Recombinant Human Putative serine protease 41 (PRSS41)

What is PRSS41 and where is it primarily expressed?

PRSS41, also known as Testis Serine Protease 1 (TESSP1), is a member of the serine protease family. It is a GPI-anchored protein primarily expressed in the testis with significant roles in reproductive biology. Research has also detected expression in the minor salivary gland and lungs . PRSS41 is encoded by a gene located on chromosome 16p13.3, specifically at coordinates 2848485-2855133 in the GRCh37 reference genome . Functionally, PRSS41 is required for the progression of meiosis during spermatogenesis, making it crucial for male reproductive research .

How can I detect PRSS41 gene copy number variations in research samples?

Gene-specific Copy Number Variation (CNV) probes are available for PRSS41 detection using Fluorescence In Situ Hybridization (FISH) techniques. These probes typically target the flanking regions of the PRSS41 gene and come with hybridization reagents . The methodology involves:

• Using a labeled probe with selected dye color (available options include OR, RE, GO, GR, and AQ with different absorbance and emission spectra)

• Hybridizing the probe to chromosome preparations

• Visualizing signals to detect amplifications or deletions

These probes can detect both amplification and deletion events affecting the PRSS41 gene, providing valuable data on genetic alterations in research samples .

What expression systems are recommended for producing recombinant PRSS41?

While the search results don't specifically address PRSS41 expression systems, we can draw parallels from methodologies used for other serine proteases. For bacterial expression systems, E. coli BL-21(DE3)pLysE has been successfully used for high-level expression of serine proteases in the same family . The recommended procedure includes:

• Transformation of the expression vector containing the PRSS41 coding sequence into the host strain

• Culture growth at 37°C with appropriate antibiotics (typically ampicillin at 50 μg/ml and chloramphenicol at 34 μg/ml)

• Induction with IPTG (2 mM) at OD560 of approximately 0.3

• Additional growth for 3 hours post-induction

• Purification using affinity chromatography, such as Ni-NTA agarose for His-tagged recombinant proteins

This approach typically yields sufficient quantities of recombinant protein for research applications, though optimization may be necessary for specific experimental needs.

How can I design experiments to investigate PRSS41's role in spermatogenesis?

Investigating PRSS41's role in spermatogenesis requires a multi-faceted approach combining molecular, cellular, and physiological techniques:

• Gene Expression Analysis: Implement RNA sequencing to identify genes with increased transcript levels and altered DNA methylation patterns that may interact with PRSS41

• Protein-Protein Interaction Studies: Identify binding partners of PRSS41 using techniques such as:

  • Yeast two-hybrid screening

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity labeling approaches

• Functional Assays for Meiotic Progression:

  • Immunohistochemistry of testicular sections to localize PRSS41 during different stages of spermatogenesis

  • Assessment of meiotic markers in PRSS41-deficient models

  • Protease activity assays using fluorogenic substrates to characterize enzymatic function

• Genetic Modification Approaches:

  • CRISPR/Cas9-mediated knockout or knockin models

  • Conditional gene targeting to study stage-specific effects

  • Rescue experiments with wild-type and mutant forms of PRSS41

Since PRSS41 is specifically required for meiotic progression during spermatogenesis , these approaches would help elucidate its precise molecular mechanisms and potential therapeutic applications.

What are the best methods for characterizing the enzymatic activity of recombinant PRSS41?

Characterizing the enzymatic activity of recombinant PRSS41 requires specialized assays that account for its serine protease nature:

• Substrate Specificity Profiling:

  • Utilize peptide libraries with diverse sequences

  • Employ fluorogenic or chromogenic substrates with different cleavage sites

  • Analyze cleavage products by mass spectrometry to determine preferred recognition motifs

• Kinetic Parameters Determination:

  • Measure initial velocities at varying substrate concentrations

  • Calculate Km, kcat, and catalytic efficiency (kcat/Km)

  • Assess the effects of pH, temperature, and ionic strength on activity

• Inhibitor Screening:

  • Test broad-spectrum serine protease inhibitors (e.g., PMSF, aprotinin)

  • Evaluate substrate-based inhibitors

  • Screen for specific small-molecule inhibitors

• Post-translational Modification Analysis:

  • Investigate the role of the GPI anchor in enzymatic activity

  • Assess potential regulatory phosphorylation or glycosylation sites

  • Compare native versus recombinant protein activity profiles

These methodological approaches would provide comprehensive characterization of PRSS41's enzymatic properties, enabling further functional studies in reproductive biology.

