Recombinant Bacillus thuringiensis subsp. konkukian UPF0344 protein BT9727_1053 (BT9727_1053)

What is the molecular structure of BT9727_1053 protein?

The BT9727_1053 protein is a 121 amino acid protein with the sequence: MVHMHITAWALGLILFFVAYSLYSAGRKGKGVHMGLRLMYIIIIVTGFMLYMGIMKTATSNMHMWYGLKMIAGILVIGGMEMVLVKMSKNKATGAVWGLFIVALVAVFYLGLKLPIGWQVF . This protein is classified as a UPF0344 family protein derived from Bacillus thuringiensis subspecies konkukian. The recombinant form typically includes an N-terminal His-tag to facilitate purification processes . Structural analysis suggests it may contain transmembrane domains based on the hydrophobic amino acid stretches present in its sequence.

What expression systems are optimal for producing BT9727_1053?

While BT9727_1053 has been successfully expressed in E. coli systems as documented in the product specifications , researchers should consider several methodological approaches when optimizing expression. The E. coli system offers advantages of high yield and relatively straightforward purification through the His-tag affinity chromatography. For experimental design considerations, researchers should evaluate:

When using E. coli, optimization of induction temperature (typically 16-30°C), IPTG concentration, and induction time are critical parameters that should be experimentally determined for maximum soluble protein yield.

How should BT9727_1053 protein be stored for optimal stability?

The BT9727_1053 protein requires specific storage conditions to maintain its structural integrity and biological activity. According to product specifications, the protein should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles . Methodologically, researchers should:

• Centrifuge the vial briefly before opening to bring contents to the bottom

• Reconstitute the lyophilized 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)

• Store working aliquots at 4°C for up to one week to minimize degradation

• For long-term storage, maintain at -20°C/-80°C in Tris/PBS-based buffer with 6% trehalose at pH 8.0

This storage protocol preserves protein integrity while minimizing structural alterations that could affect experimental outcomes.

What functional domains exist within the BT9727_1053 protein structure?

The BT9727_1053 protein belongs to the UPF0344 protein family, which contains several predicted functional regions. Based on sequence analysis of the 121 amino acid sequence , researchers can identify potential functional domains using bioinformatic approaches similar to those employed in de novo protein design . The sequence analysis reveals:

• Hydrophobic regions suggesting membrane association (amino acids 8-28 and 50-70)

• Conserved charged residues (particularly the Arginine-Lysine rich region at positions 25-30) that may be involved in protein-protein interactions

• Potential binding sites based on the conserved motifs within the UPF0344 family

Researchers investigating domain function should consider employing site-directed mutagenesis to systematically alter these regions and assess impact on protein function. Circular dichroism (CD) spectroscopy, size-exclusion chromatography combined with multi-angle light scattering (SEC-MALS), and NMR spectroscopy are recommended methodological approaches for characterizing structural changes .

How does BT9727_1053 compare functionally to other proteins in Bacillus thuringiensis?

While Bacillus thuringiensis is known for producing insecticidal crystal proteins (delta endotoxins) , the BT9727_1053 protein belongs to a different functional category (UPF0344). Using comparative analysis methodologies:

Research methodologies to establish functional relationships should include co-immunoprecipitation experiments (similar to those used in NME1-DNM2 interaction studies ), protein-protein interaction assays, and comparative genomics to identify conserved gene neighborhoods that might suggest functional associations.

What role might BT9727_1053 play in bacterial physiology?

Based on sequence analysis and the presence of hydrophobic regions, BT9727_1053 likely functions as a membrane-associated protein. Methodological approaches to investigate its physiological role should include:

• Gene knockout or CRISPR interference studies to assess phenotypic changes

• Fluorescent tagging for localization studies using confocal microscopy

• RNA-seq analysis of knockout strains to identify dysregulated pathways

• Membrane fraction isolation and proteomic analysis to identify interaction partners

The hydrophobic nature of the amino acid sequence (MVHMHITAWALGLILFFVAYSLYSAGRKGKGVHMGLRLMYIIIIVTGFMLYMGIMKTATSNMHMWYGLKMIAGILVIGGMEMVLVKMSKNKATGAVWGLFIVALVAVFYLGLKLPIGWQVF) suggests potential roles in:

• Membrane stability or organization

• Small molecule transport

• Signal transduction

• Cell envelope stress response

Researchers should design experiments that systematically test these hypotheses using both in vitro and in vivo approaches.

What purification strategies yield the highest purity of BT9727_1053?

The recombinant BT9727_1053 protein contains an N-terminal His-tag specifically designed for affinity purification . An optimal purification workflow should include:

For membrane-associated proteins like BT9727_1053, consider adding mild detergents (0.1% Triton X-100 or 0.5% CHAPS) during lysis and initial purification steps to improve solubility. Validate purification success using SDS-PAGE with expected molecular weight around 15-16 kDa (including His-tag).

How can researchers optimize structural studies of BT9727_1053?

