Recombinant Bovine Suppressor of tumorigenicity 7 protein-like (ST7L)

What is Bovine ST7L protein and how does it relate to the ST7 family?

ST7L (Suppressor of Tumorigenicity 7-Like), also known as FAM4B or ST7R, was identified through its structural and functional similarity to the ST7 tumor suppressor gene. While ST7 is located in chromosome 7q31 region in humans, ST7L is found in a different chromosomal location but shares significant sequence homology with ST7 . The protein belongs to the LDLR superfamily, with ST7 specifically designated as LRP12 .

In bovine systems, ST7L maintains high conservation with human ST7L, with the extracellular domain (ECD) sharing approximately 98% amino acid sequence homology between bovine and human versions . This high degree of conservation suggests important biological functions that have been preserved throughout mammalian evolution.

What is the molecular structure and key domains of ST7L protein?

Based on its similarity to ST7, the ST7L protein likely contains several key structural domains:

• Signal sequence for cellular targeting

• Extracellular domain containing:

  • CUB domains (complement protein subcomponents C1r/C1s, Uegf, Bone morphogenetic protein 1)

  • LDLR (Low-Density Lipoprotein Receptor) class A domains

• Transmembrane domain

• Cytoplasmic domain containing motifs implicated in endocytosis and signal transduction

These structural features suggest ST7L functions as a type I transmembrane protein with potential roles in cell signaling and protein-protein interactions in bovine tissues.

What are the known functional roles of ST7L in normal bovine physiology?

While limited information is available specifically for bovine ST7L, inferences can be made from studies on ST7. The protein is widely expressed in normal tissues, with particularly high expression in heart and skeletal muscle . Its classification as part of a tumor suppressor family suggests potential roles in:

• Cell cycle regulation

• Cell adhesion and migration

• Protein trafficking and endocytosis

• Extracellular matrix interactions

• Signal transduction pathways

Research indicates ST7 expression may be associated with downregulated expression of extracellular matrix molecules involved in remodeling, such as SPARC, IGFBP5, and matrix metalloproteinases . Similar functions may exist for bovine ST7L, though specific confirmation through targeted research is needed.

How do post-translational modifications affect the binding properties and stability of recombinant bovine ST7L?

The recombinant bovine ST7L protein requires careful analysis of post-translational modifications that may influence its binding characteristics and stability. Based on related research with ST7/LRP12, when the recombinant protein is immobilized at optimal concentrations (approximately 0.5 μg/mL), it demonstrates specific binding interactions with other proteins such as LRPAP with a binding affinity placing optimal responses at approximately 0.6-3 μg/mL .

Stability considerations include:

Researchers should validate the activity of bovine ST7L after reconstitution to ensure that binding properties remain intact for experimental applications.

What genomic variations exist in bovine ST7L across different cattle breeds, and how might these impact protein function?

While the search results don't provide specific information on bovine ST7L genomic variations, research approaches should consider potential breed-specific polymorphisms. For human ST7 (and likely ST7L), genomic sequencing has indicated the possibility of up to 18 splicing isoforms . Similar splice variant diversity may exist in bovine populations.

A comprehensive research approach would include:

• Comparative genomic analysis across cattle breeds

• RNA-Seq to identify differential expression of splice variants

• Functional characterization of isoforms using recombinant protein studies

• Association studies correlating genetic variations with production traits or disease susceptibility

These variations could affect protein domain structure, subcellular localization, binding partners, and ultimately biological function in bovine tissues.

What are the key differences in signaling pathways between ST7 and ST7L in bovine systems compared to other mammalian species?

Understanding the divergence in signaling mechanisms between ST7 and ST7L across species requires comparative functional studies. The high conservation of these proteins (95-98% amino acid sequence homology across species) suggests preserved core functions , but species-specific variations likely exist.

Key considerations for comparative research include:

• Analysis of protein-protein interaction networks in bovine cells using immunoprecipitation followed by mass spectrometry

• Phosphoproteomic analysis to identify differential activation of downstream targets

• Receptor binding studies to determine ligand specificity differences

• Gene knockout or silencing studies to assess compensatory mechanisms

Species-specific differences may be particularly evident in:

• Tissue expression patterns

• Interaction with extracellular matrix components

• Response to cellular stress or pathogenic challenges

• Developmental timing of expression

What are the optimal expression systems for producing functional recombinant bovine ST7L protein?

When selecting an expression system for bovine ST7L, researchers should consider the complexity of this type I transmembrane protein with multiple domains and potential post-translational modifications.

For most research applications requiring full-length functional bovine ST7L, mammalian expression systems are recommended for proper folding and post-translational modifications . The recombinant protein can be expressed with affinity tags (commonly His-tag at the C-terminus) to facilitate purification while minimizing impact on protein function .

What are the validated methods for assessing ST7L protein-protein interactions and functional activity?

Several methodological approaches can be employed to characterize bovine ST7L interactions and activity:

• Binding Assays:

  • ELISA-based binding studies with potential interaction partners

  • Surface Plasmon Resonance (SPR) for real-time binding kinetics

  • Pull-down assays using tagged recombinant ST7L

• Functional Analysis:

  • Cell-based assays measuring effects on extracellular matrix remodeling

  • Analysis of downstream signaling pathway activation

  • Co-localization studies using fluorescently-tagged proteins

• Validation Methods:

  • Specific antibody blocking experiments

  • Competition assays with known ligands

  • Mutagenesis of key binding domains

For recombinant bovine ST7L specifically, approaches validated for ST7/LRP12 can be adapted, such as immobilization assays that measure binding to related proteins at concentrations between 0.6-3 μg/mL .

How should researchers address stability and storage challenges when working with recombinant bovine ST7L?

Maintaining stability of recombinant bovine ST7L requires careful handling and storage protocols:

• Formulation Considerations:

  • Carrier-free preparations are recommended for applications where BSA could interfere

  • For general cell culture or ELISA applications, BSA-containing formulations enhance stability

  • Lyophilized formulations in PBS or specialized protein stability buffers show optimal long-term stability

• Reconstitution Protocol:

  • For optimal activity, reconstitute lyophilized protein at recommended concentration (200 μg/mL for ST7/LRP12)

  • Use appropriate diluent based on the original formulation buffer

  • Allow complete solubilization before use

• Storage Recommendations:

  • Store lyophilized protein at -20°C to -80°C

  • For reconstituted protein, prepare single-use aliquots

  • Avoid repeated freeze-thaw cycles

  • Use manual defrost freezers rather than auto-defrost models

• Quality Control:

  • Verify protein integrity by SDS-PAGE

  • Validate activity using functional assays after reconstitution

  • Monitor batch-to-batch consistency

What experimental controls are essential when studying ST7L in cellular and molecular research?

Rigorous experimental design for bovine ST7L research requires appropriate controls:

• Positive Controls:

  • Well-characterized ST7L from related species (human or mouse)

  • Known binding partners or functional readouts

  • Commercial preparations with validated activity

• Negative Controls:

  • Vehicle-only treatments

  • Irrelevant proteins of similar size and structure

  • Heat-denatured ST7L to control for non-specific effects

• Expression Controls:

  • qRT-PCR to verify target gene expression

  • Western blotting to confirm protein expression levels

  • Immunofluorescence to verify cellular localization

• Specificity Controls:

  • Antibody validation using knockout/knockdown systems

  • Competitive binding with unlabeled protein

  • Dose-response curves to demonstrate specificity

• System Validation:

  • Demonstration of expected cellular responses in well-characterized systems

  • Comparison with published literature on related proteins

  • Reproducibility across different cell types or experimental conditions