Recombinant Penaeus monodon Peptide tyrosine phenylalanine 2

What is Peptide Tyrosine Phenylalanine 2 in Penaeus monodon and how is it characterized?

Peptide Tyrosine Phenylalanine 2 is a bioactive peptide identified in the black tiger shrimp (Penaeus monodon). While direct information about this specific peptide is limited in the current literature, characterization of peptides in P. monodon typically follows standard protocols involving extraction from tissue samples (commonly hemocytes), purification, and sequence analysis using mass spectrometry techniques. Similar to other bioactive peptides identified in P. monodon, such as the crustin-like antimicrobial peptides and serine proteinase inhibitors, it likely has a distinct amino acid sequence with functional domains that determine its biological activity .

Characterization methods often include:

• RNA extraction and cDNA library creation from hemocytes

• Sequence identification and analysis

• Determination of the amino acid composition

• Identification of functional domains

• Phylogenetic analysis to establish evolutionary relationships

What expression systems are most efficient for producing recombinant peptides from Penaeus monodon?

The Escherichia coli expression system is most commonly used for recombinant production of P. monodon peptides due to its simplicity, cost-effectiveness, and high yield. Based on successful applications with other P. monodon peptides, the procedure typically involves:

• Cloning the coding sequence of the peptide into a suitable expression vector (e.g., pET28b)

• Adding an N-terminal hexa-histidine tag for easier purification

• Transforming the construct into an appropriate E. coli strain

• Optimizing expression conditions (temperature, IPTG concentration, induction time)

• Purifying the recombinant protein using affinity chromatography

For example, the crustin-like antimicrobial peptide from P. monodon was successfully expressed using pET28b with an N-terminal hexa-histidine tag in E. coli, resulting in a functional recombinant protein with strong antimicrobial activity . Similarly, a 5-domain Kazal-type serine proteinase inhibitor (SPIPm2) was successfully expressed in the E. coli system, yielding a 32-kDa recombinant protein that maintained its inhibitory activity against various serine proteases .

How can I verify the purity and identity of recombinant Penaeus monodon peptides?

Verification of recombinant peptide purity and identity involves a multi-step approach:

• SDS-PAGE analysis to assess protein size and purity

• Western blot analysis using specific antibodies (if available)

• Mass spectrometry to confirm the precise molecular weight and sequence

• Activity assays to confirm functional properties

For instance, in the case of recombinant SPIPm2 from P. monodon, researchers verified its identity using SDS-PAGE and its activity was confirmed through inhibitory spectrum assays against various serine proteases including trypsin (89% inhibition), chymotrypsin (70% inhibition), and subtilisin (8% inhibition) . Similar approaches would be applicable for verifying recombinant Peptide Tyrosine Phenylalanine 2.

What are the common challenges in expressing recombinant peptides from Penaeus monodon?

Common challenges include:

• Codon optimization: Shrimp codon usage differs from E. coli, potentially leading to poor expression

• Protein folding: Many peptides contain multiple cysteine residues forming disulfide bonds, which may not form correctly in bacterial systems

• Protein solubility: Recombinant peptides often form inclusion bodies

• Cytotoxicity: Some antimicrobial peptides may be toxic to the host cells

• Proteolytic degradation: Host proteases may degrade the recombinant peptide

For example, when expressing crustin-like antimicrobial peptides from P. monodon, researchers needed to optimize conditions to overcome potential toxicity to the host cells and ensure proper folding of the peptide with its 12 conserved cysteine residues . Similarly, when expressing insulin-like androgenic gland hormone (IAG) from P. monodon, researchers faced challenges related to proper protein folding and biological activity .

How do tissue-specific expression patterns of Peptide Tyrosine Phenylalanine 2 compare with other neuropeptides in Penaeus monodon?