Structural characterization of BT9727_1053 requires a multi-technique approach similar to that used in de novo protein design studies :

• Circular Dichroism (CD) Spectroscopy:

  • Prepare protein at 0.1-0.2 mg/mL in phosphate buffer (pH 7.4)

  • Scan range: 190-260 nm

  • Temperature range studies (20-90°C) to assess thermal stability

  • Expected outcome: Characteristic αβ-protein spectrum if properly folded

• Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Use to determine oligomeric state and homogeneity

  • Calibrate column with appropriate molecular weight standards

  • Expected outcome: Single peak corresponding to monomeric protein (~16 kDa)

• NMR Spectroscopy:

  • Prepare 15N-labeled protein for 1H-15N HSQC experiments

  • Concentration: 0.2-0.5 mM in appropriate buffer with 5-10% D2O

  • Expected outcome: Well-dispersed peaks indicating folded structure

• X-ray Crystallography:

  • Initial screening using commercial sparse matrix screens

  • Optimization of promising conditions

  • Consider membrane protein-specific crystallization approaches

For membrane proteins like BT9727_1053, researchers should also consider using detergent micelles or nanodiscs to mimic the membrane environment during structural studies.

What experimental design considerations should be made when studying protein-protein interactions involving BT9727_1053?

To investigate potential interaction partners of BT9727_1053, researchers should employ a systematic approach:

• Yeast Two-Hybrid Screening:

  • Consider membrane-based Y2H systems for membrane-associated proteins

  • Use both N-terminal and C-terminal fusion constructs to minimize steric hindrance

• Co-Immunoprecipitation:

  • Similar to the two-way co-immunoprecipitation method described for NME1 and DNM2

  • Use anti-His antibodies for BT9727_1053 pulldown

  • Perform both forward and reverse co-IP to confirm interactions

• Proximity-Based Labeling:

  • Fuse BT9727_1053 to BioID or APEX2

  • Express in Bacillus thuringiensis or E. coli

  • Identify biotinylated proteins as potential interaction partners

• Surface Plasmon Resonance:

  • Immobilize purified BT9727_1053 on sensor chip

  • Flow potential interaction partners over surface

  • Determine binding kinetics and affinity constants

Data analysis should include appropriate controls and statistical validation to ensure reproducibility and significance of identified interactions.

How can BT9727_1053 be utilized in protein structure prediction and validation studies?

The BT9727_1053 protein represents an interesting candidate for structure prediction studies due to its classification in the UPF0344 family. Methodologically, researchers can:

• Employ AI-based protein structure prediction tools (like AlphaFold2) to generate theoretical models

• Validate predictions through experimental structure determination

• Compare predicted vs. experimental structures to refine prediction algorithms

This approach contributes to the broader goal of improving accuracy in protein structure prediction as outlined in full-length protein research . The relatively small size (121 amino acids) of BT9727_1053 makes it a manageable target for both computational prediction and experimental validation.

What considerations should be made when designing antibodies against BT9727_1053?

Designing effective antibodies against BT9727_1053 requires strategic epitope selection:

Researchers should:

• Perform epitope prediction using bioinformatic tools

• Avoid hydrophobic transmembrane regions

• Consider generating antibodies against synthetic peptides corresponding to predicted extracellular regions

• Validate antibody specificity using both wildtype and knockout/knockdown samples

Both polyclonal and monoclonal approaches have merit, with monoclonals offering higher specificity but potentially limited epitope recognition.

What are common challenges in working with BT9727_1053 and how can they be addressed?

Membrane-associated proteins like BT9727_1053 present specific technical challenges:

For experimental design, researchers should include appropriate controls and perform pilot studies to identify optimal conditions before proceeding to large-scale experiments.

How can researchers validate the biological activity of recombinant BT9727_1053?

Since the specific biological function of BT9727_1053 is not well-characterized, researchers must employ multiple approaches to validate activity:

• Structural integrity verification:

  • Circular dichroism to confirm proper folding

  • Thermal shift assays to assess stability

  • Size exclusion chromatography to confirm monomeric state

• Functional assays based on predicted activity:

  • Membrane binding assays if membrane association is predicted

  • Lipid interaction studies using liposome flotation assays

  • Electrophysiology if channel/transport function is suspected

• Comparison to native protein:

  • Express and purify native protein from B. thuringiensis

  • Compare biochemical properties to recombinant version

  • Assess differences due to post-translational modifications

Without established activity assays, researchers should design experiments based on structural predictions and sequence homology to related proteins with known functions.

What emerging technologies could advance understanding of BT9727_1053 function?

Several cutting-edge methodologies could significantly enhance our understanding of BT9727_1053:

• Cryo-electron microscopy:

  • Particularly valuable for membrane proteins

  • Can reveal structure in native-like lipid environments

  • May identify structural features not apparent in crystallography

• AlphaFold2 and related AI prediction tools:

  • Improving accuracy of protein structure prediction

  • Can predict structures of protein complexes

  • May identify potential binding partners

• Single-molecule studies:

  • FRET to study conformational changes

  • Optical tweezers to assess mechanical properties

  • Single-particle tracking in live cells

• Integrative structural biology:

  • Combining multiple techniques (NMR, X-ray, cryo-EM, mass spectrometry)

  • Provides comprehensive structural and dynamic information

  • Particularly valuable for challenging proteins like BT9727_1053

These approaches align with the future directions in protein research described in the literature, which emphasize improved computational tools and databases to support full-length protein research and innovation .

How might comparative studies between BT9727_1053 and related proteins inform functional understanding?

Comparative studies represent a powerful approach to uncover protein function:

Researchers should particularly focus on comparing BT9727_1053 with other UPF0344 family members across different bacterial species, noting patterns of conservation that might indicate functional importance. This approach has proven valuable in understanding protein function in numerous research contexts.