Recombinant Anabaena variabilis UPF0754 membrane protein Ava_1421 (Ava_1421)

What is Anabaena variabilis UPF0754 membrane protein Ava_1421?

Anabaena variabilis UPF0754 membrane protein Ava_1421 is a full-length protein (411 amino acids) from the cyanobacterium Anabaena variabilis. The protein is identified by UniProt ID Q3MD92 and is classified as a membrane protein within the UPF0754 family. The recombinant version is typically expressed in E. coli with an N-terminal His tag to facilitate purification and characterization. The complete amino acid sequence begins with MDWSHLWLYVSPPI and continues through the full 411-amino acid sequence .

What expression systems are most effective for Ava_1421 production?

E. coli expression systems are primarily used for recombinant Ava_1421 production. Based on optimization studies with other Anabaena variabilis proteins, the E. coli BL21(DE3) strain combined with the pET expression system (such as pET28a) has proven effective. For membrane proteins like Ava_1421, expression conditions typically need to be carefully optimized, including lower temperatures (25-30°C), moderate inducer concentrations (0.5 mM IPTG), and specific media formulations like Terrific Broth (TB) that can increase protein yield and activity compared to standard LB media .

What are the recommended storage conditions for recombinant Ava_1421?

Recombinant Ava_1421 should be stored at -20°C to -80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles. Working aliquots can be stored at 4°C for up to one week. The protein is typically stored in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0. For long-term storage, addition of 5-50% glycerol (final concentration) is recommended before aliquoting. The standard recommended final concentration of glycerol is 50% .

What is the recommended procedure for reconstituting lyophilized Ava_1421?

For optimal reconstitution of lyophilized Ava_1421, briefly centrifuge the vial prior to opening to bring the contents to the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. To enhance stability, add glycerol to a final concentration of 5-50% (with 50% being standard) and aliquot for long-term storage at -20°C/-80°C. Avoid repeated freeze-thaw cycles as this can significantly reduce protein activity and stability .

How can I verify the purity and integrity of recombinant Ava_1421?

The purity of recombinant Ava_1421 can be verified using SDS-PAGE, with commercial preparations typically showing greater than 90% purity. For a more comprehensive analysis, researchers should consider:

• Western blotting using anti-His tag antibodies to confirm identity

• Size exclusion chromatography to assess oligomeric state and aggregation

• Mass spectrometry to verify molecular weight and potential post-translational modifications

• Circular dichroism (CD) spectroscopy to evaluate secondary structure integrity

• Dynamic light scattering to assess homogeneity

For membrane proteins like Ava_1421, additional assays to verify proper folding may be necessary, particularly if functional studies are planned.

How can I optimize IPTG concentration for maximum soluble expression of Ava_1421?

To optimize IPTG concentration for maximal soluble expression of Ava_1421, employ a systematic approach testing multiple concentrations (0.1, 0.5, and 1.0 mM IPTG) while maintaining other parameters constant. Based on studies with other Anabaena variabilis proteins, lower IPTG concentrations (around 0.5 mM) often yield higher specific activity despite potentially lower total protein yields. This is likely due to improved protein folding at slower induction rates .

Methodology:

• Inoculate expression cultures from overnight starter cultures

• Grow cultures to mid-log phase (OD600 ~ 0.6-0.8)

• Add different IPTG concentrations to parallel cultures

• Collect samples at fixed time points (3, 6, and 18 hours)

• Analyze both total protein expression by SDS-PAGE and specific activity through functional assays

• Calculate specific activity (μmol/min/mg) for each condition

For Anabaena variabilis proteins, maximum protein quantity often occurs at higher IPTG concentrations (1 mM), but maximum specific activity typically occurs at moderate concentrations (0.5 mM) .

What culture media composition yields optimal expression of correctly folded Ava_1421?

