Recombinant UPF0344 protein BA_1155/GBAA_1155/BAS1072 (BA_1155, GBAA_1155, BAS1072)

What is UPF0344 protein BA_1155/GBAA_1155/BAS1072 and what organism does it originate from?

UPF0344 protein BA_1155/GBAA_1155/BAS1072 is a member of the uncharacterized protein family (UPF) 0344, specifically found in Bacillus anthracis . The protein is classified as "hypothetical" because its exact function remains to be fully characterized through experimental validation . This protein consists of 121 amino acids with a molecular mass of approximately 13.5 kDa . It is encoded by the gene designated as BA_1155 in the Bacillus anthracis genome, with alternative nomenclature including GBAA_1155 and BAS1072 depending on the specific strain and annotation system . The UPF0344 family represents a collection of related proteins found across various bacterial species, with homologs present in other organisms such as Staphylococcus aureus (e.g., SA0830, SaurJH1_0988) .

What is the sequence and predicted structural characteristics of this protein?

The full amino acid sequence of UPF0344 protein BA_1155/GBAA_1155/BAS1072 is: "MVHMHITAWALGLILFFVAYSLYSAGRKGKGVHMGLRLMYIIIIVTGFMLYMGIMKTATSNMHMWYGLKMIAGILVIGGMEMVLVKMSKNKATGAVWGLFIVALVAVFYLGLKLPIGWQVF" . This sequence contains a high proportion of hydrophobic residues, suggesting potential membrane association or integration. Although no experimental structure exists for this specific protein, computational structure prediction methods like AlphaFold have been used to model structures of homologous UPF0344 proteins . For example, the UPF0344 protein SA0830 from Staphylococcus aureus has a computed model with a global pLDDT (predicted Local Distance Difference Test) score of 79.79, indicating relatively confident structural predictions for most regions of the protein . Similar modeling approaches could be applied to BA_1155 to understand its potential three-dimensional organization. The protein sequence analysis suggests multiple transmembrane regions, which corresponds with the predicted structural features seen in homologous proteins.

What expression systems are optimal for recombinant UPF0344 protein BA_1155/GBAA_1155/BAS1072 production?

Several expression systems have been evaluated for recombinant production of UPF0344 protein BA_1155/GBAA_1155/BAS1072, with Escherichia coli and yeast demonstrating the highest yields and shortest production times . The E. coli-based expression system offers advantages of rapid growth, well-established protocols, and cost-effectiveness, making it suitable for initial characterization studies. Yeast expression systems provide eukaryotic processing machinery that can be beneficial if certain post-translational modifications are required . For studies requiring mammalian-like modifications or when protein activity depends on specific glycosylation patterns, insect cells with baculovirus expression systems or mammalian cell expression may be preferable despite their longer production times . Cell-free expression systems have also been utilized to produce this protein with purity levels greater than 85% as determined by SDS-PAGE . The choice of expression system should be guided by research objectives, whether focused on structural studies (where high yield of properly folded protein is crucial) or functional characterization (where retention of native activity is paramount).

How do sequence variations between UPF0344 family proteins impact structure-function relationships?

Sequence alignment analysis of UPF0344 family proteins from various bacterial species reveals conserved regions that likely contribute to core structural elements and critical functional aspects. The UPF0344 proteins found in Bacillus anthracis (BA_1155) and Staphylococcus aureus strains (SA0830, SaurJH1_0988) show significant sequence similarity, suggesting evolutionary conservation of function . Computational structure models of S. aureus UPF0344 proteins demonstrate confidence levels (pLDDT global scores) of approximately 79.76-79.79, indicating reliable prediction for most of the protein structure . Variations in sequence typically occur in loop regions or terminal domains, which might confer species-specific functions or interaction capabilities. When designing experiments to investigate function, researchers should consider these conserved and variable regions to identify potential binding sites, catalytic domains, or protein-protein interaction surfaces. Techniques such as site-directed mutagenesis targeting conserved residues can help determine their contribution to structure stability and functional activity.

What are the challenges in maintaining UPF0344 protein BA_1155/GBAA_1155/BAS1072 stability during purification?

Purification of UPF0344 protein BA_1155/GBAA_1155/BAS1072 presents several challenges related to its apparent membrane-associated nature and hydrophobic characteristics. The high proportion of hydrophobic residues in its sequence suggests potential membrane integration, which can lead to solubility issues when extracted from cellular contexts . Researchers have achieved greater than 85% purity using SDS-PAGE assessment, indicating that purification is feasible but may require optimization . Effective purification strategies typically involve careful selection of detergents for membrane protein extraction, followed by chromatographic techniques such as affinity chromatography (if a tag is incorporated into the recombinant protein), ion exchange, and size exclusion chromatography. Buffer composition significantly impacts protein stability, with considerations for pH, ionic strength, and addition of stabilizing agents such as glycerol or specific metal ions depending on protein characteristics. Temperature management during purification is critical, with many researchers performing steps at 4°C to minimize degradation. Protein aggregation can be monitored through dynamic light scattering or analytical ultracentrifugation during purification development.