Understanding tissue-specific expression requires comprehensive transcriptomic analysis. Based on studies of other peptides in P. monodon and related species, expression patterns can be analyzed using:

• Tissue-specific RT-PCR or qPCR

• RNA-Seq data across different tissues

• Northern blot analysis

• In situ hybridization for spatial localization

Previous studies have shown that many bioactive peptides in P. monodon exhibit tissue-specific expression patterns. For example, SPIPm2 is exclusively expressed in hemocytes, as demonstrated through RT-PCR analysis . Similarly, the crustin-like antimicrobial peptide (Crus-likePm) is abundantly expressed in hemocytes and is highly up-regulated after Vibrio harveyi injection .

Comparative analysis could be performed using an approach similar to that used for neuropeptides in Litopenaeus vannamei, where expression profiles were analyzed using RNA-seq data from various tissues. The expression abundance was quantified by calculating the fragment per kilobase of transcript per million mapped reads (FPKM) and visualized as a heatmap, with red indicating high expression levels and blue denoting lower expression levels .

What regulatory elements control the expression of peptides in Penaeus monodon, and how can they be manipulated for enhanced recombinant production?

The genomic organization of peptide genes in P. monodon typically includes regulatory elements in the 5'-flanking regions. For instance, the Crus-likePm gene consists of two exons and one intron, with the 5'-flanking regions containing multiple putative transcription factor binding sites .

To enhance recombinant production:

• Identify promoter elements through genomic analysis

• Analyze transcription factor binding sites using bioinformatic tools

• Engineer expression constructs with optimal regulatory elements

• Consider inducible promoter systems for controlled expression

• Manipulate culture conditions to activate specific regulatory pathways

A methodological approach would involve:

• Isolating the genomic DNA corresponding to the target peptide

• Analyzing the 5' regulatory region

• Identifying key transcription factor binding sites

• Testing various promoter constructs in expression systems

• Optimizing culture conditions based on regulatory insights

How does the structural modeling of Peptide Tyrosine Phenylalanine 2 inform its potential biological functions?

Structural modeling of peptides provides crucial insights into their functional properties. For P. monodon peptides, this typically involves:

• Sequence-based prediction of secondary and tertiary structures

• Homology modeling based on related peptides with known structures

• Molecular dynamics simulations to assess stability and flexibility

• Docking studies to predict protein-protein interactions

• Structure-function correlation analysis

For example, protein structure modeling was used in the study of recombinant Pm-IAG to understand its biological activity . Similar approaches could be applied to Peptide Tyrosine Phenylalanine 2 to predict:

• Functional domains

• Potential binding partners

• Mechanism of action

• Evolutionary relationships with similar peptides in other species

What are the methodological considerations for studying the effects of recombinant Peptide Tyrosine Phenylalanine 2 on shrimp endocrine pathways?

Investigating the effects on endocrine pathways requires a comprehensive approach:

• In vivo trials: Similar to studies with recombinant Pm-IAG, trials would involve:

  • Careful experimental design with appropriate controls

  • Defined dosing regimens

  • Monitoring physiological parameters over time

  • Genotypic and phenotypic analysis

• Transcriptomic analysis: RNA-Seq to identify differentially expressed genes in response to peptide administration, looking for:

  • Changes in hormone receptor expression

  • Alterations in signaling pathway components

  • Downstream effects on target genes

• Metabolomic analysis: To detect changes in metabolite profiles, particularly those related to hormone signaling pathways. This approach was useful in identifying the role of dopamine and its derivatives in ovarian maturation in P. monodon .

• Receptor binding studies: To identify potential receptors and characterize binding affinities, which would be crucial for understanding the mechanism of action.

How can contradictory results in functional studies of recombinant Penaeus monodon peptides be reconciled through experimental design?

Contradictory results are common in peptide studies and can be addressed through:

• Comparative analysis of experimental conditions: Detailed documentation of all experimental parameters to identify potential sources of variation. For instance, the retrospective comparative analysis performed for IAG studies in P. monodon helped reconcile contrasting results by identifying penaeid-specific duplication in IAG and its receptor .

• Sequence and structural analysis: Identifying potential sequence variations or isoforms that might explain functional differences. The identification of duplications in IAG and its receptor in P. monodon, which were not present in paleomonids, suggested neo-functionalization that affected the hormone's activity .

