Recombinant Lactobacillus johnsonii Arginine--tRNA ligase (argS), partial

What is the function of Arginine--tRNA ligase (argS) in Lactobacillus johnsonii?

Arginine--tRNA ligase (argS) in L. johnsonii is an essential enzyme that catalyzes the attachment of arginine to its cognate tRNA (tRNAArg). This aminoacylation process is critical for protein synthesis, as charged tRNAs deliver amino acids to the ribosome during translation. In L. johnsonii, argS plays a particularly important role in the regulation of arginine metabolism, which contributes to the bacterium's ability to survive in various environmental conditions including the acidic environment of the gastrointestinal tract.

The enzyme functions by a two-step reaction:

• Activation of arginine with ATP to form arginyl-adenylate

• Transfer of the activated arginine to the 3' end of tRNAArg

Unlike simpler organisms, L. johnsonii possesses sophisticated regulatory mechanisms for arginine metabolism, as evidenced by the presence of two arginine repressors (argR1 and argR2) identified in related Lactobacillus species, suggesting complex regulation of arginine-related genes including argS .

How does recombinant L. johnsonii argS differ structurally from native argS?

Recombinant L. johnsonii argS typically contains only partial sequences of the native enzyme, as indicated by product listings for "argS, partial" . The native enzyme consists of multiple domains, including:

• A catalytic domain that binds ATP and arginine

• An anticodon binding domain that recognizes tRNAArg

• Structural elements that maintain proper enzyme conformation

Recombinant versions often focus on preserving the catalytic core while modifying other aspects to facilitate expression, purification, or specific research applications. Modifications may include:

• Addition of affinity tags (His-tag, GST, etc.)

• Codon optimization for expression in different host systems

• Removal of membrane-associated regions if present

• Potential alterations in glycosylation or other post-translational modifications

Researchers must consider these structural differences when interpreting experimental results with recombinant argS, particularly for kinetic or binding studies .

What methods are most effective for confirming the identity and purity of recombinant L. johnsonii argS?

For reliable identification and purity assessment of recombinant L. johnsonii argS, researchers should employ multiple complementary techniques:

For recombinant argS expressed in mammalian cell systems (as mentioned in product listings ), particular attention should be paid to glycosylation patterns and other post-translational modifications that may affect enzyme function .

What expression systems are most suitable for producing recombinant L. johnsonii argS?

The choice of expression system for recombinant L. johnsonii argS depends on research objectives and downstream applications. Based on current research practices:

Mammalian cell expression systems:

• Provide proper protein folding and post-translational modifications

• Yield functionally active argS with native-like properties

• Allow for secretion of properly folded protein

• Typically used for structural studies and functional assays

E. coli expression systems:

• Offer high yield and cost-effectiveness

• Suitable for preliminary studies and antigen production

• May require refolding protocols due to inclusion body formation

• Work best for partial argS constructs rather than full-length protein

Lactobacillus-based expression systems:

• Provide a more native-like environment for L. johnsonii proteins

• Can be engineered using vectors like those developed for L. johnsonii expression

• May better preserve species-specific post-translational modifications

• Allow for potential surface display of argS if fused to appropriate anchoring domains

The methodology for constructing recombinant L. johnsonii strains typically involves electroporation with specific parameters (2.1 kV for 3 ms), followed by selection with appropriate antibiotics (e.g., chloramphenicol at 10 μg/mL) .

What are the critical challenges in expressing functional recombinant L. johnsonii argS?

Researchers face several significant challenges when expressing functional recombinant L. johnsonii argS:

• Maintaining enzymatic activity:

  • Preserving the complex tertiary structure required for catalysis

  • Ensuring proper coordination of metal ions essential for function

  • Preventing loss of activity during purification steps

• Codon optimization challenges:

  • L. johnsonii has a low GC content (34.67%) , requiring careful codon optimization

  • Balancing expression efficiency with maintaining protein folding kinetics

  • Avoiding rare codons that might cause translational pausing

• Structural integrity:

  • Preventing protein aggregation and inclusion body formation

  • Maintaining proper disulfide bond formation if present

  • Ensuring correct domain orientation for substrate binding

• Purification complications:

  • Developing protocols that yield high purity without compromising activity

  • Removing contaminating nucleic acids that may interfere with activity assays

  • Preventing protein degradation during multiple purification steps

• Stability concerns:

  • Maintaining protein stability during storage (shelf-life limitations of 6-12 months)

  • Preventing freeze-thaw damage (repeated freezing and thawing not recommended)

  • Optimizing buffer conditions for long-term stability

How can researchers optimize the yield and activity of recombinant L. johnsonii argS?