What protocols are recommended for expression and purification of correctly folded recombinant UPF0344 protein?

For optimal expression of recombinant UPF0344 protein BA_1155/GBAA_1155/BAS1072, a systematic approach comparing multiple systems is recommended. When using E. coli expression, BL21(DE3) or its derivatives are preferred due to reduced protease activity and compatibility with T7 promoter-based expression vectors . Expression should be optimized by testing various induction conditions, including IPTG concentration (0.1-1.0 mM), temperature (16-37°C), and induction duration (4-24 hours). Lower temperatures (16-25°C) often favor proper folding of membrane-associated proteins. For purification, a multi-step process is typically effective: 1) Cell lysis using methods gentle enough to maintain protein structure, such as sonication in short pulses or pressure-based systems; 2) Primary capture using affinity chromatography if a tag (His6, GST, etc.) is incorporated; 3) Intermediate purification via ion exchange chromatography; 4) Polishing step using size exclusion chromatography to remove aggregates and ensure homogeneity. Throughout purification, protein folding can be monitored using circular dichroism spectroscopy to assess secondary structure content. Thermal shift assays can help identify optimal buffer conditions that maximize stability. For membrane-associated proteins like UPF0344, inclusion of appropriate detergents (e.g., DDM, CHAPS, or Triton X-100) at concentrations above their critical micelle concentration is essential for maintaining solubility without denaturing the protein.

How can researchers verify the function and activity of purified UPF0344 protein?

Verifying the function and activity of UPF0344 protein BA_1155/GBAA_1155/BAS1072 requires a multifaceted approach given its uncharacterized nature. Initial structural integrity assessment through circular dichroism spectroscopy provides information about secondary structure elements, while thermal shift assays can confirm proper folding by measuring denaturation temperatures. For functional characterization, researchers should consider the predicted membrane association and design experiments accordingly. Liposome binding assays can determine if the protein interacts with specific lipid compositions, potentially revealing preferences for particular membrane environments. Protein-protein interaction studies using pull-down assays, co-immunoprecipitation, or yeast two-hybrid systems may identify binding partners that provide functional context. Given the presence of UPF0344 proteins in studies related to antibiotic resistance in Staphylococcus aureus, researchers might investigate whether BA_1155 influences susceptibility to various antibiotics through complementation studies in knockout strains . Electrophysiology techniques could assess potential ion channel or transporter functions if the protein spans the membrane. Crystallization trials or cryo-electron microscopy may provide structural insights to guide functional hypotheses. RNA-sequencing analysis of gene expression changes in response to protein overexpression or deletion can indicate pathways affected by UPF0344 protein activity.

What experimental design considerations are important when studying potential roles of UPF0344 proteins in antibiotic resistance?

When investigating potential roles of UPF0344 proteins in antibiotic resistance, experimental design should account for several critical factors. First, establish genetic manipulation systems in Bacillus anthracis or model organisms to create knockout, knockdown, and overexpression strains of the BA_1155 gene . Antibiotic susceptibility testing should include minimum inhibitory concentration (MIC) determination using standardized methods (broth microdilution or disk diffusion) across a panel of antibiotics representing different classes and mechanisms of action. RNA-sequencing analysis comparing wild-type and mutant strains under antibiotic stress can reveal transcriptional networks associated with the UPF0344 protein . Complementation studies are essential to confirm phenotypes are specifically due to UPF0344 protein function; these should include the wild-type gene and site-directed mutants targeting conserved residues. Time-kill kinetics provide information on how the presence or absence of the protein affects the rate of bacterial death upon antibiotic exposure. Membrane integrity assays using fluorescent dyes can determine if the protein influences cell envelope properties that affect antibiotic penetration. Synergy testing with combinations of antibiotics may uncover specific resistance mechanisms. Protein localization studies using fluorescent fusion proteins can confirm membrane association and potentially identify specific subcellular regions of activity . All experiments should include appropriate controls, including complemented strains and homologous proteins from other species to assess conservation of function.

How should researchers interpret conflicting experimental results when characterizing UPF0344 proteins?

