Recombinant Saccharomyces cerevisiae Uncharacterized protein C1Q_03362 (C1Q_03362)

What is Recombinant Saccharomyces cerevisiae Uncharacterized protein C1Q_03362?

Recombinant Full Length Saccharomyces cerevisiae Uncharacterized protein C1Q_03362 (C1Q_03362) is a 72-amino acid protein (UniProt ID: C7GSI6) that is produced recombinantly with an N-terminal His tag expressed in E. coli. The full amino acid sequence is: MSKHKHEWTESVANSGPASILSYCASSILMTVTNKFVVNLDNFNMNFVMLFVQSLVCTVTLCILRIVGVANF. The protein is typically supplied as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE .

What are the recommended storage conditions for C1Q_03362?

The protein should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles which can compromise protein integrity. For working solutions, store aliquots at 4°C for up to one week. The lyophilized protein is typically stored in Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 .

How should C1Q_03362 protein be reconstituted for experimental use?

For optimal reconstitution:

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

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

• Add glycerol to a final concentration of 5-50% (50% is recommended as default)

• Prepare multiple aliquots for long-term storage at -20°C/-80°C

What is the basic experimental workflow for studying C1Q_03362 expression patterns?

The basic workflow involves:

• Cell culturing of S. cerevisiae under various conditions

• RNA extraction and quality control

• Microarray or RNA-seq analysis to detect expression levels

• Data normalization and statistical analysis

• Validation of expression patterns using RT-qPCR

• Comparative analysis with known genes related to cellular functions

• Correlation of expression with physiological conditions

What protein extraction and digestion methods are optimal for mass spectrometry analysis of C1Q_03362?

Based on comparative studies, two protocols have been found effective with Protocol A showing superior results:

Protocol A (Preferred - Urea/Thiourea Extraction):

• Resuspend 100 mg wet cell pellets in lysis buffer (6 M urea, 2 M thiourea, 50 mM ammonium bicarbonate, protease inhibitors)

• Disrupt cells via multiple rounds of sonication on ice

• Reduce proteins with 10 mM dithiothreitol at room temperature for one hour

• Alkylate with 20 mM iodoacetamide at room temperature for 30 minutes in dark

• First digest with Lys-C for three hours

• Dilute 10-fold with 20 mM ammonium bicarbonate (pH 8.5)

• Further digest overnight at 37°C with MS-grade trypsin (enzyme:protein ratio ~1:50)

• Acidify tryptic peptides with 5% formic acid to pH ≤3

• Desalt using Poros Oligo R3 reverse-phase micro-columns

This protocol has been shown to yield approximately 40% more protein identifications than alternative methods .

How can researchers design quantitative proteomic experiments to study C1Q_03362?

For robust quantitative proteomics:

• Experimental Design:

  • Use minimum three independent biological replicates per condition

  • Include at least three technical replicates per biological sample

  • Include appropriate controls (wild-type, vector-only, etc.)

• Mass Spectrometry Analysis:

  • Implement label-free quantitative (LFQ) mass spectrometric approaches

  • Analyze samples on high-resolution instruments (e.g., Q-Exactive)

  • Process data using established platforms (e.g., MaxQuant)

• Data Processing:

  • Convert LFQ values to log2 scale and normalize by subtraction of means

  • Retain proteins detected in at least 5 sample runs among 9+ in any condition

  • Perform missing value imputation using normal distribution (width 0.3, downshift 1.8)

  • Apply statistical analysis (Student's t-test) to identify significantly changed proteins

  • Consider proteins with p-value <0.05 and fold-change >2 as significantly responsive

How can transcriptomic data mining be applied to understand C1Q_03362 function in S. cerevisiae?