• Tissue-specific and developmental stage considerations: Ensuring that the same tissues and developmental stages are compared across studies. For example, the expression of neuropeptides can vary significantly across different developmental stages, as shown in L. vannamei .

• Methodological standardization: Developing standardized protocols for:

  • Peptide expression and purification

  • Activity assays

  • Dosing regimens in vivo

  • Data analysis and interpretation

What is the optimal experimental design for testing the biological activity of recombinant Peptide Tyrosine Phenylalanine 2?

An optimal experimental design would include:

• Preparation Phase:

  • Expression and purification of the recombinant peptide

  • Quality control testing (purity, identity, activity)

  • Preparation of appropriate controls (negative control, positive control with known activity)

• In vivo Testing Framework:

  Group	Treatment	Number of Animals	Duration	Sampling Points
  1	Negative Control (Buffer)	30	4 weeks	Weekly
  2	Low Dose rPeptide	30	4 weeks	Weekly
  3	Medium Dose rPeptide	30	4 weeks	Weekly
  4	High Dose rPeptide	30	4 weeks	Weekly
  5	Positive Control	30	4 weeks	Weekly

• Assessment Parameters:

  • Physiological responses (growth, molting, reproductive development)

  • Molecular markers (gene expression changes)

  • Biochemical parameters (hormone levels, metabolite profiles)

  • Histological examination of target tissues

This design is inspired by the in vivo testing approaches used for recombinant Pm-IAG, where experimental animals were carefully selected, and various parameters were monitored to assess biological activity .

How can RNA-Seq analysis be optimized to identify genes regulated by Peptide Tyrosine Phenylalanine 2 in Penaeus monodon?

Optimizing RNA-Seq analysis for identifying regulated genes involves:

• Sample Collection and Preparation:

  • Collect tissues from treated and control animals

  • Ensure proper preservation to maintain RNA integrity

  • Extract high-quality total RNA using optimized protocols

• Library Preparation and Sequencing:

  • Construct stranded RNA-Seq libraries

  • Include biological replicates (minimum 3 per condition)

  • Sequence to sufficient depth (30-50 million reads per sample)

• Data Analysis Pipeline:

  • Quality control and filtering of raw reads

  • Alignment to the P. monodon reference genome using HISAT2

  • Transcript assembly using StringTie

  • Quantification of gene expression as FPKM (Fragment Per Kilobase of transcript per Million mapped reads)

  • Differential expression analysis comparing treated vs. control samples

  • Functional annotation and pathway analysis

• Validation:

  • Confirm key findings using qRT-PCR

  • Validate at the protein level where possible

This approach is similar to the RNA-Seq analysis methods used to identify neuropeptide genes in L. vannamei, which successfully mapped expression profiles across different tissues .

What methodological approaches can be used to investigate the role of Peptide Tyrosine Phenylalanine 2 in shrimp immune response?

Investigation of immune response roles would include:

• In vitro Immune Assays:

  • Hemocyte culture with recombinant peptide

  • Assessment of antimicrobial activity against relevant pathogens

  • Measurement of immune-related enzyme activities (phenoloxidase, lysozyme)

  • Quantification of reactive oxygen species production

• Immune Challenge Studies:

  • Pre-treatment with recombinant peptide followed by pathogen challenge

  • Monitoring survival rates and disease progression

  • Sampling for immune parameter analysis at defined time points

• Gene Expression Analysis:

  • qRT-PCR for immune-related genes

  • RNA-Seq for global transcriptome changes

  • Focus on pathways related to antimicrobial peptide production, pattern recognition, and inflammatory response

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation to identify binding partners

  • Yeast two-hybrid screening for interacting proteins

  • Analysis of signaling pathway activation

Similar methodological approaches have been used to study the crustin-like antimicrobial peptide in P. monodon, which showed strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, including V. harveyi .

What statistical approaches are most appropriate for analyzing dose-response relationships of recombinant Peptide Tyrosine Phenylalanine 2?