When confronted with conflicting experimental results while characterizing UPF0344 proteins, researchers should systematically evaluate several factors. Expression system differences may significantly impact protein function – results from E. coli-expressed protein might differ from those using yeast or mammalian systems due to variations in post-translational modifications, folding machinery, or membrane composition . Protein tags can influence activity and localization, so comparing tagged and untagged versions or placing tags at different termini is advisable. Buffer composition effects should be examined, as pH, salt concentration, and presence of specific ions or detergents may drastically alter protein behavior. Experimental technique limitations must be considered – for instance, in vitro assays may not recapitulate the native membrane environment necessary for proper function. Species-specific differences might explain divergent results when studying homologs from different organisms like B. anthracis versus S. aureus. Control experiments should be rigorously evaluated, including positive and negative controls appropriate for each assay. Protein concentration effects can be significant, with high concentrations potentially causing aggregation or non-specific interactions that don't occur at physiological levels. Technical replication across multiple independent experiments and biological replication using different protein preparations helps distinguish systematic errors from genuine biological phenomena. Statistical analysis should be applied appropriately to determine if differences are significant or within experimental variation.

What computational tools are most effective for predicting UPF0344 protein structure and function?

Several computational tools prove particularly valuable for predicting structure and function of UPF0344 proteins like BA_1155/GBAA_1155/BAS1072. For structural prediction, AlphaFold has demonstrated high accuracy, producing models of homologous proteins with confidence scores (pLDDT) around 79.76-79.79, indicating reliable prediction for most regions . RoseTTAFold provides complementary structural predictions that can be compared with AlphaFold results to increase confidence. For transmembrane topology prediction, TMHMM, MEMSAT, and Phobius can identify membrane-spanning regions, which is crucial given the likely membrane association of UPF0344 proteins. Functional annotation can be approached through several methods: InterProScan integrates multiple protein signature databases to identify domains and predict function; CATH and SCOP2 classify protein structures into evolutionary and functional groups based on structural similarities; ConSurf identifies evolutionarily conserved residues that may be functionally important by analyzing sequence alignments. Molecular dynamics simulations using GROMACS or NAMD can investigate protein stability and conformational changes in membrane environments. Protein-protein interaction predictions using STRING or STITCH may reveal functional associations with characterized proteins. Coevolution analysis using tools like EVcouplings can identify residue pairs that have coevolved, suggesting structural contacts or functional relationships. These computational approaches should be used in combination, with results from multiple tools compared to build confidence in predictions.

What advanced structural biology techniques are recommended for detailed characterization of UPF0344 protein?

Advanced structural biology techniques offer powerful approaches for detailed characterization of UPF0344 protein BA_1155/GBAA_1155/BAS1072. X-ray crystallography remains a gold standard for high-resolution structure determination, though membrane proteins like UPF0344 present crystallization challenges requiring specialized approaches such as lipidic cubic phase crystallization or the use of crystallization chaperones. Cryo-electron microscopy (cryo-EM) has revolutionized membrane protein structural biology, allowing visualization without crystallization, though the relatively small size of UPF0344 proteins (13.5 kDa) may necessitate strategies like fusion to larger scaffolds or antibody fragments . Nuclear magnetic resonance (NMR) spectroscopy is particularly valuable for investigating dynamics and conformational changes; for membrane proteins, solid-state NMR or solution NMR with detergent micelles or nanodiscs can be employed. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) provides insights into protein dynamics and solvent accessibility, helping identify potential functional regions. Small-angle X-ray scattering (SAXS) offers lower-resolution structural information but can examine protein behavior in solution under various conditions. Cross-linking mass spectrometry (XL-MS) can identify spatial relationships between protein regions and interaction partners. Förster resonance energy transfer (FRET) microscopy allows examination of conformational changes and interactions in real-time, potentially in living cells. Atomic force microscopy (AFM) provides topographical information and mechanical properties, particularly useful for membrane-associated proteins.

How does UPF0344 protein BA_1155/GBAA_1155/BAS1072 compare to homologous proteins in other bacterial species?

The UPF0344 protein family shows remarkable conservation across various bacterial species, with informative similarities and differences. The table below presents a comparative analysis of key characteristics:

Sequence analysis reveals that while the core structural elements remain conserved across species, there are notable variations particularly in the N-terminal and C-terminal regions that may confer species-specific functions . The high confidence scores (pLDDT) for S. aureus homologs suggest these proteins adopt well-defined structures despite being classified as hypothetical . The membrane-associated nature appears consistent across all family members, indicating a likely role in membrane-related processes. The conservation pattern suggests essential cellular functions that have been maintained throughout bacterial evolution, while species-specific variations may reflect adaptation to different ecological niches or physiological requirements. Functional studies in one species could potentially inform understanding of homologs in other bacteria, though validation experiments would be necessary to confirm conservation of function.