Recombinant Kluyveromyces lactis Nuclear rim protein 1 (NUR1)

What are the key physicochemical properties of recombinant NUR1 protein relevant to laboratory handling?

Understanding the physical and chemical properties of recombinant NUR1 is essential for experimental design and implementation. The table below summarizes key properties that researchers should consider when working with this protein :

What expression systems are optimal for producing functional recombinant K. lactis NUR1?

When using E. coli, codon optimization may be necessary to accommodate differences in codon usage between K. lactis and E. coli. The addition of an N-terminal His-tag enables straightforward purification via metal affinity chromatography while minimally affecting protein structure when appropriate linker sequences are incorporated .

How should researchers design optimal reconstitution protocols for lyophilized NUR1 protein?

Reconstitution of lyophilized NUR1 requires careful consideration of buffer conditions and handling techniques. The recommended protocol includes:

• Centrifuge the vial briefly before opening to collect all material at the bottom

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

• Add glycerol to a final concentration of 5-50% for long-term stability (50% is standard)

• Prepare multiple small aliquots to minimize freeze-thaw cycles

• Store working aliquots at 4°C (stable for up to one week)

• Maintain long-term storage at -20°C/-80°C

How does the K. lactis PDR1 gene regulation system compare to NUR1 regulation?

While direct information about NUR1 regulation is limited in current research, examining parallels with the well-characterized PDR1 system provides valuable insights. The K. lactis PDR1 gene encodes a zinc finger Zn(2)Cys(6)-containing transcription factor involved in multidrug resistance (MDR) . Molecular characterization reveals several important regulatory features:

• KlPDR1 functions as a critical transcriptional regulator that controls expression of membrane efflux pumps

• Deletion of KlPDR1 generates strains hypersusceptible to toxins like oligomycin, antimycin A, and azole antifungals

• PDR-responsive elements in promoters of target genes mediate the regulatory effects

• Overexpression of KlPDR1 from multicopy plasmids increases drug tolerance

Similar regulatory mechanisms may apply to NUR1, particularly given its potential role at the nuclear rim where it may interact with transcriptional machinery.

What experimental approaches are recommended for studying protein-protein interactions involving NUR1?

To investigate the functional interactions of NUR1 with other cellular proteins, researchers should consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies against the His-tag or NUR1 itself to pull down protein complexes, followed by mass spectrometry identification of binding partners

• Yeast two-hybrid assays: Particularly valuable in yeast systems, creating fusion constructs of NUR1 with DNA-binding and activation domains

• Proximity-dependent biotin labeling: Methods like BioID or APEX can identify proteins in proximity to NUR1 in vivo

• Pull-down assays: Using recombinant His-tagged NUR1 as bait with K. lactis cell lysates as prey

• Fluorescence resonance energy transfer (FRET): For analyzing direct protein interactions in live cells

What gene manipulation strategies are most effective for studying NUR1 function in K. lactis?

Gene deletion and modification techniques specifically optimized for K. lactis provide powerful tools for studying NUR1 function. The recommended approach utilizes the one-step gene replacement procedure, as outlined below :

How can researchers effectively analyze gene expression changes related to NUR1 manipulation?

Based on methodologies used for similar K. lactis studies, RNA analysis techniques provide valuable insights into NUR1's role in gene regulation :

• Northern Blot Analysis: To determine relative mRNA levels of NUR1 and potentially regulated genes

• Real-Time Quantitative PCR: For precise quantification of expression changes

• RNA-Seq: For genome-wide transcriptional profiling following NUR1 deletion or overexpression

• Reporter Gene Assays: Using constructs containing promoter regions of interest fused to reporter genes like GFP or luciferase

The KlPDR1 research demonstrated that Northern blot analysis effectively identified changes in expression of the ATP-binding cassette transporter gene (KlPDR5) in response to gene deletion, suggesting similar approaches would be valuable for NUR1 studies .

What approaches can determine subcellular localization and dynamic behavior of NUR1?

As a nuclear rim protein, NUR1's localization is central to understanding its function. Multiple complementary techniques should be employed:

• Fluorescent Protein Tagging: Creating NUR1-GFP fusions for live-cell imaging

• Immunofluorescence Microscopy: Using antibodies against the His-tag or NUR1 itself

• Subcellular Fractionation: Biochemical separation of nuclear envelope, nucleoplasm, and cytoplasm

• Super-Resolution Microscopy: Techniques like STORM or PALM for precise localization within the nuclear rim

• Electron Microscopy: Immunogold labeling for ultrastructural localization

How can researchers address solubility issues with recombinant NUR1 protein?

Membrane-associated proteins like NUR1 often present solubility challenges. Consider the following strategies:

• Buffer Optimization: Screen various pH conditions and salt concentrations

• Detergent Selection: Test mild non-ionic detergents (e.g., DDM, CHAPS)

• Co-expression with Chaperones: Particularly when using E. coli expression systems

• Truncation Constructs: Express specific domains rather than the full-length protein

• Fusion Tags: MBP or SUMO tags can enhance solubility compared to His-tag alone

What are the key considerations for successful transformation of K. lactis with NUR1 constructs?

Transformation efficiency in K. lactis can be optimized using these research-validated approaches:

• Use electroporation with specific settings (1.0 kV, 25 μF, 400Ω) in 0.2-cm cuvettes

• Harvest cells in mid-logarithmic phase (OD600 of 0.8-1.2)

• Thorough washing steps with ice-cold water and 1M sorbitol

• Pre-chill electroporation cuvettes and recovery media

• Consider using multicopy vectors based on the 2μ-like plasmid pKD1 from K. drosophilarum for high expression levels

• For secreted constructs, fusion with the 'pre'-region of K. lactis killer toxin has demonstrated highly efficient secretion