Recombinant Alkaliphilus metalliredigens UPF0316 protein Amet_0954 (Amet_0954)

What are the general characteristics of Recombinant Alkaliphilus metalliredigens UPF0316 protein Amet_0954?

Recombinant Alkaliphilus metalliredigens UPF0316 protein Amet_0954 (also known as A6TLV6) is a full-length protein consisting of 173 amino acids. The protein's amino acid sequence is: MELVLGYLFIFVARVTDVGMGTVRMIMVVKGKRIQAAAIGFVESIIYILAIGKVLEALDNPVNILVYATGFAAGNYVGIYIEERMALGNIIAQVMCDHNVMQLVDLLRDAGFGVTVVEGYGRTGIRHLLNVSLQRKNLSKLYNVLDTHDHKAFITVTDARSIRGGYFTSVKKK . The protein is typically expressed with an N-terminal His-tag in E. coli expression systems, which facilitates its purification using affinity chromatography techniques. The protein belongs to the UPF0316 family, a group of proteins with relatively unknown functions that are being investigated for their potential roles in bacterial metabolism .

What expression systems are optimal for Amet_0954 production?

While E. coli is the most commonly used expression system for Amet_0954, as evidenced in the available literature, researchers have multiple options. A systematic approach to expression system selection should consider:

• Bacterial expression (E. coli): The standard BL21(DE3) strain is commonly used, providing high yield with relatively simple protocols. The BL21(DE3) system is particularly effective because it contains the T7 RNA polymerase gene under the control of the lacUV5 promoter, which can be induced by IPTG for high-level protein expression .

• Alternative expression systems: For researchers encountering challenges with bacterial expression, other systems are available:

  • Yeast systems (SMD1168, GS115, X-33)

  • Insect cell lines (Sf9, Sf21, High Five)

  • Mammalian cell lines (293, 293T, CHO)

The selection should be based on experimental requirements, such as post-translational modifications, solubility needs, and downstream applications.

How should Amet_0954 be stored to maintain stability?

Proper storage of Amet_0954 is critical for maintaining its structural integrity and biological activity. The recommended storage protocol involves:

• Long-term storage: Store at -20°C to -80°C in aliquots to avoid repeated freeze-thaw cycles. The protein is typically provided as a lyophilized powder, which enhances stability during shipping and long-term storage .

• Working solutions: For ongoing experiments, store working aliquots at 4°C for up to one week. This approach minimizes protein degradation from repeated freezing and thawing .

• Buffer composition: The recommended storage buffer is Tris/PBS-based with 6% Trehalose at pH 8.0. The addition of trehalose acts as a cryoprotectant, helping to preserve protein structure during freeze-thaw cycles .

• Reconstitution procedure: When reconstituting the lyophilized protein, it is advised to:

  • Briefly centrifuge the vial to bring contents to 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% (50% is standard) before aliquoting for long-term storage

What experimental design considerations are crucial when conducting functional studies with Amet_0954?

When designing experiments to characterize the functional properties of Amet_0954, researchers should implement a comprehensive approach:

• Experimental controls: Include both positive and negative controls in all functional assays. For proteins with unclear functions like Amet_0954, comparing its activity with well-characterized members of the UPF0316 family can provide valuable insights into potential functions.

• Statistical design: Implement robust statistical designs such as those measured by the Experimental Design Ability Test (EDAT). A well-designed experiment should follow the 2 (group: experimental/control) × 2 (test: pre-test/post-test) ANOVA structure to identify significant interactions and effects .

• Parameter optimization: Systematically vary key experimental parameters including:

  • pH range (particularly important given the alkaliphilic nature of the source organism)

  • Temperature conditions

  • Salt concentration

  • Presence of potential cofactors

• Activity verification: Given the limited knowledge of UPF0316 family functions, multiple assay types should be employed to identify potential activities:

  • Enzymatic activity screens

  • Binding assays with various substrates

  • Protein-protein interaction studies

Researchers who implemented similar experimental design strategies in other contexts demonstrated significantly improved outcomes, with experimental groups showing greater improvement on composite EDAT scores (pre-test: M = 3.760, SE = 0.102 to post-test: M = 5.429, SE = 0.105) compared to control groups (pre-test: M = 3.588, SE = 0.091 to post-test: M = 4.765, SE = 0.095) .

How can researchers effectively perform site-directed mutagenesis studies on Amet_0954?