To maximize yield and activity of recombinant L. johnsonii argS, consider the following optimization strategies:

Expression optimization:

• Test multiple expression systems (mammalian, bacterial, yeast)

• Optimize induction conditions (temperature, inducer concentration, timing)

• Consider co-expression with molecular chaperones to improve folding

• Employ fusion partners that enhance solubility (MBP, SUMO, etc.)

Purification strategies:

• Implement multi-step purification protocols (affinity, ion exchange, size exclusion)

• Use affinity tags that can be cleaved post-purification

• Consider on-column refolding for proteins expressed in inclusion bodies

• Optimize buffer conditions throughout purification process

Activity preservation:

• Add stabilizing agents like glycerol (recommended 5-50% final concentration)

• Include cofactors or metal ions required for catalytic activity

• Store at appropriate temperature (-20°C/-80°C for longer periods)

• Divide into single-use aliquots to avoid freeze-thaw cycles

Quality control:

• Implement rigorous purity checks with SDS-PAGE (>85% purity standard)

• Verify activity using aminoacylation assays

• Confirm folding status via circular dichroism or thermal shift assays

• Test batch-to-batch consistency of enzymatic parameters

How can recombinant L. johnsonii argS be used to study arginine metabolism regulation?

Recombinant L. johnsonii argS provides valuable tools for investigating the complex regulation of arginine metabolism:

Regulatory studies:

• Investigate potential interactions between argS and the two arginine repressors (argR1 and argR2) found in Lactobacillus species

• Analyze the impact of arginine concentration on argS activity and expression

• Study the ARG box DNA sequence elements that may regulate argS expression

Expression analysis:

• Use recombinant argS to generate antibodies for detecting native expression levels

• Compare recombinant argS activity with native enzyme to assess regulatory modifications

• Investigate post-translational modifications that may regulate enzyme activity

Metabolic integration:

Research has shown that in related Lactobacillus species, arginine-dependent repression involves both argR1 and argR2 gene products, suggesting the active repressor may be a heterooligomeric complex affecting argS expression .

What experimental approaches can determine the kinetic parameters of recombinant L. johnsonii argS?

To determine the kinetic parameters of recombinant L. johnsonii argS, researchers should employ these methodological approaches:

ATP-PPi exchange assay:

• Measures the first step of the aminoacylation reaction (activation of arginine)

• Allows determination of Km for arginine and ATP

• Provides data on the catalytic efficiency (kcat/Km) of the enzyme

tRNA aminoacylation assay:

• Measures the complete reaction (charging of tRNAArg with arginine)

• Monitors the formation of Arg-tRNAArg over time

• Can be performed using radioactive arginine or more modern fluorescent methods

Steady-state kinetic analysis:

• Determine Km, Vmax, and kcat for all substrates

• Analyze the effect of pH, temperature, and ionic strength on activity

• Investigate potential allosteric regulators of enzyme activity

Inhibition studies:

• Test competitive inhibitors that may provide insights into active site structure

• Evaluate product inhibition patterns to elucidate reaction mechanism

• Assess the effects of antibiotics that target aminoacyl-tRNA synthetases

A standardized experimental workflow should include careful enzyme concentration determination, substrate preparation (especially pure tRNAArg), and appropriate controls to account for potential contaminants in the recombinant enzyme preparation.

How does L. johnsonii argS differ from argS in related bacterial species?

Comparative analysis reveals important differences between L. johnsonii argS and argS enzymes from related bacteria:

L. johnsonii's argS likely evolved specific adaptations for function in low pH environments, given the organism's ecological niche in the gastrointestinal tract. The regulation of argS in L. johnsonii is likely complex, as suggested by genomic studies of related Lactobacillus species showing that arginine-dependent repression requires both argR1 and argR2 gene products .

