Recombinant Lactobacillus plantarum Sulfate adenylyltransferase (sat)

What is Sulfate adenylyltransferase (sat) and what is its role in L. plantarum?

Sulfate adenylyltransferase (sat) is an enzyme (EC 2.7.7.4) also known as ATP-sulfurylase or Sulfate adenylate transferase that plays a critical role in sulfur metabolism. In Lactobacillus plantarum, this enzyme catalyzes the first step in the activation of inorganic sulfate, converting sulfate and ATP to adenosine 5'-phosphosulfate (APS) and pyrophosphate. This reaction is essential for incorporating sulfur into cellular components such as sulfur-containing amino acids, which are crucial for protein structure and function .

The enzymatic activity of sat is fundamental to L. plantarum's ability to synthesize key metabolites that may contribute to its probiotic effects, including its potential therapeutic applications in inflammatory conditions such as colitis .

How does the recombinant version of L. plantarum sat differ from the native enzyme?

The recombinant version of L. plantarum Sulfate adenylyltransferase is produced through heterologous expression systems such as E. coli, yeast, or baculovirus-infected insect cells. The primary sequence of the recombinant protein corresponds to the native enzyme found in L. plantarum strain ATCC BAA-793/NCIMB 8826/WCFS1, consisting of 391 amino acids .

While the amino acid sequence remains identical to the native protein, the recombinant version may exhibit differences in post-translational modifications depending on the expression system used. When expressed in baculovirus systems, the protein may retain more of the post-translational modifications necessary for proper folding and activity compared to bacterial expression systems .

What expression systems are most effective for producing recombinant L. plantarum sat?

Several expression systems can be used to produce recombinant L. plantarum Sulfate adenylyltransferase, each with distinct advantages:

The choice of expression system should be guided by the specific research requirements, balancing protein yield, activity preservation, and post-translational modification needs.

What are the optimal conditions for reconstitution and storage of recombinant L. plantarum sat?

Based on manufacturer recommendations for commercially available recombinant L. plantarum sat, the following protocol should be followed:

• Reconstitution procedure:

  • Briefly centrifuge the vial prior to opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (50% is recommended)

  • Aliquot for long-term storage

• Storage conditions:

  • For liquid formulations: Store at -20°C/-80°C with an expected shelf life of 6 months

  • For lyophilized formulations: Store at -20°C/-80°C with an expected shelf life of 12 months

  • Avoid repeated freezing and thawing cycles

  • Working aliquots can be stored at 4°C for up to one week

These conditions help maintain the structural integrity and enzymatic activity of the protein for experimental applications.

How can researchers verify the activity and purity of recombinant L. plantarum sat preparations?

To ensure the quality of recombinant L. plantarum sat preparations for experimental use, researchers should implement several verification methods:

• Purity assessment:

  • SDS-PAGE analysis should show a single predominant band at the expected molecular weight, with purity greater than 85%

  • Western blot analysis using antibodies specific to the sat protein or associated tags can confirm identity

• Activity verification:

  • Enzymatic assay measuring the conversion of sulfate and ATP to APS and pyrophosphate

  • The activity can be measured spectrophotometrically by coupling the reaction to pyrophosphatase and monitoring inorganic phosphate release

  • Comparison with a known standard can provide quantitative activity measurements

• Mass spectrometry:

  • To confirm the exact molecular weight and potential post-translational modifications

  • Peptide mapping to verify the amino acid sequence matches the expected sequence from L. plantarum strain ATCC BAA-793/NCIMB 8826/WCFS1

What controls should be included when using recombinant L. plantarum sat in experimental studies?

When designing experiments with recombinant L. plantarum sat, include the following controls:

• Negative controls:

  • Heat-inactivated enzyme (treatment at 95°C for 10 minutes) to control for non-enzymatic effects

  • Buffer-only samples to account for background reactions

  • Non-transformed L. plantarum to distinguish effects of the recombinant protein from those of the host organism

• Positive controls:

  • Commercial sat from a verified source with known activity

  • Well-characterized recombinant sat from another species for comparative analysis

• Expression system controls:

  • Empty vector-transformed expression host to account for any effects from the expression system itself

  • Non-relevant recombinant protein expressed in the same system to control for general recombinant protein effects

• Stability controls:

  • Fresh vs. stored enzyme preparations to assess stability over time

  • Different storage conditions to optimize preservation of activity

How does recombinant L. plantarum affect gut microbiota composition and diversity?

