Recombinant Acinetobacter sp. UPF0339 protein ACIAD0721 (ACIAD0721)

What is ACIAD0721 and from which organism is it derived?

ACIAD0721 is a protein belonging to the UPF0339 family, specifically from the Duplicated subfamily. It is derived from Acinetobacter baylyi (strain ATCC 33305 / BD413 / ADP1), a Gram-negative bacterium known for its natural competence and transformation capabilities . This specific protein has a length of 111 amino acids and a molecular mass of approximately 12.2 kDa . The protein is considered part of the small proteins family in Acinetobacter sp. ADP1, a versatile bacterium that has gained significant attention as a model organism for genetic analysis and genome engineering .

Why is Acinetobacter sp. ADP1 considered an ideal model organism for recombinant protein studies?

Acinetobacter sp. ADP1 possesses two key characteristics that make it an exceptional model organism:

• Natural competence for DNA uptake, which extends to both plasmid DNA and linear fragments .

• Strong natural tendency towards homology-directed recombination .

These properties allow genetic manipulation by simple addition of linear PCR products to small volumes of growing cell culture, followed by incubation and plating on selective media . Importantly, ADP1 is approximately 10–100 times as competent as calcium chloride-treated E. coli, eliminating the need for complex transformation protocols . Additionally, the close relationship between E. coli and ADP1, combined with the available whole-genome sequence of ADP1, allows the application of existing knowledge about gene function and metabolism from E. coli studies to ADP1 .

What expression systems are recommended for recombinant production of ACIAD0721?

For recombinant expression of ACIAD0721, a prokaryotic expression system using E. coli BL21(DE3) with the pET-28a(+) vector is recommended based on successful expression strategies for similar proteins . This approach involves:

• Cloning the gene segment encoding the mature peptide part of ACIAD0721 based on designed primers

• Constructing the recombinant prokaryotic expression plasmid using pET-28a(+) vector

• Transforming the construct into competent BL21(DE3) cells

• Expression induction using IPTG under optimized conditions

• Purification via affinity chromatography using a Ni-NTA column to obtain the recombinant protein with a His-tag sequence

This expression system has been successfully used for similar proteins from Acinetobacter species and allows for high-yield production of soluble recombinant protein .

How should experimental controls be designed for studies involving recombinant ACIAD0721?

When designing experiments with recombinant ACIAD0721, robust controls are essential to ensure reliable results. The experimental design should include:

• Negative controls:

  • Empty vector-transformed E. coli BL21(DE3) cells processed identically to the recombinant strain

  • Purification from non-induced cultures to account for leaky expression

  • Buffer-only controls for activity assays

• Positive controls:

  • Well-characterized proteins from the same family or with similar predicted functions

  • Commercial enzymes with relevant activities for comparative analysis

• Validation controls:

  • Western blotting with anti-His antibodies to confirm expression and purification

  • Mass spectrometry analysis to verify protein identity and integrity

The experimental design should follow randomized controlled double-blind principles where applicable to eliminate human bias, with treatments and controls randomly assigned to minimize systematic errors . This approach is particularly important when testing potential functions or activities of ACIAD0721, as its precise biological role remains to be fully characterized.

What purification strategies yield the highest purity for recombinant ACIAD0721?

For optimal purification of recombinant ACIAD0721, a multi-step purification strategy is recommended:

• Initial capture: Ni-NTA affinity chromatography utilizing the His-tag incorporated into the recombinant construct is the primary purification step. Optimization of imidazole concentration in washing and elution buffers is crucial for high purity .

• Secondary purification: Size exclusion chromatography (SEC) to separate the target protein from aggregates and contaminating proteins of different molecular weights.

• Optional polishing step: Ion exchange chromatography can be employed if higher purity is required.

The purification protocol should be optimized to achieve >70% purity, which is sufficient for most functional studies . Key factors affecting purification efficiency include:

Monitoring purification success using SDS-PAGE at each step is essential, with Western blotting as a confirmatory technique .

