The protein is synthesized using codon-optimized K. lactis DNA cloned into E. coli expression vectors. Key production features include:
Vector system: pET-based plasmids for high-yield cytoplasmic expression
Purification: Immobilized metal affinity chromatography (IMAC) leveraging the His-tag
Yield: Milligram quantities per liter of bacterial culture (exact titers proprietary)
While KLLA0D04334g's specific biological role remains uncharacterized, its production leverages K. lactis' validated protein synthesis machinery. This yeast strain is industrially significant for:
Post-translational modification fidelity comparable to higher eukaryotes
GRAS (Generally Recognized As Safe) status for biopharmaceutical applications
Current documented uses include:
Antigen production: Used in ELISA development for antibody validation
Protein interaction studies: Serves as bait/prey in yeast two-hybrid screens
Structural biology: Crystallization trials due to small size and solubility (>1 mg/mL in reconstitution buffer)
Feature | K. lactis System | S. cerevisiae System |
---|---|---|
Glycosylation Pattern | Minimal/human-like | Hypermannosylation |
Methanol Requirement | No | No |
Industrial Scalability | FDA-approved for enzymes | Limited to smaller scales |
Endotoxin levels: <1.0 EU/μg (LAL assay)
Stability: Maintains integrity for 6 months at -80°C; avoid >3 freeze-thaw cycles
No published crystal structures or enzymatic activity data exist for KLLA0D04334g
Pathway associations remain unannotated in KEGG/UniPathway databases
Commercial availability restricted to research use (not for human consumption)
This protein represents a niche tool for yeast molecular biology studies, with its primary utility stemming from K. lactis' robust protein production heritage rather than characterized biological functions. Further studies require functional genomics approaches to elucidate its native role in K. lactis physiology.
KEGG: kla:KLLA0D04334g
STRING: 284590.XP_453255.1
The UPF0495 protein KLLA0D04334g is a small protein (72 amino acids) from Kluyveromyces lactis with the sequence: MRPTQFVLNAAKKKSGFSVPVELTPLFLAMGVALASGTWFSYKKFFHDDSLRVSRKNPEQSALDKVLNQKAE . The protein belongs to the UPF0495 family of uncharacterized proteins. Structural analysis suggests it contains mostly alpha-helical regions with hydrophobic patches, indicating potential membrane association. The protein contains a relatively high proportion of basic amino acids (lysine) in its N-terminal region, which may facilitate interactions with negatively charged cellular components such as nucleic acids or phospholipids.
To effectively study this protein's structure, researchers should consider comparative modeling approaches with homologous proteins, circular dichroism spectroscopy for secondary structure determination, and potentially X-ray crystallography or NMR for high-resolution structural analysis.
Research approaches to elucidate its function should include:
Gene knockout or knockdown studies to observe phenotypic changes
Protein-protein interaction studies (yeast two-hybrid, co-immunoprecipitation)
Transcriptomic analysis under various growth conditions
Subcellular localization studies using fluorescently-tagged variants
Comparative genomic analyses with homologous proteins in related yeast species
For optimal expression of KLLA0D04334g in K. lactis, researchers should consider a chromosomal integration approach using the LAC4 locus. Based on established K. lactis expression systems, the following methodology is recommended:
Clone the KLLA0D04334g gene into an appropriate expression vector (such as pKLAC2) under the control of the strong LAC4 promoter .
Linearize the plasmid with SacII or BstXI to create an expression cassette for homologous recombination .
Transform K. lactis GG799 strain (an industrial isolate with no auxotrophies) with the linearized expression cassette .
Select transformants on nitrogen-free minimal medium containing acetamide, which enriches for cells with multiple tandem integration events .
Optimize culture conditions: typically, growth at 30°C in rich medium with glucose, followed by induction with lactose or galactose.
Expression yields can be significantly enhanced by including additional copies of the KlGAL4 transactivator gene, which has been shown to increase transcription rates of target genes in K. lactis .