Terrific Broth (TB) typically yields higher specific activity for Anabaena variabilis recombinant proteins compared to standard LB media. For Ava_1421, as a membrane protein, consider the following media optimization methodology:

• Compare minimal media (M9), standard media (LB), and enriched media (TB, 2xYT)

• Supplement with additives that can enhance membrane protein folding:

  • Glycerol (0.5-1%) to stabilize membrane proteins

  • Specific ions (Mg²⁺, Ca²⁺) that may assist in protein folding

  • Mild detergents at sub-CMC concentrations

• Test the addition of rare codon supplements or using Rosetta strains for expression

• Analyze both yield and activity metrics for each condition

For other Anabaena variabilis proteins, TB media has shown approximately 22% higher specific activity compared to LB media (1.65±0.1 vs. 1.35±0.1 μmol/min/mg) .

What temperature and induction duration parameters maximize active Ava_1421 yield?

For membrane proteins like Ava_1421, lower expression temperatures often produce more correctly folded protein despite lower total expression. Based on optimization studies with other Anabaena variabilis proteins, consider the following methodology:

• Test multiple temperatures (25°C, 30°C, and 37°C)

• Evaluate different induction periods (3, 6, and 18 hours)

• Analyze both total protein yield by SDS-PAGE and functional activity

• Create a temperature-time matrix to identify optimal combinations

Research with similar Anabaena variabilis proteins suggests that while highest band intensity on SDS-PAGE may be observed at 37°C, the specific activity is often significantly higher at 25°C. For longer induction times (18 hours), protein expressed at 37°C shows decreased specific activity, likely due to aggregation, while protein expressed at 25°C maintains high activity even after extended induction .

How can I determine the membrane topology of Ava_1421?

Determining the membrane topology of Ava_1421 requires a multi-technique approach:

• Computational prediction: Use algorithms like TMHMM, Phobius, or TOPCONS to predict transmembrane regions and orientation.

• Cysteine scanning mutagenesis:

  • Generate mutants with single cysteine residues at various positions

  • Label with membrane-impermeable and membrane-permeable thiol-reactive reagents

  • Analyze labeling patterns to determine which regions are accessible from which side of the membrane

• Fusion protein approach:

  • Create fusion constructs with reporter proteins (GFP, PhoA, LacZ) at various positions

  • Analyze reporter activity to determine cytoplasmic or periplasmic localization of different regions

• Protease protection assays:

  • Express Ava_1421 in a membrane system

  • Treat with proteases in the presence or absence of membrane permeabilization

  • Analyze protected fragments by immunoblotting to identify regions shielded by the membrane

• Cryo-EM or X-ray crystallography:

  • For definitive structure determination if high-resolution analysis is desired

The amino acid sequence provided (MDWSHLWLYVSPPILGGIIGYFTNDIAIKMLFRPYRAIYIGGRRVPFTPGLIPRNQERLA...) suggests hydrophobic regions consistent with transmembrane domains, which should be experimentally verified .

What strategies can overcome expression challenges for Ava_1421 as a membrane protein?

Membrane proteins like Ava_1421 present unique expression challenges that can be addressed through multiple strategies:

• Fusion tags optimization:

  • Test N-terminal vs. C-terminal His-tags

  • Evaluate alternative tags (MBP, SUMO, Trx) that can enhance solubility

  • Consider dual tagging strategies for improved purification

• Strain engineering:

  • Use C41(DE3) or C43(DE3) strains specifically designed for membrane protein expression

  • Consider strains with altered membrane compositions or additional chaperones

• Induction strategies:

  • Implement auto-induction media to avoid IPTG shock

  • Use lower temperatures (16-25°C) and longer expression times (18-24 hours)

  • Test stepped temperature protocols (initial growth at 37°C, then shift to 16-25°C upon induction)

• Stabilization approaches:

  • Add specific lipids or mild detergents to the culture medium

  • Include chemical chaperones like glycerol (5-10%) or specific ions

• Alternative expression systems:

  • Consider cell-free expression systems with added lipids/nanodiscs

  • Evaluate eukaryotic systems (yeast, insect cells) for complex membrane proteins

Based on optimization of other Anabaena variabilis proteins, moderate shaking speeds (150 rpm) combined with longer induction times at lower temperatures (25°C for 18 hours) using 0.5 mM IPTG in TB media would be a recommended starting point .