What computational approaches can be used to predict the structure of ACIAD0721?

Several complementary computational approaches can be employed to predict the structure of ACIAD0721:

• Homology modeling: Using structures of proteins with high sequence similarity as templates. For UPF0339 family proteins, available crystal structures from related bacteria can serve as templates .

• Threading methods: This approach is particularly useful when sequence identity with known structures is low (<30%). The threading score (Z) is evaluated against randomly reshuffled sequences to determine the probability of structural similarity .

• Direct Coupling Analysis (DCA): This method leverages evolutionary covariation of amino acids to predict residue-residue interactions. Since substitution of one interacting amino acid would impact another, covariation patterns can reveal potential 3D interactions .

• Machine learning approaches: Recent breakthroughs in computational structure prediction have employed machine learning algorithms that combine multiple sources of information .

• Experimentally-driven structure modeling: Integrating experimental data such as limited proteolysis, surface exposure mapping, or distance constraints from cross-linking studies can significantly improve structure prediction accuracy .

For ACIAD0721, given its relatively small size (111 amino acids), combining homology modeling with validation through experimental approaches like circular dichroism (CD) spectroscopy would provide the most reliable structural predictions.

How can the functional role of ACIAD0721 be experimentally determined?

Determining the functional role of ACIAD0721 requires a multi-faceted experimental approach:

• Knockout/knockdown studies:

  • Generate ACIAD0721 deletion mutants in Acinetobacter sp. ADP1 using its natural competence and homologous recombination capabilities

  • Perform phenotypic characterization including growth curves, stress response, and metabolic profiling

  • Complementation studies to verify phenotype specificity

• Protein interaction studies:

  • Pull-down assays using His-tagged ACIAD0721 as bait

  • Bacterial two-hybrid system to identify protein-protein interactions

  • Co-immunoprecipitation followed by mass spectrometry

• Biochemical activity assays:

  • Test for enzymatic activities based on structural predictions

  • Substrate specificity determination

  • Kinetic parameter analysis

• Expression analysis:

  • Transcriptomics to identify conditions that modulate ACIAD0721 expression

  • Proteomics to identify co-regulated proteins

  • Promoter analysis to identify regulatory elements

• Localization studies:

  • GFP fusion proteins to determine subcellular localization

  • Fractionation studies to identify compartment-specific distribution

This systematic approach would provide complementary lines of evidence to elucidate ACIAD0721's functional role in Acinetobacter sp. ADP1 .

What are the challenges in working with small proteins like ACIAD0721?

Working with small proteins like ACIAD0721 (12.2 kDa) presents several challenges:

• Expression and purification issues:

  • Potential instability or rapid degradation during expression

  • Difficult detection on standard SDS-PAGE without specialized techniques

  • Potential loss during dialysis or ultrafiltration due to membrane cutoff limitations

• Structural characterization challenges:

  • Limited secondary structure elements making circular dichroism interpretation difficult

  • Challenges in obtaining sufficient diffraction-quality crystals for X-ray crystallography

  • Potential aggregation during concentration for NMR studies

• Functional analysis complications:

  • Difficulty in distinguishing between direct effects of protein deletion versus indirect metabolic consequences

  • Potential redundancy with other UPF0339 family members masking phenotypes

  • Limited comparative genomic information due to annotation inconsistencies

• Technical considerations:

  • Need for specialized tags or fusion partners that don't interfere with function

  • Careful optimization of buffer conditions to maintain stability

  • Potential non-specific interactions during binding studies due to surface charge effects

Addressing these challenges requires careful experimental design, multiple complementary approaches, and proper controls to validate findings .

How can ACIAD0721 be used in experimental evolution studies?