Recombinant L. plantarum has been shown to significantly alter gut microbiota composition and enhance microbial diversity. Studies have demonstrated:

These findings highlight the potential of recombinant L. plantarum as a tool for modulating gut microbiota composition, which may have significant implications for treating gut-related disorders.

What methodologies are most effective for studying interactions between recombinant L. plantarum sat and the gut microbiome?

To effectively study the interactions between recombinant L. plantarum sat and the gut microbiome, researchers should consider the following methodological approaches:

What immunological parameters should be measured when assessing the impact of recombinant L. plantarum on gut immunity?

When evaluating the immunomodulatory effects of recombinant L. plantarum, researchers should assess the following immunological parameters:

• Antibody production:

  • Serum IgG and IgG1 levels as indicators of systemic immune response

  • Fecal secretory IgA (sIgA) levels to assess mucosal immunity

  • Specific antibodies against proteins expressed by the recombinant L. plantarum

• Cellular immune responses:

  • Quantification of CD4+ T cell populations in gut-associated lymphoid tissues

  • Analysis of IgA+ B cell enrichment in intestinal tissues

  • Assessment of T cell differentiation patterns (Th1, Th2, Th17, Treg)

• Cytokine profiling:

  • Measurement of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α)

  • Analysis of regulatory cytokines (IL-10, TGF-β)

  • Evaluation of cytokines involved in specific T cell responses (IFN-γ, IL-4, IL-17)

• Barrier function assessment:

  • Intestinal permeability tests

  • Expression of tight junction proteins

  • Mucin production and intestinal mucus layer integrity

How effective is recombinant L. plantarum in mitigating colitis in experimental models?

Recombinant L. plantarum has demonstrated significant efficacy in mitigating colitis in experimental models, particularly in dextran sulfate sodium (DSS)-induced colitis in mice. Research findings indicate:

• Reduction in inflammatory markers:

  • L. plantarum therapy significantly reduced inflammatory colon lesions in DSS-challenged mice

  • Histological examination showed decreased inflammatory cell infiltration and preserved mucosal architecture

• Microbial community reprogramming:

  • Administration of L. plantarum increased the relative abundance of beneficial Actinobacteria in the colon

  • The microbial community changes correlated with reduced inflammation

• Metabolic alterations:

  • Serum metabolomics analysis showed increased levels of:

    • MG (18:4 (6Z, 9Z, 12Z, 15Z)/0:0/0:0)

    • Indolepyruvate

    • 1-hydroxyibuprofen

  • Decreased levels of:

    • 13-oxooctadecadienoic acid (13-oxoODE)

    • Indolylacryloylglycine

  • These metabolic changes are associated with reduced inflammation and improved colitis outcomes

The mechanism of action appears to involve both direct modulation of the intestinal immune response and indirect effects through alteration of the gut microbiota composition and metabolic activity.

What is the mechanism of action by which recombinant L. plantarum expressing protein fusions enhances immune responses?

Recombinant L. plantarum expressing protein fusions enhances immune responses through several complex mechanisms:

• Mucosal immune stimulation:

  • Recombinant L. plantarum serves as a mucosal delivery system for antigenic proteins, presenting them directly to gut-associated lymphoid tissue

  • This stimulates local immune responses more effectively than systemic delivery

• Adjuvant effects of fusion partners:

  • Fusion proteins such as P14.5-IL-33 or CTA1-p14.5-D-D incorporate adjuvant properties to enhance immunogenicity

  • IL-33 promotes DC responses to stimulate differentiation of naive T cells

  • IL-33 enhances NK and NKT cell expansion and improves Th1 and CD8+ T cell responses during infection