How can copy number variations of PRSS41 be correlated with male fertility phenotypes?

Correlating PRSS41 copy number variations (CNVs) with male fertility phenotypes requires a systematic approach integrating genomic, clinical, and functional data:

• CNV Detection Using FISH Probes:

  • Apply gene-specific PRSS41 probes available in multiple dye colors (as shown in the table below)

  • Perform quantitative analysis of signal numbers and intensities

  Number	Dye Color	Order Name	Absorbance Maximum	Emission Maximum
  1	OR	CNVFP-PRSS41-16476-OR	573nm	548nm
  2	RE	CNVFP-PRSS41-16476-RE	599nm	580nm
  3	GO	CNVFP-PRSS41-16476-GO	551nm	525nm
  4	GR	CNVFP-PRSS41-16476-GR	515nm	491nm
  5	AQ	CNVFP-PRSS41-16476-AQ	467nm	418nm

• Clinical Correlation Studies:

  • Collect comprehensive semen analysis data from subjects

  • Evaluate testicular biopsies for histological abnormalities

  • Track fertility outcomes in patients with identified CNVs

• Functional Validation in Model Systems:

  • Generate models with varying PRSS41 copy numbers

  • Assess spermatogenesis progression at cellular and molecular levels

  • Evaluate fertility outcomes in animal models

• Multi-omics Integration:

  • Correlate CNV data with transcriptomics and proteomics profiles

  • Identify compensatory mechanisms in cases of PRSS41 deletions

  • Map CNV-associated changes in regulatory networks affecting fertility

This integrative approach would establish meaningful connections between PRSS41 genetic variations and male fertility, potentially revealing diagnostic markers or therapeutic targets.

What are the optimal purification strategies for obtaining high-yield, active recombinant PRSS41?

Purifying recombinant PRSS41 to maintain its native conformation and enzymatic activity requires careful consideration of its biochemical properties:

• Affinity Chromatography Approaches:

  • For His-tagged PRSS41: Use Ni-NTA agarose matrix purification under native or denaturing conditions (8M urea) depending on protein solubility

  • For GPI-anchored forms: Consider detergent solubilization followed by immunoaffinity chromatography

• Optimization Protocol:

  • Assess solubility in bacterial expression systems (soluble supernatant vs. inclusion bodies)

  • For inclusion bodies: Implement proper refolding protocols using step-wise dialysis

  • Test different buffer compositions to maintain stability and activity

• Quality Control Assays:

  • SDS-PAGE and Western blotting with specific antibodies to confirm purity and identity

  • Size exclusion chromatography to ensure monomeric state

  • Circular dichroism to verify proper protein folding

  • Activity assays with model substrates to confirm functional integrity

• Storage Considerations:

  • Determine optimal pH, buffer composition, and additives for long-term stability

  • Evaluate freeze-thaw stability and appropriate storage temperature

  • Consider lyophilization options if applicable

These methodological considerations are essential for obtaining functional recombinant PRSS41 suitable for downstream applications in reproductive biology research.

How can I develop specific antibodies against human PRSS41 for research applications?

Developing specific antibodies against human PRSS41 requires strategic planning to ensure specificity, especially considering the homology with other serine proteases:

• Antigen Design Strategies:

  • Select unique epitopes with low homology to other serine proteases

  • Consider both full-length recombinant protein and synthetic peptides

  • Use bioinformatics tools to identify surface-exposed regions

• Antibody Production Protocol:

  • Immunize rabbits with purified recombinant PRSS41 (50-100 μg per immunization)

  • Follow a standard immunization schedule with appropriate adjuvants

  • Collect sera and evaluate antibody titers by ELISA

• Specificity Testing:

  • Perform immunoblotting against recombinant PRSS41 and related serine proteases

  • Test reactivity against tissue lysates where PRSS41 is expressed (testis, minor salivary gland, lung)

  • Include appropriate controls to rule out cross-reactivity

• Application-Specific Validation:

  • Verify antibody performance in immunohistochemistry, immunofluorescence, and flow cytometry

  • Test functionality in immunoprecipitation assays

  • Validate in knockout models or with siRNA knockdown controls

Despite potential sequence homology with other serine proteases, proper antigen design can yield specific antibodies, as demonstrated in related studies where antisera against homologous proteins (66% sequence identity) showed specificity for their respective immunizing antigens .