How can I assess the structural integrity of purified Ava_1421?

Assessing the structural integrity of purified Ava_1421 requires multiple complementary approaches:

• Circular Dichroism (CD) Spectroscopy:

  • Far-UV CD (190-260 nm) to analyze secondary structure content

  • Near-UV CD (250-350 nm) to evaluate tertiary structure fingerprint

  • Thermal denaturation monitored by CD to assess stability

• Fluorescence Spectroscopy:

  • Intrinsic tryptophan fluorescence to monitor tertiary structure

  • ANS binding to detect exposed hydrophobic patches

  • FRET-based approaches if multiple fluorophores are introduced

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

  • Determine oligomeric state

  • Detect aggregation or fragmentation

  • Assess homogeneity of the preparation

• Differential Scanning Calorimetry/Fluorimetry:

  • Measure thermal stability (Tm)

  • Evaluate effects of different buffers and additives on stability

• Protease Resistance Assay:

  • Limited proteolysis to assess compact folding

  • Compare digestion patterns with denatured controls

For membrane proteins like Ava_1421, additional considerations include reconstitution into appropriate membrane mimetics (detergent micelles, nanodiscs, or liposomes) before structural assessment to ensure native-like conformation.

What membrane mimetic systems are most suitable for functional studies of Ava_1421?

Selecting appropriate membrane mimetic systems for Ava_1421 functional studies requires systematic evaluation:

• Detergent Screening:

  • Mild non-ionic detergents (DDM, LMNG, C12E8)

  • Zwitterionic detergents (CHAPSO, Fos-choline)

  • Comparison matrix evaluating protein stability, homogeneity, and activity

• Nanodiscs:

  • MSP1D1 or MSP1E3D1 scaffold proteins

  • Lipid composition optimization (POPC, POPE, POPG mixtures)

  • Testing various protein:MSP:lipid ratios

• Liposomes:

  • Composition matched to native Anabaena variabilis membranes

  • Size control through extrusion

  • Various reconstitution methodologies (detergent dialysis, direct incorporation)

• Amphipols:

  • A8-35 or PMAL-C8

  • Transition from detergent to amphipol

• Bicelles:

  • DMPC/CHAPSO mixtures

  • Various q-ratios (lipid:detergent)

Each system should be evaluated using:

• Protein stability over time (monitored by activity or structural methods)

• Functional assays specific to UPF0754 family proteins

• Homogeneity assessment by DLS or analytical ultracentrifugation

• Structural integrity verification by CD or fluorescence

For initial studies, DDM or LMNG detergents often provide a good starting point for membrane protein stabilization while maintaining functional properties.

How does Ava_1421 compare structurally and functionally to other UPF0754 family proteins?

Comparative analysis of Ava_1421 with other UPF0754 family proteins requires a multi-faceted approach:

• Sequence Alignment Analysis:

  • Multiple sequence alignment of UPF0754 family members

  • Identification of conserved motifs and variable regions

  • Phylogenetic analysis to establish evolutionary relationships

• Structural Comparison:

  • Homology modeling based on related structures

  • Secondary structure prediction comparison

  • Hydropathy profile analysis to compare transmembrane topology

• Functional Domain Mapping:

  • Identification of conserved functional residues

  • Comparison of predicted binding sites or active centers

  • Analysis of post-translational modification sites

• Expression Pattern Comparison:

  • Analysis of gene expression data across different conditions

  • Comparison of regulation mechanisms

  • Co-expression network analysis

• Experimental Verification:

  • Mutagenesis of conserved residues to test functional hypotheses

  • Cross-complementation studies in model organisms

  • Comparative biochemical characterization

The 411-amino acid sequence of Ava_1421 contains characteristic hydrophobic regions typical of membrane proteins, and comparative analysis with other UPF0754 family members would help elucidate its specific role within Anabaena variabilis .

What approaches can be used to identify potential interaction partners of Ava_1421?