  • CTA1-DD is an artificial adjuvant composed of the enzymatically active CTA1 subunit of cholera toxin and the D domain dimer of Staphylococcus aureus protein A

• Microbiota-mediated immune modulation:

  • Recombinant L. plantarum alters the gut microbiota composition, which in turn influences immune function

  • Increased levels of IgG and IgG1 in serum and sIgA in feces indicate both systemic and mucosal immune activation

  • Enrichment of CD4+ T cells and IgA+ B cells demonstrates enhancement of both cellular and humoral immunity

• Metabolic immunomodulation:

  • Changes in bacterial metabolites influence immune cell function

  • Altered metabolites can affect signaling pathways in immune cells, leading to enhanced immune responses

How can recombinant L. plantarum sat be optimized for therapeutic applications in inflammatory bowel disease?

Optimizing recombinant L. plantarum sat for therapeutic applications in inflammatory bowel disease (IBD) requires consideration of several factors:

• Expression system optimization:

  • Selection of expression systems that maintain structural integrity and enzymatic activity

  • Consideration of post-translational modifications that may affect in vivo function

  • Development of stable expression constructs with controlled release mechanisms

• Delivery formulation:

  • Encapsulation techniques to protect the recombinant bacteria during gastric transit

  • Controlled release formulations to target specific regions of the intestine

  • Lyophilized preparations for improved stability and shelf life

• Dosing regimen design:

  • Determination of optimal bacterial load (studies typically use 1 × 10^9 CFU)

  • Establishment of effective administration schedules (e.g., initial administration for 3 consecutive days followed by booster administrations)

  • Assessment of long-term administration safety and efficacy

• Combination therapeutic approaches:

  • Co-administration with other probiotics or prebiotics to enhance colonization and effect

  • Integration with conventional IBD treatments for synergistic effects

  • Consideration of patient-specific microbiome profiles for personalized approaches

What genetic modifications can enhance the efficacy of recombinant L. plantarum sat in research and therapeutic applications?

Advanced genetic modifications can significantly enhance the effectiveness of recombinant L. plantarum sat for various applications:

• Promoter optimization:

  • Selection of strong, inducible promoters for controlled expression

  • Development of environmental-responsive promoters (pH, temperature, or microbiota-activated) for site-specific expression

  • Implementation of dual-promoter systems for balanced expression of sat and fusion partners

• Protein engineering approaches:

  • Structure-guided mutagenesis to enhance catalytic efficiency

  • Domain shuffling with other enzymes for novel functionalities

  • Addition of secretion signals for extracellular delivery of the enzyme

  • Development of fusion proteins with immunomodulatory molecules such as:

    • IL-33 for enhanced T cell responses

    • CTA1-DD adjuvant components for stronger immune activation

• Genome integration strategies:

  • Chromosomal integration for stable expression without antibiotic selection

  • Multi-copy integration for higher protein yields

  • Site-specific integration to minimize disruption of essential functions

• Regulatory circuit design:

  • Implementation of quorum-sensing systems for density-dependent expression

  • Creation of feedback loops to maintain optimal enzyme levels

  • Development of kill-switches for biocontainment in clinical applications

How does protein structure influence the functional properties of recombinant L. plantarum sat?

The structure-function relationship of recombinant L. plantarum Sulfate adenylyltransferase is complex and influences its catalytic activity, stability, and interactions:

• Key structural elements:

  • The full-length protein consists of 391 amino acids with multiple functional domains

  • The amino acid sequence includes regions critical for ATP binding, sulfate binding, and catalysis

  • The protein sequence from L. plantarum strain ATCC BAA-793/NCIMB 8826/WCFS1 contains numerous conserved residues that are essential for function

• Functional implications of structural features:

  • ATP-binding domain conformation affects catalytic efficiency

  • Surface-exposed residues influence protein-protein interactions and complex formation

  • Secondary structure elements determine thermal stability and pH tolerance

• Post-translational modifications:

  • Expression in different host systems results in varying patterns of post-translational modifications

  • These modifications can alter protein folding, enzymatic activity, and immunogenicity

  • Baculovirus expression systems provide many necessary post-translational modifications for proper folding

• Structure-based predictions:

  • Computational modeling can predict how mutations might affect enzyme function

  • Structural analysis can guide the design of fusion proteins that maintain sat activity while incorporating additional functional domains

  • Understanding structural determinants of stability can inform storage and handling protocols

What are the challenges in scaling up production of recombinant L. plantarum sat for research purposes?