What considerations should be taken when designing CRISPR/Cas9 knockout strategies for PRSS41?

Designing effective CRISPR/Cas9 knockout strategies for PRSS41 requires careful consideration of several factors:

• Target Site Selection:

  • Analyze the PRSS41 gene structure (exon-intron boundaries)

  • Select early exons encoding critical domains of the protein

  • Use in silico tools to identify guide RNAs with high on-target and low off-target scores

  • Consider targeting conserved catalytic residues of the serine protease domain

• Delivery Methods for Reproductive Research:

  • For cell lines: Standard transfection or viral delivery methods

  • For animal models: Zygote microinjection or embryonic stem cell modification

  • For primary testicular cells: Optimize nucleofection or viral transduction protocols

• Validation Strategy:

  • Design PCR-based genotyping assays to detect intended modifications

  • Sequence the target region to confirm exact genetic alterations

  • Verify protein knockout by Western blotting using validated antibodies

  • Assess functional consequences through spermatogenesis analysis

• Phenotypic Analysis Plan:

  • Comprehensive evaluation of male reproductive parameters

  • Histological assessment of testicular architecture

  • Stage-specific analysis of meiotic progression

  • Fertility testing in animal models

Since PRSS41 is required for meiotic progression during spermatogenesis , knockout models would be particularly valuable for understanding its precise role in reproductive biology and potentially informing male fertility treatments.

How might PRSS41 research contribute to understanding male infertility disorders?

PRSS41 research has significant potential to advance our understanding of male infertility, particularly in cases with unexplained meiotic arrest:

• Diagnostic Applications:

  • Development of genetic screening panels including PRSS41 CNVs

  • Identification of PRSS41 mutations or variants in infertile men

  • Correlation of PRSS41 expression levels with specific infertility phenotypes

• Mechanistic Insights:

  • Elucidation of molecular pathways regulated by PRSS41 during spermatogenesis

  • Identification of PRSS41 substrates essential for meiotic progression

  • Understanding the role of proteolytic processing in sperm development

• Translational Potential:

  • Design of targeted therapies for specific forms of male infertility

  • Development of non-hormonal male contraceptives targeting PRSS41

  • Creation of diagnostic biomarkers based on PRSS41 activity or levels

• Comparative Biology Approaches:

  • Exploration of PRSS41 function across species with different reproductive strategies

  • Investigation of evolutionary conservation of PRSS41-dependent pathways

  • Identification of compensatory mechanisms in species with PRSS41 variants

Given that PRSS41 is required for the progression of meiosis during spermatogenesis , these research directions could significantly impact our approach to diagnosing and treating certain forms of male infertility.

What are the current technical challenges in studying PRSS41 and potential solutions?

Researchers face several technical challenges when studying PRSS41, each requiring specific methodological solutions:

• Protein Expression and Purification Challenges:

  • Challenge: Maintaining the native conformation of GPI-anchored PRSS41

  • Solution: Develop mammalian expression systems with proper post-translational modification machinery or use truncated constructs focusing on the catalytic domain

• Specificity in Functional Assays:

  • Challenge: Distinguishing PRSS41 activity from other serine proteases

  • Solution: Design highly specific substrates based on unique cleavage preferences and use PRSS41 knockout controls

• Tissue-Specific Expression Limitations:

  • Challenge: Restricted expression pattern primarily in testis

  • Solution: Develop organoid models of testicular tissue or stage-specific isolation of spermatogenic cells

• In Vivo Functional Analysis:

  • Challenge: Complexity of studying meiosis in living systems

  • Solution: Combine advanced imaging techniques with cell-specific reporters to track PRSS41 activity during spermatogenesis

• Structural Biology Hurdles:

  • Challenge: Obtaining crystal structures of membrane-associated proteases

  • Solution: Use cryo-electron microscopy or computational modeling approaches validated with experimental data

Addressing these technical challenges will accelerate progress in understanding PRSS41 biology and its potential applications in reproductive medicine.