Identifying interaction partners of Ava_1421 requires multiple complementary approaches:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Express His-tagged Ava_1421 in native or heterologous systems

  • Perform crosslinking to capture transient interactions

  • Affinity purify protein complexes

  • Identify partners by LC-MS/MS

  • Validate with reciprocal pulldowns

• Yeast Two-Hybrid (Y2H) or Membrane Y2H:

  • Create bait constructs with Ava_1421 or domain fragments

  • Screen against cDNA libraries from Anabaena variabilis

  • Validate positive interactions through secondary screens

• Proximity Labeling:

  • Fuse Ava_1421 to BioID or APEX2

  • Express in appropriate system

  • Identify labeled proteins by streptavidin pulldown and MS

• Co-immunoprecipitation with Targeted Antibodies:

  • Generate antibodies against Ava_1421

  • Perform IP from native membranes

  • Identify co-precipitated proteins

• Computational Prediction:

  • Use protein-protein interaction databases

  • Apply co-evolution analysis

  • Perform structural docking with candidate partners

• Functional Screening:

  • Genetic interaction mapping through synthetic lethality screening

  • Suppressor screens to identify functional relationships

For membrane proteins like Ava_1421, special consideration must be given to maintaining the native membrane environment during interaction studies, possibly using membrane-mimetic systems or in-membrane approaches.

How can I address low solubility issues when expressing recombinant Ava_1421?

Addressing low solubility of recombinant Ava_1421 requires a systematic troubleshooting approach:

• Expression Condition Optimization:

  • Reduce temperature to 16-25°C during induction

  • Lower IPTG concentration to 0.1-0.5 mM

  • Use TB media instead of LB for better expression

  • Test induction at different cell densities (OD600 0.4-0.8)

  • Reduce shaking speed to 150 rpm to minimize stress

• Vector and Construct Modifications:

  • Test different fusion tags (MBP, SUMO, Trx) known to enhance solubility

  • Create truncated constructs removing flexible or aggregation-prone regions

  • Optimize codon usage for E. coli

  • Include purification tags at both N- and C-termini

• Host Strain Selection:

  • Use specialized strains like C41(DE3) or C43(DE3) for membrane proteins

  • Try Rosetta strains for rare codon optimization

  • Test SHuffle strains if disulfide bonds are present

• Solubilization Strategies:

  • Screen different detergents (DDM, LMNG, LDAO, etc.)

  • Test various detergent concentrations (1-5x CMC)

  • Include stabilizing additives (glycerol, specific salts, arginine)

  • Optimize pH and ionic strength

Based on optimization studies with other Anabaena variabilis proteins, expressing at 25°C for 18 hours with 0.5 mM IPTG in TB media at 150 rpm shaking speed has shown significant improvements in soluble protein yield and activity .

What methods can distinguish between properly folded and misfolded Ava_1421?

Distinguishing between properly folded and misfolded Ava_1421 requires multiple analytical approaches:

• Thermal Stability Assays:

  • Differential Scanning Fluorimetry (DSF) using SYPRO Orange

  • Circular Dichroism thermal melt curves

  • Comparative melting temperatures between different preparations

• Limited Proteolysis:

  • Controlled digestion with proteases (trypsin, chymotrypsin)

  • Analysis of digestion patterns by SDS-PAGE

  • Properly folded protein shows resistance to proteolytic degradation

• Size Exclusion Chromatography:

  • Analysis of elution profiles

  • Comparison with known standards

  • Detection of aggregates or oligomeric states

• Intrinsic Fluorescence Spectroscopy:

  • Monitoring tryptophan fluorescence spectra

  • Comparison with denatured controls

  • Red-shift in emission maximum indicates exposed tryptophans in misfolded protein

• Activity Assays:

  • Development of functional assays specific to Ava_1421

  • Correlation between activity and other folding metrics

• Detergent Binding:

  • Analysis of detergent:protein ratio by analytical ultracentrifugation

  • Abnormal detergent binding can indicate misfolding

For membrane proteins like Ava_1421, proper folding is often critically dependent on the lipid or detergent environment, making comparative studies in different membrane mimetics particularly valuable.

What are effective strategies for site-directed mutagenesis of Ava_1421 to investigate structure-function relationships?