Comparative genomic hybridization (CGH) studies between L. johnsonii and related species like L. taiwanensis and L. gasseri show varying degrees of gene conservation (83% within L. johnsonii strains, but only 51% and 47% for L. taiwanensis and L. gasseri respectively) , suggesting potential differences in argS regulation and function across these related species.

How can recombinant L. johnsonii argS be integrated into engineered probiotic applications?

Recombinant L. johnsonii argS offers several possibilities for engineered probiotic applications:

Expression system optimization:

• Utilize expression vectors like pPG612, which has been successfully used for recombinant protein expression in L. johnsonii

• Consider inducible promoters that respond to environmental signals in the GI tract

• Engineer secretion signals for efficient export or surface display of fusion proteins

Therapeutic development:

• Engineer L. johnsonii strains with modified argS expression to enhance survival in specific GI environments

• Develop strains with argS modifications that could alter local arginine metabolism in therapeutic contexts

• Create bifunctional fusion proteins combining argS with other therapeutic proteins

Vaccine delivery applications:

• L. johnsonii has been demonstrated to partially survive gastric conditions, making it a potential oral vaccine delivery vehicle

• Engineered L. johnsonii expressing cell surface fusion proteins has induced both systemic IgG responses and local mucosal immune responses

• ArgS could be used as a fusion partner or expression regulator in such systems

Recent research has shown that engineered L. johnsonii strains expressing bovine GM-CSF reduced inflammation in a mouse model of postpartum endometritis , suggesting that similar engineering approaches could be applied to argS-based recombinant strains for various therapeutic applications.

What methods can assess the impact of mutations on recombinant L. johnsonii argS function?

To evaluate how mutations affect recombinant L. johnsonii argS function, researchers should employ a multi-faceted experimental approach:

Site-directed mutagenesis strategies:

• Target conserved catalytic residues based on structural predictions

• Modify potential regulatory sites identified through sequence comparison

• Create chimeric proteins with domains from related species' argS enzymes

Functional assessment techniques:

• Develop high-throughput aminoacylation assays to screen multiple mutants

• Compare kinetic parameters (Km, kcat) between wild-type and mutant enzymes

• Analyze substrate specificity changes using various tRNA substrates

Structural analysis methods:

• Use circular dichroism to assess secondary structure changes

• Employ thermal shift assays to evaluate stability alterations

• If possible, determine crystal structures of wild-type and mutant proteins

In vivo validation:

• Create L. johnsonii strains expressing mutant argS variants

• Assess growth characteristics under various conditions

• Evaluate stress responses and survival rates in simulated GI environments

Studies of arginine repressors in related Lactobacillus species have shown that mutations in specific domains (DNA binding domain, oligomerization domain) can abolish arginine repression . Similar domain-specific analyses of argS could provide valuable insights into structure-function relationships.

How can researchers investigate potential interactions between argS and the arginine repressor system in L. johnsonii?

Investigating interactions between argS and the arginine repressor system requires sophisticated experimental approaches:

Genetic approaches:

• Create knockout/knockdown strains of argR1 and argR2 in L. johnsonii

• Analyze argS expression levels in these modified strains

• Construct reporter gene fusions to monitor argS promoter activity

Protein-protein interaction studies:

• Perform co-immunoprecipitation experiments with tagged argS and argR proteins

• Use bacterial two-hybrid systems to detect direct interactions

• Employ surface plasmon resonance to measure binding kinetics

DNA-protein interaction analysis:

• Conduct chromatin immunoprecipitation to identify argR binding sites near the argS gene

• Perform electrophoretic mobility shift assays with purified argR proteins and argS promoter fragments

• Use DNase footprinting to precisely map binding sites

Regulatory network mapping:

• Perform RNA-Seq analysis under varying arginine concentrations

• Integrate data with metabolomic profiling of arginine-related metabolites

• Develop mathematical models of the arginine regulatory network

Research in L. plantarum has identified specific ARG box sequences in the intergenic regions of arginine biosynthesis operons that serve as binding sites for arginine repressors . Similar regulatory elements may control argS expression in L. johnsonii and could be targeted in these investigations.