The purification of KLLA0D04334g requires careful consideration of its biochemical properties to maintain native conformation. An effective purification strategy includes:
Initial clarification: Harvest cells and lyse using either mechanical disruption (glass beads) or enzymatic methods (zymolyase treatment followed by gentle lysis).
Capture phase: Utilize affinity chromatography if a tag is incorporated (His, FLAG, or other affinity tags), or ion exchange chromatography exploiting the protein's charge characteristics.
Intermediate purification: Size exclusion chromatography to separate the target protein based on molecular size.
Polishing: Hydrophobic interaction chromatography to remove remaining contaminants.
Throughout purification, maintain a Tris-based buffer system with 50% glycerol as used in commercial preparations . The addition of protease inhibitors and performing purification at 4°C is crucial to prevent protein degradation. For long-term storage, aliquot the purified protein and store at -20°C or preferably -80°C to avoid repeated freeze-thaw cycles.
Purification Step | Method | Buffer Conditions | Expected Recovery |
---|---|---|---|
Capture | Affinity/Ion Exchange | Tris-HCl pH 7.5, 150 mM NaCl | 70-80% |
Intermediate | Size Exclusion | Tris-HCl pH 7.5, 150 mM NaCl | 80-90% |
Polishing | Hydrophobic Interaction | Tris-HCl pH 7.5, 1M (NH₄)₂SO₄ | 90-95% |
Storage | Flash freeze | Tris-based buffer, 50% glycerol | - |
KLLA0D04334g serves as an excellent model for studying uncharacterized protein families due to several advantageous characteristics:
Compact size: At 72 amino acids, the protein is manageable for structural studies and synthetic biology applications.
Expression in K. lactis: The native host provides authentic post-translational modifications and folding environment.
Evolutionary conservation: As a member of the UPF0495 family, comparative studies with homologs can reveal functionally important regions.
Research approaches should include:
a) Systematic mutagenesis studies to identify critical residues for function and stability
b) Domain swapping with homologous proteins from other yeast species
c) Heterologous expression in different hosts to evaluate functional conservation
d) Transcriptomic and proteomic analyses of knockout strains under various stress conditions
e) Protein interaction network mapping using techniques like BioID or proximity labeling
The insights gained from studying KLLA0D04334g can be extrapolated to understand similar uncharacterized protein families, providing methodological frameworks for functional genomics in yeast systems.
Designing effective antibodies against KLLA0D04334g requires careful epitope selection and validation strategies:
Epitope selection considerations:
The small size (72 amino acids) limits the number of potential epitopes
Hydrophilic, surface-exposed regions should be prioritized
Regions with low homology to other K. lactis proteins will improve specificity
The N-terminal region (MRPTQFVLNAAKKKSGFSVP) appears to have good antigenicity characteristics
Antibody development approaches:
Develop both polyclonal antibodies against the whole protein and monoclonal antibodies against specific epitopes
Consider using synthetic peptides corresponding to predicted antigenic regions
Express the protein with different tags (His, GST) to provide alternative immunization antigens
Validation requirements:
Western blot against both recombinant protein and native K. lactis lysates
Immunoprecipitation followed by mass spectrometry
Immunofluorescence to confirm subcellular localization
Testing against knockout strains as negative controls
The resulting antibodies will be valuable tools for protein detection, localization studies, and interaction analyses in research settings.
