating with endoglycosidases if the protein contains variable glycosylation.

Protein stability issues pose challenges as proteins that aggregate or degrade quickly are poor crystallization candidates. Solutions include using differential scanning fluorimetry (DSF) to identify stabilizing buffer conditions, screening additives that enhance thermal stability, performing stability trials at target temperature, and adding ligands or inhibitors that might stabilize a particular conformation.

Construct optimization is necessary because flexible regions or unfavorable surface properties can hinder crystal packing. Solutions include designing multiple constructs with N- and C-terminal truncations based on secondary structure predictions, using disorder prediction algorithms to identify and remove flexible regions, employing surface entropy reduction by mutating clusters of high-entropy surface residues to alanines, and considering fusion partners that facilitate crystallization.

Finding conditions that promote ordered crystal growth is largely empirical. Solutions include deploying high-throughput initial screening with commercial sparse matrix screens, utilizing specialized screens if the protein contains cupin domains like other ZmGLPs , implementing automated imaging to detect microcrystals, and exploring both vapor diffusion and alternative crystallization methods.