A comprehensive data mining approach should include:

• Dataset Collection and Processing:

  • Gather microarray/RNA-seq datasets related to S. cerevisiae under various conditions

  • Normalize data and perform quality control

  • Focus on datasets with manipulated fermentation conditions (e.g., Mg²⁺ and Cu²⁺ supplementation)

• Machine Learning Implementation:

  • Apply multiple algorithms (minimum 11 recommended) from platforms like RapidMiner

  • Identify discriminative genes between conditions (e.g., improved vs. repressed ethanol production)

  • Select probe sets identified by at least 5 different algorithms

  • Validate top-ranked selective genes through Principal Component Analysis and heatmap clustering

• Functional Analysis:

  • Construct decision tree models to identify key genes (100% performance)

  • Perform gene ontology enrichment to identify related biological processes

  • Analyze pathway involvement through enrichment analysis

  • Identify regulatory networks using clustering analysis of transcription factors

This approach has successfully identified genes involved in carbohydrate metabolism, oxidative phosphorylation, and ethanol fermentation in S. cerevisiae .

What strategies can be employed to create C1Q_03362 deletion mutants for functional studies?

A systematic approach for in-frame deletion includes:

• Design and Construction:

  • Design an in-frame deletion of the coding sequence except first four and last four codons

  • Join ~900-1000 bp 5' flanking region (with first four codons) with ~900-1000 bp 3' flanking region (with last four codons) to a suicide plasmid

  • Use PCR with primers containing 25 bp 5' overlapping regions between vector arms and gene fragments

• Verification and Transfer:

  • Control PCR product quality via nanodrop and gel electrophoresis

  • Use HiFi assembly master mix for the assembly reaction

  • Transform into appropriate E. coli strain (e.g., S17-1)

  • Confirm assembled deletion alleles via restriction enzyme digestion and DNA sequencing

  • Conjugate resultant plasmids into wild-type S. cerevisiae

• Selection and Confirmation:

  • Select for transconjugants using appropriate markers

  • Test for inability to grow under specific conditions

  • Select for chromosomal deletions (e.g., resistance to 10% sucrose)

  • Confirm final mutants by PCR and DNA sequencing

How can researchers resolve contradictory findings about C1Q_03362 in the literature?

When faced with contradictory findings, implement this structured approach:

• Systematic Literature Review:

  • Collect comprehensive set of publications on C1Q_03362

  • Extract specific claims about protein function, localization, or interactions

  • Identify claims that directly contradict each other

• Contradiction Analysis:

  • Categorize contradictions by type (functional, structural, regulatory)

  • Examine experimental conditions across studies (strains, media, analysis methods)

  • Note any study characteristics that correlate with contradictory results

• Resolution Strategy:

  • Design experiments that specifically address variables differing between contradictory studies

  • Implement standardized protocols for protein handling and analysis

  • Consider multiple analytical approaches to validate findings

• Advanced Analysis:

  • Use natural language processing techniques for automated contradiction detection

  • Normalize terminology across studies to address acronym and naming variations

  • Measure inter-annotator agreement in claim identification (>90% agreement is achievable)

What bioinformatic approaches can predict potential functions of C1Q_03362?

A multi-layered bioinformatic strategy should include:

What are the considerations for optimizing the expression and purification of C1Q_03362?

For optimal expression and purification:

• Expression System Optimization:

  • Test multiple E. coli strains (BL21, Rosetta, Arctic Express)

  • Optimize induction conditions (temperature, IPTG concentration, duration)

  • Consider fusion partners (His-tag position, additional solubility tags)

  • Test expression in eukaryotic systems for proper post-translational modifications

• Purification Strategy:

  • Implement IMAC (Immobilized Metal Affinity Chromatography) as primary purification

  • Include secondary purification steps:

    • Ion exchange chromatography to remove charged contaminants

    • Size exclusion chromatography to ensure homogeneity

  • Optimize buffer conditions to maintain protein stability

  • Consider on-column refolding for inclusion body purification

• Quality Control:

  • Verify purity by multiple methods (SDS-PAGE, MS)

  • Assess protein folding (circular dichroism, fluorescence spectroscopy)

  • Validate activity through appropriate functional assays

  • Ensure batch-to-batch consistency through standardized protocols

How can researchers design experiments to investigate potential post-translational modifications of C1Q_03362?