Site-directed mutagenesis provides valuable insights into structure-function relationships for proteins like Amet_0954. A systematic approach includes:

• Target residue identification: Based on the amino acid sequence provided (MELVLGYLFIFVARVTDVGMGTVRMIMVVKGKRIQAAAIGFVESIIYILAIGKVLEALDNPVNILVYATGFAAGNYVGIYIEERMALGNIIAQVMCDHNVMQLVDLLRDAGFGVTVVEGYGRTGIRHLLNVSLQRKNLSKLYNVLDTHDHKAFITVTDARSIRGGYFTSVKKK), focus on:

  • Conserved residues within the UPF0316 family

  • Residues in predicted active or binding sites

  • Residues at key structural positions (based on secondary structure predictions)

• Mutagenesis strategy:

  • Use PCR-based methods with custom primers targeting specific codons

  • Consider creating multiple mutants in parallel to systematically analyze the contribution of different residues

  • For extensive mutations, gene synthesis services may be more cost-effective than traditional mutagenesis

• Expression and purification validation:

  • Verify that mutations do not disrupt protein folding or expression

  • Ensure purification yield and purity are comparable to wild-type protein

  • Confirm protein stability using thermal shift assays

• Functional comparison:

  • Design assays that directly compare wild-type and mutant proteins under identical conditions

  • Analyze kinetic parameters if enzymatic activity is identified

  • Examine structural changes using circular dichroism or other biophysical techniques

What approaches are recommended for structural characterization of Amet_0954?

Structural characterization of Amet_0954 is crucial for understanding its function and mechanism. A comprehensive approach includes:

• Preliminary structural analysis:

  • Secondary structure prediction from the primary sequence

  • Homology modeling based on related proteins with known structures

  • ab initio modeling for regions without homologous templates

• Experimental structure determination:

  • X-ray crystallography: Requires high-purity protein (>95%) and screening of crystallization conditions

  • NMR spectroscopy: Suitable for smaller proteins or domains, requires isotope labeling

  • Cryo-EM: Increasingly viable for smaller proteins with recent technological advances

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Thermal stability studies to determine melting temperature

  • Small-angle X-ray scattering (SAXS) for low-resolution structural information in solution

• Computational approaches:

  • Molecular dynamics simulations to study protein flexibility and conformational changes

  • Docking studies to predict potential binding partners or substrates

  • Integration of experimental data with computational models for structure refinement

What is the recommended protocol for high-yield purification of His-tagged Amet_0954?

A systematic purification protocol for His-tagged Amet_0954 should include:

• Cell lysis optimization:

  • Mechanical disruption (sonication or high-pressure homogenization)

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors

  • Addition of 0.1-1% mild detergent (Triton X-100) if protein is partially membrane-associated

• Affinity chromatography:

  • Use Ni-NTA resin with gravity flow or FPLC system

  • Washing steps: Increasing imidazole concentrations (20-50 mM) to remove weakly bound proteins

  • Elution: 250-300 mM imidazole in multiple fractions

  • Analysis of fractions by SDS-PAGE to identify protein-containing fractions

• Secondary purification:

  • Size exclusion chromatography to remove aggregates and further increase purity

  • Ion exchange chromatography if isoelectric point is favorable

  • Affinity tag removal if required for downstream applications

• Quality control:

  • Purity assessment by SDS-PAGE (aim for >90% purity)

  • Western blot confirmation using anti-His antibodies

  • Mass spectrometry verification of protein identity

This protocol typically yields 1-10 mg of purified protein per liter of bacterial culture, with final purity exceeding 90% as determined by SDS-PAGE .

How can researchers troubleshoot common issues in Amet_0954 expression and purification?

When working with recombinant Amet_0954, researchers frequently encounter several challenges. The following troubleshooting strategies address these common issues:

What methods are recommended for analyzing protein-protein interactions involving Amet_0954?

Understanding protein-protein interactions is crucial for elucidating the biological function of poorly characterized proteins like Amet_0954. A comprehensive approach includes:

• In vitro binding assays:

  • Pull-down assays using the His-tagged Amet_0954 as bait

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to analyze complex formation

• Crosslinking strategies:

  • Chemical crosslinking combined with mass spectrometry (XL-MS)

  • Photo-crosslinking with modified amino acids for capturing transient interactions

  • In vivo crosslinking to identify physiologically relevant partners

• Computational predictions:

  • Sequence-based prediction of interaction motifs

  • Structural docking to identify potential binding interfaces

  • Coevolution analysis to identify potentially interacting partners

• Functional validation:

  • Co-immunoprecipitation from native or recombinant systems

  • Bacterial two-hybrid or yeast two-hybrid screening

  • Biolayer interferometry to confirm direct binding

  • FRET or BRET assays for monitoring interactions in real-time

For bacterial proteins like Amet_0954, consideration of the native cellular environment is essential. Interactions should be validated in conditions that mimic the alkaliphilic nature of Alkaliphilus metalliredigens whenever possible.

How does Amet_0954 compare structurally and functionally to other members of the UPF0316 family?