ACIAD0721 can serve as an excellent model for experimental evolution studies, leveraging the natural competence and transformation capabilities of Acinetobacter sp. ADP1. This approach involves:

• Directed evolution setup:

  • Create a library of ACIAD0721 variants through error-prone PCR or site-directed mutagenesis

  • Transform these variants into Acinetobacter sp. ADP1 using its natural competence

  • Apply selection pressure relevant to the protein's hypothesized function

  • Perform multiple cycles of mutagenesis and selection to obtain evolved variants

• Sequence analysis of evolved variants:

  • Deep sequencing to identify enriched mutations

  • Evolutionary coupling analysis to infer residue interaction constraints

  • Comparison with wild-type sequence to identify adaptive mutations

• Structural implications:

  • Use the evolutionary data to improve structure prediction

  • Analyze co-varying residues to identify functional domains

  • Apply computational protein folding with interaction constraints to determine structural changes

This experimental evolution approach can yield valuable insights into protein function, structural constraints, and adaptive potential, as demonstrated with other antibiotic resistance proteins in similar experimental setups .

How can homologous recombination systems be optimized for studying ACIAD0721 function?

Optimizing homologous recombination systems for ACIAD0721 functional studies requires understanding the recombination pathways in Acinetobacter:

• Pathway selection based on substrate format:

  • For double-strand break repair (linear DNA integration), the RecBCD pathway is most efficient

  • For circular DNA recombination, the RecF pathway is preferred

  • Understanding these pathway differences allows optimization of DNA substrate design

• Recombination efficiency enhancement:

  • Optimize the length of homology arms (minimum 100 bp for efficient RecA-dependent recombination)

  • Utilize the natural competence of Acinetobacter sp. ADP1 during exponential growth phase

  • Design constructs that minimize the requirement for extensive homologous recombination processing

• Complex genetic manipulations:

  • For multiple modifications, sequential transformations can be performed

  • Splicing PCR can create recombinant constructs for transformation

  • Markerless deletion systems can be employed for clean gene replacements

The natural recombination capabilities of Acinetobacter sp. ADP1 make it an ideal system for these studies, as it allows genetic manipulation by simple addition of linear PCR products to growing cultures without the complex transformation protocols required for other organisms .

How do recombinant expression conditions affect the structural integrity of ACIAD0721?

The effect of recombinant expression conditions on ACIAD0721 structural integrity is a critical consideration that can significantly impact downstream applications:

• Temperature effects:

  • Expression at lower temperatures (16-25°C) often results in slower production but better folding

  • Higher temperatures may increase yield but can lead to inclusion body formation

  • Temperature shifting strategies (initial growth at 37°C followed by induction at lower temperatures) can optimize both growth and proper folding

• Induction parameters:

  • IPTG concentration significantly impacts protein folding, with lower concentrations (0.1-0.5 mM) often favoring proper folding

  • Induction duration affects both yield and structural integrity, with shorter periods sometimes preserving native structure

• Buffer composition impact:

  • pH variations (±0.5 units from optimal) can alter secondary structure elements

  • Ionic strength affects protein stability, with optimal NaCl concentration typically between 150-300 mM

  • Addition of stabilizing agents (glycerol, trehalose) may preserve structural integrity during purification

• Assessment methods:

  • Circular dichroism spectroscopy can detect secondary structure changes under different conditions

  • Differential scanning fluorimetry to determine thermal stability across different buffer conditions

  • Size exclusion chromatography to assess aggregation propensity

A systematic approach to optimizing these parameters is essential for maintaining the structural integrity of ACIAD0721, particularly given its relatively small size and potentially delicate structural features .

How can contradictory results in ACIAD0721 functional studies be reconciled?