What are the emerging applications of recombinant L. johnsonii and its proteins in microbiome research?

Recent advancements highlight several promising applications for recombinant L. johnsonii and its proteins:

Immunomodulatory applications:

• L. johnsonii strains have demonstrated the ability to modulate immune responses, reducing inflammation in various models

• Recombinant L. johnsonii expressing bovine GM-CSF showed significant therapeutic effects on cow endometritis

• L. johnsonii can decrease allergic airway inflammation upon oral administration

Pathogen antagonism:

• Certain L. johnsonii strains exhibit antagonistic effects against pathogens like Salmonella

• Engineered strains could enhance these protective effects through recombinant protein expression

• The antimicrobial properties could be leveraged in both human and animal health applications

Novel delivery mechanisms:

• Recent research has identified that L. johnsonii produces extracellular vesicles (EVs) that can deliver functional proteins to host cells

• The Sdp-SH3b2 domain contained in L. johnsonii N6.2-derived EVs has been shown to inhibit murine norovirus replication

• These natural delivery systems could be engineered to incorporate recombinant proteins like argS or argS-fusion proteins

Host-microbiome interaction studies:

• L. johnsonii has been shown to play a critical role in maintaining host homeostasis by controlling pathogen expansion, modulating metabolic pathways, and regulating immune responses

• Recombinant proteins could serve as tools to study these interactions at a molecular level

• The genome sequences of various L. johnsonii strains provide resources for identifying potential targets for recombinant expression

What are the key methodological challenges in studying argS function in complex microbial communities?

Researchers face several methodological challenges when investigating argS function in complex microbial communities:

Technical limitations:

• Difficulty in isolating active enzymes from complex microbial samples

• Challenges in distinguishing between host and microbial argS activities

• Limited sensitivity of current methods for detecting low-abundance proteins

Experimental design considerations:

• Need for methods that preserve native microbial community structure

• Challenges in creating relevant model systems that reflect in vivo conditions

• Difficulty in controlling variables in complex community experiments

Analytical challenges:

• Integrating multi-omics data (metagenomics, metatranscriptomics, metaproteomics)

• Attributing observed functions to specific community members

• Accounting for horizontal gene transfer and genetic variation

Future methodological directions:

• Development of activity-based protein profiling for aminoacyl-tRNA synthetases in complex samples

• Application of single-cell techniques to study argS expression at individual cell level

• Creation of reporter systems to monitor argS activity in mixed communities

The development of approaches like those used to analyze gene expression of L. plantarum in the human gastrointestinal tract could potentially be adapted to study argS function in complex microbial communities.

How might structural biology approaches enhance our understanding of L. johnsonii argS?

Advanced structural biology techniques offer promising avenues for deepening our understanding of L. johnsonii argS:

Cryo-electron microscopy (cryo-EM):

• Could reveal the complete three-dimensional structure of argS at near-atomic resolution

• May capture different conformational states during the catalytic cycle

• Could visualize interactions with tRNA substrates and regulatory proteins

X-ray crystallography:

• Would provide high-resolution structural data of the active site

• Could capture structures of argS bound to inhibitors or substrate analogs

• Might reveal species-specific structural features not present in other bacterial argS enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

• Could map dynamic regions of the protein under different conditions

• Would help identify regulatory sites that undergo conformational changes

• Could provide insights into protein-protein interaction interfaces

Integrative structural biology:

• Combining multiple techniques (NMR, SAXS, computational modeling)

• Would provide a comprehensive understanding of argS structure and dynamics

• Could reveal how argS interacts with the arginine repressor system

Computational approaches:

• Molecular dynamics simulations to study enzyme flexibility and substrate binding

• Homology modeling based on related argS structures from other species

• Protein-protein docking to predict interactions with regulatory proteins

Structural insights could guide the engineering of argS variants with enhanced stability or altered specificity for various biotechnological applications in L. johnsonii-based probiotics.