Effective site-directed mutagenesis strategies for Ava_1421 structure-function studies include:

• Target Selection Approach:

  • Conserved residues identified through multiple sequence alignments

  • Predicted functional sites from homology models

  • Charged or polar residues within transmembrane regions

  • Interface residues if oligomerization is suspected

• Mutation Design Principles:

  • Conservative substitutions (e.g., Leu→Ile, Asp→Glu) to test importance of specific properties

  • Alanine scanning of specific regions to identify essential residues

  • Charge reversals to test electrostatic interactions

  • Cysteine substitutions for accessibility studies or crosslinking

• Technical Implementation:

  • QuikChange or Q5 site-directed mutagenesis for single mutations

  • Gibson Assembly for multiple simultaneous mutations

  • Golden Gate Assembly for systematic mutation libraries

  • Codon optimization in primer design for improved expression

• Validation Pipeline:

  • Expression level comparison with wild-type

  • Thermal stability assessment

  • Structural integrity verification by CD or fluorescence

  • Functional assays relevant to UPF0754 family proteins

• Analysis Framework:

  • Systematic comparison of mutant properties in tabular format

  • Structure-based visualization of mutation effects

  • Correlation analysis between different measured parameters

For Ava_1421, with its 411-amino acid sequence, initial focus might be on conserved residues within predicted transmembrane regions or at interfaces between transmembrane helices .

How can molecular dynamics simulations contribute to understanding Ava_1421 membrane interactions?

Molecular dynamics (MD) simulations offer powerful insights into Ava_1421 membrane interactions through the following methodological approach:

• System Preparation:

  • Generate homology model of Ava_1421 based on related structures

  • Create diverse membrane compositions representing potential native environments

  • Prepare protein-membrane systems with appropriate hydration and ion concentrations

• Simulation Framework:

  • Equilibration protocol with gradual restraint release

  • Production runs of 100-500 ns for basic interactions

  • Microsecond-scale simulations for conformational dynamics

  • Enhanced sampling techniques (umbrella sampling, metadynamics) for energy landscapes

• Analysis Dimensions:

  • Protein-lipid interaction patterns and residence times

  • Membrane deformation near the protein

  • Hydrophobic matching between protein and bilayer

  • Water and ion penetration patterns

  • Protein conformational dynamics in membrane context

• Comparison Strategy:

  • Wild-type vs. mutant simulations

  • Different lipid compositions

  • Various membrane mimetic systems

• Integration with Experiments:

  • Validation of simulation findings with experimental approaches

  • Design of new experiments based on simulation insights

  • Iterative refinement of models based on experimental feedback

Given the membrane protein nature of Ava_1421 with its predicted transmembrane regions, MD simulations are particularly valuable for understanding how it positions within the membrane, potential conformational changes, and specific lipid interactions that may be crucial for function.

What emerging technologies could advance our understanding of Ava_1421 structure and function?

Several emerging technologies hold promise for advancing Ava_1421 research:

• Cryo-Electron Microscopy:

  • High-resolution structural determination of membrane proteins without crystallization

  • Single-particle analysis for structure in detergent micelles or nanodiscs

  • Tomography for in situ visualization

• Integrative Structural Biology:

  • Combining data from multiple experimental techniques (SAXS, NMR, FRET, crosslinking-MS)

  • Computational integration to generate comprehensive structural models

  • Validation through orthogonal methods

• AlphaFold2 and Deep Learning Approaches:

  • AI-based structure prediction specifically optimized for membrane proteins

  • Improved modeling of protein-lipid interactions

  • Functional site prediction through deep learning

• Single-Molecule Techniques:

  • Fluorescence-based approaches to monitor conformational dynamics

  • Force spectroscopy to probe mechanical properties

  • Real-time monitoring of functional cycles

• Native Mass Spectrometry:

  • Analysis of intact membrane protein complexes

  • Determination of lipid binding specificity

  • Characterization of post-translational modifications

• In-Cell Structural Biology:

  • FRET-based sensors for conformational changes in living cells

  • Genetic code expansion for site-specific probes

  • Correlative light and electron microscopy

These technologies could provide unprecedented insights into Ava_1421's native structure, dynamic behavior, and functional mechanisms beyond what conventional approaches have revealed.