A comprehensive investigation requires:

• Predictive Analysis:

  • Use bioinformatic tools to predict potential PTM sites

  • Focus on common yeast modifications (phosphorylation, glycosylation, acetylation)

  • Compare predictions across multiple algorithms

• Experimental Detection:

  • Perform specialized digestion protocols to preserve modifications

  • Implement enrichment strategies for specific PTMs

  • Use high-resolution mass spectrometry with ETD/HCD fragmentation

  • Consider targeted approaches for predicted modification sites

• Validation:

  • Generate site-specific antibodies for key modifications

  • Create site-directed mutants to abolish modification sites

  • Assess functional consequences of mutations

  • Compare modifications across different growth conditions

What methodologies are suitable for studying C1Q_03362 localization in yeast cells?

For accurate localization studies:

• Fluorescent Protein Fusion Approaches:

  • Create C-terminal and N-terminal GFP/mCherry fusions

  • Validate fusion protein function compared to wild-type

  • Perform live-cell imaging under various conditions

  • Co-localize with known compartment markers

• Immunolocalization:

  • Generate specific antibodies against C1Q_03362

  • Optimize fixation and permeabilization for yeast cells

  • Implement super-resolution microscopy techniques

  • Perform co-localization with organelle markers

• Biochemical Fractionation:

  • Perform subcellular fractionation to isolate cellular compartments

  • Validate fractions using compartment-specific markers

  • Detect C1Q_03362 in fractions via Western blotting

  • Confirm localization through multiple independent approaches

How can high-throughput screening be used to identify interaction partners of C1Q_03362?

Implement a multi-approach strategy:

• Yeast Two-Hybrid Screening:

  • Create bait constructs with different protein domains

  • Screen against genomic or cDNA libraries

  • Validate interactions through multiple reporter systems

  • Confirm with reciprocal bait-prey configurations

• Affinity Purification-Mass Spectrometry:

  • Perform tandem affinity purification of tagged C1Q_03362

  • Implement crosslinking strategies to capture transient interactions

  • Analyze by high-resolution mass spectrometry

  • Use quantitative approaches (SILAC, TMT) to distinguish specific interactors

  • Filter against common contaminant databases

• Proximity Labeling:

  • Create fusion proteins with BioID or APEX2

  • Induce proximity-dependent biotinylation

  • Purify biotinylated proteins and identify by MS

  • Validate key interactions using orthogonal methods

How can researchers troubleshoot low yield or degradation issues with C1Q_03362?

For addressing production issues:

• Low Yield Troubleshooting:

  • Verify expression construct sequence integrity

  • Optimize codon usage for E. coli expression

  • Test multiple growth media formulations

  • Adjust induction conditions (OD, temperature, duration)

  • Consider autoinduction media for gradual protein expression

• Degradation Prevention:

  • Include protease inhibitors throughout purification

  • Maintain cold temperature during all processing steps

  • Add stabilizing agents (glycerol, specific ions, reducing agents)

  • Minimize processing time between steps

  • Consider adding stabilizing binding partners during purification

What analytical methods are recommended for characterizing purified C1Q_03362?

A comprehensive characterization includes:

• Primary Structure Verification:

  • Peptide mass fingerprinting by MS

  • N-terminal sequencing

  • Intact mass analysis to confirm full-length protein

• Secondary/Tertiary Structure Analysis:

  • Circular dichroism for secondary structure elements

  • Fluorescence spectroscopy for tertiary structure integrity

  • Thermal shift assays for stability assessment

  • Limited proteolysis to identify stable domains

• Functional Characterization:

  • Activity assays (once function is established)

  • Binding studies with potential interaction partners

  • Stability studies under various buffer conditions

  • Dynamic light scattering for homogeneity assessment

How can researchers develop activity assays for an uncharacterized protein like C1Q_03362?

Develop assays through systematic approaches:

• Bioinformatic-Guided Assay Development:

  • Use structural predictions to identify potential active sites

  • Search for conserved domains with known functions

  • Design assays based on predicted biochemical activities

  • Test activity with structurally related substrates

• Unbiased Screening Approaches:

  • Screen against substrate libraries (peptides, metabolites)

  • Test for common enzymatic activities (hydrolase, transferase)

  • Assess binding to cellular extracts or fractionated components

  • Implement label-free interaction detection methods

• Phenotypic Assays:

  • Compare wild-type and knockout strains under various conditions

  • Analyze metabolic profiles of mutant strains

  • Assess transcriptional responses to gene deletion

  • Evaluate stress responses in the presence/absence of the protein