Appropriate statistical approaches include:

• Regression Analysis:

  • Nonlinear regression for dose-response curves

  • Determination of EC50/IC50 values

  • Analysis of curve parameters (slope, maximum effect)

• ANOVA-based Methods:

  • One-way ANOVA for comparing multiple dose groups

  • Repeated measures ANOVA for time-course experiments

  • Post-hoc tests (Tukey's, Dunnett's) for specific comparisons

• Mixed-Effects Models:

  • For handling repeated measurements and hierarchical data structures

  • Accounting for random effects (tank effects, genetic variation)

  • Modeling of covariance structures for time-series data

• Bayesian Approaches:

  • For complex experimental designs

  • When incorporating prior knowledge

  • For robust parameter estimation with limited sample sizes

Data presentation should include:

• Dose-response curves with confidence intervals

• Tables of statistical test results

• Visualization of time-dependent effects

How can structural bioinformatics be used to predict potential receptor interactions for Peptide Tyrosine Phenylalanine 2?

Structural bioinformatics approaches include:

• Sequence-Based Analysis:

  • Multiple sequence alignment with known receptor-binding peptides

  • Identification of conserved motifs and binding domains

  • Prediction of post-translational modifications

• Homology Modeling:

  • Construction of 3D structural models based on related peptides

  • Refinement and validation of models

  • Analysis of surface properties and potential binding sites

• Molecular Docking:

  • Virtual screening against potential receptor candidates

  • Analysis of binding modes and interaction energies

  • Identification of key residues involved in binding

• Molecular Dynamics Simulations:

  • Assessment of complex stability over time

  • Analysis of conformational changes upon binding

  • Calculation of binding free energies

This approach has been valuable in understanding receptor-ligand interactions in P. monodon, as demonstrated in the analysis of insulin-like peptides and their receptors. For example, researchers identified duplications in the IAG receptor unique to P. monodon through careful sequence and structural analysis .

What emerging technologies could enhance our understanding of Peptide Tyrosine Phenylalanine 2 function in Penaeus monodon?

Emerging technologies with significant potential include:

• CRISPR/Cas9 Gene Editing:

  • Creating knockout models to study peptide function

  • Introducing reporter constructs for in vivo visualization

  • Engineering modified peptide variants to study structure-function relationships

• Single-Cell Transcriptomics:

  • Mapping peptide and receptor expression at cellular resolution

  • Identifying cell populations responsive to peptide signaling

  • Characterizing heterogeneity in peptide effects

• Spatial Transcriptomics/Proteomics:

  • Visualizing peptide expression and activity in tissue context

  • Mapping receptor distribution across tissues

  • Correlating peptide activity with tissue architecture

• Advanced Microscopy:

  • Super-resolution imaging of peptide-receptor interactions

  • Live imaging of signaling dynamics

  • Correlative light and electron microscopy for ultrastructural context

• Metabolomics and Lipidomics:

  • Comprehensive profiling of metabolic changes induced by peptide activity

  • Identification of biomarkers associated with peptide function

  • Integration with transcriptomic data for pathway analysis

These approaches build upon methodologies that have been successful in studying other peptides in P. monodon, such as the combined transcriptomic and metabolomic analysis used to investigate ovarian maturation .

How might the function of Peptide Tyrosine Phenylalanine 2 differ across developmental stages in Penaeus monodon?

Investigating developmental differences requires:

• Stage-Specific Expression Analysis:

  • Quantitative PCR across developmental stages

  • RNA-Seq analysis of stage-specific transcriptomes

  • Protein quantification using stage-specific samples

• Functional Testing Across Stages:

  • Administration of recombinant peptide to different life stages

  • Monitoring of stage-specific responses

  • Identification of windows of sensitivity

• Receptor Expression Mapping:

  • Characterization of receptor expression dynamics across development

  • Correlation with peptide effects

  • Identification of stage-specific signaling networks

• Comparative Analysis:

  • Comparison with related species to identify conserved developmental roles

  • Analysis of evolutionary patterns in developmental function

This approach is supported by studies of neuropeptide expression across developmental stages in L. vannamei, which revealed significant variations in expression patterns . Similar developmental regulation may exist for Peptide Tyrosine Phenylalanine 2 in P. monodon.