When faced with contradictory results in ACIAD0721 functional studies, a systematic approach to data reconciliation is necessary:

• Methodological differences analysis:

  • Compare experimental conditions, including buffer compositions, protein concentrations, and assay temperatures

  • Evaluate tag interference effects (His-tag position and cleavage status)

  • Assess purity levels and potential contaminant effects across studies

• Statistical robustness evaluation:

  • Re-analyze data using standardized statistical methods

  • Consider the power of the studies to detect effects (sample sizes, replicate numbers)

  • Implement more sophisticated statistical approaches like partial least squares modeling to identify hidden variables

• Biological context consideration:

  • Examine strain differences in Acinetobacter species used across studies

  • Consider growth phase and environmental factors affecting protein function

  • Evaluate potential interaction partners present in some experimental setups but not others

• Integrated resolution approaches:

  • Design critical experiments specifically addressing the contradictions

  • Implement multiple complementary techniques to verify findings

  • Develop composite outcome measures that may be more sensitive to subtle effects

When analyzing contradictory results, it's important to recognize that proteins may have multiple functions depending on conditions, and apparent contradictions might reflect this functional diversity rather than experimental error .

What are the most common pitfalls in recombinant ACIAD0721 expression and how can they be addressed?

Common pitfalls in recombinant ACIAD0721 expression and their solutions include:

• Low expression levels:

  • Problem: Poor codon optimization for E. coli

  • Solution: Synthesize codon-optimized gene or use specialized E. coli strains containing rare tRNAs

• Inclusion body formation:

  • Problem: Improper folding leading to protein aggregation

  • Solution: Reduce induction temperature, decrease IPTG concentration, co-express with chaperones, or add folding enhancers like trehalose or arginine to the culture medium

• Protein degradation:

  • Problem: Proteolytic cleavage during expression or purification

  • Solution: Use protease-deficient strains, add protease inhibitors, optimize purification speed, maintain cold conditions (4°C)

• Poor solubility:

  • Problem: Hydrophobic interactions leading to aggregation

  • Solution: Add solubility tags (SUMO, MBP, GST), screen different buffer compositions, use detergents at low concentrations

• Low purity after affinity chromatography:

  • Problem: Non-specific binding of host proteins

  • Solution: Increase imidazole in wash buffers, add secondary purification steps, use tandem affinity tags

• Loss of activity after purification:

  • Problem: Structural changes during purification process

  • Solution: Optimize buffer conditions, add stabilizing agents, minimize freeze-thaw cycles, validate activity immediately after purification

Each of these challenges requires methodical troubleshooting with careful documentation of conditions and outcomes to establish optimal protocols for ACIAD0721 expression and purification .

How can researchers distinguish between the role of ACIAD0721 and other proteins in the UPF0339 family?

Distinguishing the specific role of ACIAD0721 from other UPF0339 family members requires a multi-level approach:

• Comparative genomic analysis:

  • Perform detailed sequence alignment of all UPF0339 family members in Acinetobacter

  • Identify unique sequence motifs or domains specific to ACIAD0721

  • Analyze gene neighborhood and operon structures for functional context clues

• Targeted genetic approaches:

  • Create single and combinatorial knockout mutants of all UPF0339 family members

  • Perform complementation studies with each individual gene

  • Utilize promoter exchange experiments to express each gene under control of ACIAD0721 promoter

• Biochemical specificity determination:

  • Conduct in vitro activity assays with purified proteins under identical conditions

  • Perform substrate specificity profiling for each family member

  • Analyze binding partner differences through pulldown experiments followed by mass spectrometry

• Expression pattern differentiation:

  • Use RT-qPCR to determine expression profiles under various growth conditions

  • Implement reporter gene fusions to visualize expression patterns

  • Perform chromatin immunoprecipitation to identify different transcriptional regulators

• Structural differentiation:

  • Compare predicted or determined structures of family members

  • Identify structural features unique to ACIAD0721

  • Map conservation patterns onto structural models to identify functionally important regions

This systematic approach helps delineate the unique role of ACIAD0721 while understanding potential functional overlap or complementation within the UPF0339 family .

How might ACIAD0721 be involved in antibiotic resistance mechanisms in Acinetobacter species?

