LACTB E.Coli, His Active

What is LACTB E.Coli, His Active protein?

LACTB E.Coli, His Active is a recombinant protein produced in E. coli containing 379 amino acids (residues 20-377) with a molecular mass of 41.8kDa. It is expressed with an N-terminal His tag and purified using proprietary chromatographic techniques. LACTB is a member of the penicillin-binding protein and β-lactamase superfamily, which has been validated in vivo to play a role in obesity development .

What is the evolutionary origin of LACTB?

LACTB and its eukaryotic homologs are closely related to low molecular weight penicillin-binding proteins class B from the β-proteobacterial division. This evolutionary relationship provides insight into the mitochondrial origins and suggests that LACTB shares structural and functional features with bacterial enzymes involved in peptidoglycan synthesis and maintenance .

Where is LACTB localized in cells?

LACTB is a soluble protein localized specifically in the mitochondrial intermembrane space. Research has shown that the LACTB preprotein undergoes proteolytic processing, revealing an N-terminal tetrapeptide motif that is also found in a set of apoptosis-inducing proteins. This localization is critical for understanding its physiological function in mitochondrial organization and metabolism regulation .

What expression systems are optimal for producing functional LACTB?

E. coli expression systems have been successfully used to produce recombinant LACTB with proper folding and activity. Specifically, N-terminal fusion tags such as GST and His have yielded significant quantities of properly folded LACTB protein. When designing expression constructs, it's important to consider that the protein contains 379 amino acids (residues 20-377) and requires appropriate folding conditions to maintain its active conformation .

What purification strategies yield high-purity LACTB preparations?

High-purity LACTB preparations can be achieved using affinity chromatography targeting the His-tag, followed by additional proprietary chromatographic techniques. When designing a purification protocol, researchers should consider that LACTB is a soluble protein with specific folding requirements. Sequential purification steps may be necessary to remove contaminating proteins and ensure the final preparation contains functional protein suitable for downstream applications .

How can researchers verify the proper folding and activity of purified LACTB?

Verification of properly folded LACTB can be assessed through multiple approaches including circular dichroism spectroscopy to evaluate secondary structure elements, thermal shift assays to assess protein stability, and functional activity assays based on its predicted enzymatic activity. Previous research has successfully employed these methods to confirm that GST-LACTB fusion protein expressed in E. coli was properly folded and retained its structural integrity .

How can researchers study LACTB's role in mitochondrial organization?

To study LACTB's role in mitochondrial organization, researchers can employ fluorescence microscopy with tagged LACTB constructs to visualize its polymerization into filaments within the mitochondrial intermembrane space. Electron microscopy has successfully revealed that LACTB forms stable filaments ranging from twenty to several hundred nanometers in length. These filaments define a distinct microdomain in the intermembrane space, suggesting a role in compartmentalization .

What experimental approaches can determine LACTB's involvement in obesity pathways?

To investigate LACTB's involvement in obesity pathways, researchers have utilized transgenic mouse models with altered LACTB expression. Gene expression profiling of these models has revealed associations with metabolic pathways. Additional approaches include analyzing LACTB expression in response to insulin stimulation in skeletal muscle, as research has shown that insulin increases LACTB mRNA expression. Cell-based assays measuring metabolic parameters after LACTB modulation can also provide insights into its metabolic functions .

How should experiments be designed to study LACTB's enzymatic activity?

Experimental designs to study LACTB's enzymatic activity should consider its relationship to the penicillin-binding protein and β-lactamase superfamily. Assays should incorporate appropriate substrates based on this relationship and monitor product formation under varying conditions. Researchers should also consider that LACTB is an active-site serine enzyme, which may inform the selection of enzyme inhibitors or activators. Control experiments with mutated active site residues can help confirm the specificity of observed enzymatic activities .

How does LACTB polymerization affect its function in mitochondrial microcompartmentalization?

LACTB's polymerization into filaments creates physical structures that potentially compartmentalize the mitochondrial intermembrane space. Research investigating this phenomenon should consider how these filaments interact with other mitochondrial proteins and membranes. Advanced imaging techniques such as super-resolution microscopy combined with proximity labeling approaches can help map the molecular neighborhood of LACTB filaments. Understanding the regulation of LACTB polymerization and its reversibility under various physiological conditions would provide insight into its dynamic role in organizing mitochondrial subcompartments .

What is the molecular mechanism linking LACTB to obesity and metabolic regulation?