Scaling up production of recombinant L. plantarum sat presents several technical challenges that researchers must address:

• Expression system limitations:

  • E. coli and yeast systems offer higher yields but may lack critical post-translational modifications

  • Insect and mammalian cell systems provide better modifications but have lower yields and higher costs

  • Balancing protein quality with production quantity remains a significant challenge

• Purification obstacles:

  • Achieving >85% purity while maintaining enzymatic activity requires optimized purification protocols

  • Selective extraction methods must be developed to isolate sat without co-purifying host proteins

  • Scale-up of chromatography steps without compromising resolution or recovery

• Activity preservation:

  • Preventing denaturation during concentration and buffer exchange steps

  • Identifying appropriate stabilizers for long-term storage

  • Developing lyophilization protocols that maintain refolding capacity upon reconstitution

• Quality control considerations:

  • Implementing robust activity assays suitable for batch testing

  • Developing standards for acceptable lot-to-lot variation

  • Establishing protocols to verify structural integrity and post-translational modification consistency across production batches

How might recombinant L. plantarum sat contribute to the development of novel gut microbiome-based therapeutics?

Recombinant L. plantarum sat has significant potential for developing innovative microbiome-based therapeutics:

• Precision microbiome modulation:

  • Recombinant L. plantarum expressing sat could selectively alter sulfur metabolism within the gut microbiome

  • This metabolic modulation could shift microbial community structure toward beneficial compositions for specific disease states

  • The approach offers more targeted intervention compared to broad-spectrum probiotics

• Immunomodulatory applications:

  • Development of dual-function therapeutics that express both sat and immunomodulatory proteins

  • Design of sat fusion proteins that enhance mucosal immunity while preserving enzymatic activity

  • Creation of vaccine delivery platforms using L. plantarum sat as an adjuvant carrier

• Metabolic engineering approaches:

  • Engineering of sat variants that produce specific beneficial metabolites

  • Development of strains with enhanced production of compounds like indolepyruvate that have anti-inflammatory properties

  • Creation of synthetic microbial consortia including recombinant L. plantarum sat for complex metabolic functions

• Personalized medicine applications:

  • Integration of patient microbiome analysis with tailored recombinant L. plantarum sat therapy

  • Development of diagnostic-therapeutic combinations that adjust bacterial functionality based on host needs

  • Patient-specific dosing regimens based on microbiome composition and metabolic profiles

What analytical techniques are emerging for studying the interactions between recombinant bacterial enzymes and host metabolism?

Emerging analytical techniques are revolutionizing our understanding of recombinant bacterial enzyme interactions with host metabolism:

• Multi-omics integration approaches:

  • Combined analysis of metagenomics, transcriptomics, proteomics, and metabolomics data

  • Network analysis to identify key nodes in host-microbe metabolic interactions

  • Machine learning algorithms to predict metabolic outcomes from microbiome perturbations

• Advanced metabolomic technologies:

  • Untargeted metabolomics to discover novel metabolites affected by recombinant L. plantarum sat

  • Stable isotope labeling to track metabolic fluxes between microbes and host

  • Spatial metabolomics to localize metabolic activities within specific intestinal regions

• Single-cell technologies:

  • Single-cell RNA sequencing of host immune cells responding to recombinant bacteria

  • Imaging mass cytometry to visualize host-microbe interactions at the tissue level

  • Single-cell metabolomics to characterize cell-specific responses to bacterial metabolites

• In situ visualization techniques:

  • Engineered biosensors to monitor enzyme activity in the gut environment

  • Fluorescence resonance energy transfer (FRET) systems to detect protein-protein interactions

  • Intravital microscopy to observe bacterial colonization and host responses in real-time