How can comparative genomics inform functional hypotheses about Ava_1421?

Comparative genomics provides powerful frameworks for generating functional hypotheses about Ava_1421:

• Phylogenetic Profiling:

  • Analysis of UPF0754 protein distribution across species

  • Correlation with specific metabolic capabilities or environmental adaptations

  • Identification of co-evolving gene families

• Synteny Analysis:

  • Examination of gene neighborhood conservation

  • Identification of consistently co-localized genes suggesting functional relationships

  • Operon structure analysis in prokaryotic genomes

• Gene Expression Correlation:

  • Meta-analysis of transcriptomic data across conditions

  • Identification of genes with similar expression patterns

  • Regulatory network reconstruction

• Genetic Association Analysis:

  • Identification of genetic variants associated with phenotypic differences

  • Analysis of selective pressure on different protein domains

  • Detection of horizontal gene transfer events

• Methodological Implementation:

  • Database integration (KEGG, STRING, UniProt)

  • Custom bioinformatic pipeline development

  • Machine learning approaches to predict functional relationships

For Ava_1421, comparative genomics could reveal if this membrane protein is associated with specific cyanobacterial adaptations, stress responses, or metabolic pathways, informing targeted experimental designs to test these hypotheses.

What are the key considerations for ensuring reproducibility in Ava_1421 research?

Ensuring reproducibility in Ava_1421 research requires attention to multiple dimensions:

• Detailed Methodological Reporting:

  • Complete experimental protocols with all parameters specified

  • Exact buffer compositions including pH and additives

  • Temperature, time, and other environmental factors

  • Specific reagent sources and catalog numbers

  • Instrument settings and calibration procedures

• Expression and Purification Documentation:

  • Precise genetic construct sequences including all tags

  • Expression strain genotypes

  • Detailed purification protocol with column types and flow rates

  • Quality control metrics for each preparation

  • Storage conditions and stability data

• Data Management Practices:

  • Raw data preservation in non-proprietary formats

  • Clear data processing workflows with version-controlled code

  • Separation of raw data from analysis results

  • Comprehensive metadata capture

• Validation Approaches:

  • Multiple independent protein preparations

  • Replicate experiments with statistical analysis

  • Multiple complementary techniques for key findings

  • Positive and negative controls for all assays

• Open Science Practices:

  • Deposition of sequences in public databases

  • Sharing of protocols on platforms like protocols.io

  • Consideration of pre-registration for hypothesis-driven studies

  • Data availability statements in publications

For membrane proteins like Ava_1421, special attention to detergent/lipid compositions and membrane mimetic systems is critical for reproducibility, as small changes can significantly affect protein behavior.

How should researchers address potential contamination issues in Ava_1421 preparations?

Addressing contamination in Ava_1421 preparations requires systematic prevention and detection:

• Preventive Measures:

  • Dedicated equipment and reagents for recombinant protein work

  • Sterile technique for all buffer preparations

  • Regular cleaning of chromatography systems

  • Autoclaved or filter-sterilized buffers

  • Use of protease inhibitors throughout purification

• Detection Methodology:

  • High-resolution SDS-PAGE with silver staining

  • Western blotting with specific antibodies

  • Mass spectrometry for protein identification

  • Endotoxin testing for preparations intended for biological assays

  • Microbial contamination tests for long-term storage

• Quantitative Assessment:

  • Densitometry analysis of SDS-PAGE gels

  • Calculation of specific activity ratios

  • Statistical comparison between different preparations

• Purification Optimization:

  • Multi-step purification strategies (IMAC followed by SEC or ion exchange)

  • Selective precipitation steps

  • Detergent exchange protocols for membrane proteins

  • On-column washing optimization

• Quality Control Standards:

  • Establishment of acceptance criteria for purity

  • Regular validation of purification protocols

  • Benchmarking against reference preparations