The UPF0316 family remains largely uncharacterized, presenting an opportunity for innovative comparative research:

• Sequence comparison:

  • Multiple sequence alignment of UPF0316 family proteins reveals conserved motifs that might indicate functional sites

  • Phylogenetic analysis to understand evolutionary relationships among family members

  • Identification of species-specific adaptations that might relate to ecological niches

• Structural comparison:

  • Homology modeling based on any available structures in the family

  • Prediction of secondary structure elements and their conservation

  • Identification of potential active sites or binding pockets common to family members

• Functional prediction:

  • Gene neighborhood analysis to identify potential metabolic pathways

  • Co-expression patterns with proteins of known function

  • Presence of recognized domains or motifs that might indicate function

• Experimental validation:

  • Complementation studies in knockout strains

  • Comparative biochemical assays across multiple family members

  • Structural studies to confirm predicted similarities and differences

This comparative approach is particularly valuable for UPF0316 proteins as it can reveal conserved features that may indicate essential biological functions across different bacterial species.

What considerations are important when designing experiments to determine the physiological role of Amet_0954?

Determining the physiological role of a poorly characterized protein like Amet_0954 requires a multi-faceted experimental approach:

• Genetic approaches:

  • Gene knockout or knockdown studies in Alkaliphilus metalliredigens or model organisms

  • Complementation assays to confirm phenotypes

  • Overexpression studies to identify potential gain-of-function effects

  • CRISPR-Cas9 genome editing for precise modifications

• Transcriptomic and proteomic analysis:

  • RNA-Seq to identify co-regulated genes under various conditions

  • Proteomics to identify changes in protein abundance in response to environmental stressors

  • Phosphoproteomics or other post-translational modification analyses if relevant

• Biochemical characterization:

  • Enzymatic activity screening with diverse substrates

  • Metabolite profiling in wild-type versus mutant strains

  • In vitro reconstitution of potential pathways

• Localization studies:

  • Fluorescent protein fusions to determine subcellular localization

  • Immunogold electron microscopy for high-resolution localization

  • Fractionation studies to biochemically determine protein compartmentalization

When designing these experiments, researchers should consider the unique environmental adaptations of Alkaliphilus metalliredigens, particularly its alkaliphilic nature, which may influence protein function and interactions in ways not observed in neutrophilic organisms.

How should researchers interpret contradictory results in Amet_0954 functional studies?

When faced with contradictory results in functional studies of poorly characterized proteins like Amet_0954, a systematic analytical approach is essential:

• Methodological comparison:

  • Evaluate differences in experimental conditions (pH, temperature, buffer composition)

  • Assess protein preparation methods (purification strategy, tag position, presence/absence of tag)

  • Compare detection methods and their sensitivity/specificity

  • Analyze statistical approaches and significance thresholds

• Integrative analysis:

  • Combine results from multiple experimental approaches

  • Weight evidence based on methodological rigor

  • Consider evolutionary context and conservation patterns

  • Integrate structural information with functional data

• Reproducibility assessment:

  • Implement standardized protocols across laboratories

  • Use biological and technical replicates to establish variability

  • Document detailed experimental procedures following best practices in experimental design

• Alternative hypothesis generation:

  • Develop models that might explain seemingly contradictory results

  • Consider context-dependent functionality (environmental factors, binding partners)

  • Examine potential moonlighting functions (multiple distinct roles)

Statistical analysis similar to that used in experimental design studies can be valuable here, with proper attention to interaction effects between experimental conditions (F(1, 1164) = 17.369, p = 0.013) .

What bioinformatic tools and databases are most useful for Amet_0954 research?

A comprehensive bioinformatic analysis of Amet_0954 should utilize multiple tools and databases:

• Sequence analysis tools:

  • BLAST for identifying homologs across species

  • MUSCLE or Clustal Omega for multiple sequence alignments

  • HMMER for profile-based searches

  • SignalP/TMHMM for predicting cellular localization signals

• Structural prediction resources:

  • AlphaFold or RoseTTAFold for ab initio structure prediction

  • SWISS-MODEL for homology modeling

  • PDB for accessing experimentally determined structures of related proteins

  • DSSP for secondary structure prediction

• Functional annotation databases:

  • InterPro for domain and family identification

  • UniProt for curated protein information (Amet_0954 is listed under accession A6TLV6)

  • KEGG for pathway mapping

  • STRING for protein-protein interaction networks

• Specialized resources:

  • CAZy for carbohydrate-active enzymes

  • BRENDA for enzyme information

  • MetaCyc for metabolic pathways

  • SEED for genomic context analysis

When using these resources, researchers should critically evaluate predictions and, where possible, integrate information from multiple sources to develop robust hypotheses for experimental testing.