Understanding the expression patterns of KLLA0D04334g under various stress conditions provides valuable insights into its potential functions. While comprehensive transcriptomic data specific to this gene is limited in the literature, general principles for such analysis include:
Design of stress experiments:
Heat shock (37°C, 42°C)
Oxidative stress (H₂O₂, paraquat)
Osmotic stress (high salt, sorbitol)
Nutrient limitation (carbon, nitrogen starvation)
Cell wall stress (Congo red, calcofluor white)
DNA damage (UV, MMS exposure)
Expression analysis methodologies:
RT-qPCR for targeted expression analysis
RNA-seq for genome-wide context
Reporter constructs (e.g., KLLA0D04334g promoter driving GFP expression)
Western blotting with specific antibodies to analyze protein levels
Data interpretation framework:
Coexpression analysis with known stress-response genes
Comparison with homologous genes in related yeasts
Correlation with physiological parameters (growth rate, viability)
Integration with other -omics data (proteomics, metabolomics)
Preliminary observations suggest that proteins in the UPF0495 family may be involved in membrane adaptation to environmental changes, potentially responding to alterations in membrane fluidity or integrity during stress conditions.
Investigating protein-protein interactions (PPIs) involving KLLA0D04334g requires multi-faceted approaches due to its small size and potential membrane association:
In vivo interaction studies:
Yeast two-hybrid (Y2H) screening using KLLA0D04334g as bait
Split-ubiquitin membrane Y2H for membrane-associated interactions
Bimolecular fluorescence complementation (BiFC) to visualize interactions in living cells
Proximity-dependent biotin identification (BioID) to capture transient interactions
In vitro interaction studies:
Co-immunoprecipitation followed by mass spectrometry
Pull-down assays using recombinant tagged KLLA0D04334g
Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) for measuring binding kinetics
Crosslinking mass spectrometry to identify interaction interfaces
Computational predictions:
Structure-based docking simulations
Coevolution analysis to identify potential interaction partners
Network analysis using existing PPI databases from related yeast species
Understanding these interactions will provide critical insights into the biological pathways involving KLLA0D04334g and guide further functional studies.
Comparative analysis of KLLA0D04334g with homologs in other yeast species provides evolutionary context and functional insights:
Identification of homologs:
BLAST searches against fungal genomes
Profile-based searches using hidden Markov models
Analysis of syntenic regions in related yeast genomes
Sequence conservation patterns:
Multiple sequence alignment to identify conserved residues
Analysis of selection pressure using dN/dS ratios
Identification of species-specific adaptations
Structural conservation:
Homology modeling of related UPF0495 family members
Comparison of predicted secondary structure elements
Analysis of conserved surface patches that may indicate functional sites
The UPF0495 protein family appears to be conserved across many fungal species, suggesting an important basic cellular function. Variations in sequence conservation patterns between different yeast clades may indicate functional specialization in response to specific ecological niches or metabolic requirements.
Determining the subcellular localization of KLLA0D04334g presents several methodological challenges due to its small size and potential membrane association:
Challenges with fluorescent protein tagging:
The small size (72 amino acids) means that fluorescent protein tags (typically >200 amino acids) may disrupt localization or function
Membrane association may be affected by tag placement
K. lactis-specific codon optimization may be necessary for optimal expression
Solutions and alternative approaches:
Use small epitope tags (HA, FLAG, Myc) followed by immunofluorescence
Create both N- and C-terminal fusions to compare localization patterns
Employ split GFP systems where only a small fragment is fused to the target
Use fluorescent nanobodies for live-cell imaging
Complement with biochemical fractionation followed by Western blotting
Advanced imaging techniques:
Super-resolution microscopy (STORM, PALM) for precise localization
Correlative light and electron microscopy (CLEM) for ultrastructural context
Live-cell imaging to track dynamic localization changes
Localization Method | Advantages | Limitations | Best For |
---|---|---|---|
Epitope tagging + Immunofluorescence | Minimal interference with protein function | Requires fixation | Initial localization studies |
Fluorescent protein fusion | Live-cell visualization | May disrupt protein function | Dynamic localization studies |
Biochemical fractionation | Quantitative distribution | Low spatial resolution | Membrane association studies |
Super-resolution microscopy | High precision localization | Complex sample preparation | Detailed suborganelle localization |
Electron microscopy | Ultrastructural context | Labor intensive | Definitive membrane integration studies |