The molecular mechanism connecting LACTB to obesity involves complex metabolic pathways. Researchers exploring this link should consider designing experiments that analyze changes in lipid metabolism, energy expenditure, and mitochondrial function in response to LACTB modulation. Metabolomic analyses of tissues with varying LACTB expression levels can identify affected metabolic pathways. Integration of transcriptomic and proteomic data from models with altered LACTB expression may help construct a comprehensive model of how LACTB influences metabolic regulation and contributes to obesity development .

How does LACTB's response to immune challenges inform its evolutionary role?

Studies have shown that LACTB is modulated by immune responses triggered by viral or fungal infections. Research investigating this aspect should examine the signaling pathways connecting immune activation to LACTB regulation and the functional consequences of this regulation. Comparative analyses across species can provide evolutionary insights into how LACTB's immune-responsive characteristics may relate to its ancestral bacterial functions. This research direction could reveal novel connections between immunity, metabolism, and mitochondrial organization .

What strategies can overcome challenges in expressing soluble, active LACTB?

Researchers facing difficulties with LACTB expression should consider optimizing expression conditions including temperature, induction parameters, and host strain selection. Lower expression temperatures (16-20°C) often improve protein folding for complex proteins. Fusion partners beyond His-tags, such as GST or MBP, may enhance solubility. Previous research successfully used GST-LACTB fusion constructs to recover significant quantities of properly folded protein. Co-expression with chaperones may also facilitate correct folding of LACTB during recombinant expression .

How can researchers distinguish between direct and indirect effects of LACTB in complex physiological systems?

To distinguish direct from indirect effects of LACTB, researchers should implement multiple complementary approaches. Acute versus chronic modulation of LACTB activity can help separate primary from secondary effects. In vitro reconstitution experiments with purified components can establish direct biochemical activities. Proximity-based labeling approaches can identify direct interaction partners of LACTB in its native mitochondrial environment. Additionally, time-course experiments following LACTB activation or inhibition can help establish the sequence of events and separate causal relationships from downstream consequences .

What analytical methods are most appropriate for studying LACTB filament formation?

LACTB filament formation can be studied using a combination of microscopy and biochemical approaches. Electron microscopy has successfully revealed LACTB filaments ranging from twenty to several hundred nanometers. Light scattering techniques can monitor polymerization kinetics in solution. For in situ studies, super-resolution microscopy with appropriately tagged LACTB constructs can visualize filament dynamics in living cells. Biochemical fractionation approaches can isolate polymerized versus monomeric LACTB from mitochondrial preparations for subsequent analysis. These methods should be selected based on the specific research question regarding filament structure, dynamics, or function .

How should researchers interpret contradictory findings regarding LACTB function across different experimental systems?

When faced with contradictory findings, researchers should conduct a systematic comparison of experimental conditions, including organism models, cell types, LACTB expression levels, and detection methods. Physiological context matters significantly for LACTB function, as its role in metabolic regulation may vary depending on tissue type and metabolic state. Researchers should consider that LACTB's expression is higher in tissues with high metabolic rates, suggesting context-dependent functions. Integration of results across multiple experimental approaches, including in vitro biochemical assays, cell culture models, and in vivo systems, provides the most comprehensive understanding of LACTB's multifaceted functions .

What statistical approaches are appropriate for analyzing LACTB's effects on complex metabolic pathways?

Complex metabolic effects of LACTB require sophisticated statistical approaches. Multivariate analyses such as principal component analysis or partial least squares discriminant analysis can identify patterns in metabolomic or transcriptomic datasets. Time-series analyses may be necessary to capture dynamic metabolic responses. Power calculations should account for biological variability in metabolic parameters, often requiring larger sample sizes than standard biochemical assays. Pathway enrichment analyses can help identify metabolic networks affected by LACTB modulation, providing a systems-level understanding of its function rather than focusing on individual metabolites or genes .

How can researchers correlate LACTB structure with its diverse cellular functions?

Structure-function correlations for LACTB require integration of structural biology data with functional assays. Researchers have created structural models indicating that LACTB shares characteristic features with penicillin-binding proteins and β-lactamases. Mutational analyses targeting specific domains or the active site serine residue can define regions essential for different LACTB functions. Researchers should consider that LACTB's ability to polymerize into filaments represents a structural feature with important functional implications. Cross-linking studies combined with mass spectrometry can identify regions involved in filament formation versus enzymatic activity, helping to dissect LACTB's multiple functional roles .