While ACIAD0721's direct role in antibiotic resistance remains to be fully characterized, several research avenues can explore this potential connection:

• Expression correlation analysis:

  • Compare ACIAD0721 expression levels between antibiotic-susceptible and resistant Acinetobacter strains

  • Monitor expression changes in response to antibiotic exposure

  • Correlate expression with specific resistance phenotypes

• Functional genomics approaches:

  • Generate ACIAD0721 knockout mutants and assess changes in minimum inhibitory concentrations (MICs) for various antibiotics

  • Perform overexpression studies to determine if enhanced levels confer increased resistance

  • Conduct transposon mutagenesis screens to identify genetic interactions with known resistance determinants

• Structural association studies:

  • Investigate potential structural similarities with known resistance proteins

  • Perform molecular docking studies with antibiotics to assess binding potential

  • Analyze if ACIAD0721 belongs to any protein families associated with drug efflux or modification

• Clinical correlations:

  • Examine sequence variations in ACIAD0721 across clinical isolates with different resistance profiles

  • Determine if specific mutations correlate with resistance to particular antibiotics

  • Analyze gene expression in clinical isolates under antibiotic pressure

Given the rise of multidrug-resistant Acinetobacter baumannii as a critical global health threat, understanding potential contributions of proteins like ACIAD0721 to resistance mechanisms could be valuable for developing novel therapeutic strategies .

What novel experimental approaches could advance our understanding of ACIAD0721 function?

Several cutting-edge experimental approaches could significantly advance our understanding of ACIAD0721 function:

• CRISPR interference (CRISPRi) for gene regulation:

  • Implement tunable repression of ACIAD0721 expression

  • Study dose-dependent phenotypic effects

  • Identify genetic interactions through CRISPRi-based screens

• Protein proximity labeling:

  • Utilize BioID or APEX2 fusion proteins to identify proximal interacting partners in vivo

  • Map the spatial interactome of ACIAD0721 within the cell

  • Identify transient or weak interactions often missed by traditional methods

• Single-cell analysis techniques:

  • Apply single-cell RNA-seq to identify cell-to-cell variation in ACIAD0721 expression

  • Implement microfluidic approaches to study phenotypic heterogeneity

  • Utilize time-lapse microscopy with fluorescent reporters to track dynamic responses

• Structural mass spectrometry:

  • Employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe structural dynamics

  • Utilize cross-linking mass spectrometry (XL-MS) to identify interaction interfaces

  • Implement limited proteolysis-MS to identify flexible regions and binding sites

• 3Dseq approach:

  • Apply experimental evolution combined with deep sequencing

  • Analyze covariation patterns to infer structural constraints

  • Use evolutionary coupling analysis to predict residue interactions

  • Implement computational protein folding with these constraints to determine structure

These innovative approaches would provide multidimensional data to elucidate ACIAD0721's function, expanding beyond traditional genetic and biochemical methods .

How can systems biology approaches integrate ACIAD0721 into the broader cellular network of Acinetobacter?

Systems biology offers powerful frameworks to contextualize ACIAD0721 within the broader cellular network:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and ACIAD0721 mutant strains

  • Implement network analysis to identify perturbed pathways

  • Develop mathematical models to predict system-wide effects of ACIAD0721 perturbation

• Protein-protein interaction network mapping:

  • Perform systematic yeast two-hybrid or bacterial two-hybrid screens

  • Implement affinity purification-mass spectrometry under various conditions

  • Construct interaction networks specific to different environmental conditions

• Flux balance analysis:

  • Incorporate ACIAD0721-related data into genome-scale metabolic models

  • Predict metabolic flux changes upon ACIAD0721 deletion or overexpression

  • Identify potential metabolic bottlenecks or rewiring associated with ACIAD0721 function

• Comparative systems analysis:

  • Compare network positions of UPF0339 family proteins across bacterial species

  • Identify conserved network motifs associated with these proteins

  • Analyze how evolutionary changes in these proteins correlate with network adaptations

• Multi-scale modeling:

  • Develop models that connect molecular interactions to cellular phenotypes

  • Integrate temporal dynamics of protein expression and activity

  • Predict emergent properties related to ACIAD0721 function in different environmental contexts