Recombinant Bovine Integrin alpha-3 (ITGA3), partial

What is Recombinant Bovine Integrin alpha-3 (ITGA3)?

Recombinant Bovine Integrin alpha-3 (ITGA3) is a laboratory-produced version of the bovine integrin alpha-3 subunit. Integrins are heterodimeric integral membrane proteins composed of alpha and beta chains that function as cell surface adhesion molecules. The alpha-3 subunit typically pairs with beta-1 to form integrin α3β1, which serves as a receptor for multiple extracellular matrix proteins including fibronectin, laminin, collagen, epiligrin, thrombospondin, and CSPG4 . When designated as "partial," this indicates that the recombinant protein contains a specific fragment rather than the complete sequence of the native bovine ITGA3 protein.

What are the primary functions of Integrin alpha-3 in biological systems?

In biological systems, Integrin alpha-3 primarily functions as part of the α3β1 heterodimer, mediating critical cellular processes through interaction with extracellular matrix components. These functions include:

• Cell adhesion to extracellular matrix components, particularly laminin

• Signal transduction through interactions with multiple ligands

• Participation in adhesion, formation of invadopodia, and matrix degradation processes

• Promotion of cell invasion through its role as a docking site for proteins such as FAP (seprase) at invadopodia plasma membranes

• Mediation of endothelial cell migration in conjunction with other proteins such as LGALS3

• Potential involvement in pathogen recognition and cellular entry mechanisms

What expression systems are commonly used to produce Recombinant Bovine ITGA3?

Multiple expression systems are utilized for producing Recombinant Bovine ITGA3, each offering distinct advantages for different research applications:

These various expression platforms are commercially available from suppliers such as CUSABIO, which offers recombinant bovine ITGA3 (partial) from all five expression systems listed above .

How should I design experiments to study ITGA3 binding specificity to different ligands?

To study ITGA3 binding specificity to different ligands, a systematic approach using multiple complementary methods is recommended:

• Solid-phase binding assays:

  • Coat microplates with purified ligands (laminin, fibronectin, collagen)

  • Incubate with recombinant bovine ITGA3 (preferably the α3β1 heterodimer)

  • Detect binding using anti-ITGA3 antibodies and appropriate secondary antibodies

  • Include appropriate controls such as the GRGDSP peptide, which has been shown to inhibit penton base binding to α3β1 integrin

• Competition assays:

  • Test binding in the presence of potential competitive peptides or antibodies

  • Consider that different peptide motifs may have varying effects on ITGA3 binding

  • Research indicates that while the RGD motif is involved in ITGA3 binding, it may only be part of multiple binding sites in the complete interface

• Surface Plasmon Resonance (SPR):

  • Immobilize either ITGA3 or its ligands on sensor chips

  • Measure real-time binding kinetics and calculate association/dissociation rates

  • Determine equilibrium dissociation constants (KD) for each ligand

• Cell-based assays:

  • Express recombinant bovine ITGA3 in cells lacking endogenous expression

  • Assess cell adhesion to different matrix proteins

  • Confirm specificity using function-blocking antibodies against ITGA3

What are the optimal conditions for maintaining the structural integrity of Recombinant Bovine ITGA3 during purification?

Maintaining the structural integrity of Recombinant Bovine ITGA3 during purification requires careful attention to several parameters:

• Buffer composition:

  • Use physiological pH (7.2-7.4) with appropriate buffering agents

  • Include divalent cations (1-2 mM Ca²⁺ and Mg²⁺) which are essential for integrin function

  • Add 150-300 mM NaCl to maintain ionic strength

  • Consider adding 5-10% glycerol as a stabilizing agent

• Temperature control:

  • Perform all purification steps at 4°C to minimize proteolysis

  • Avoid repeated freeze-thaw cycles; store aliquots at -80°C for long-term storage

• Protease inhibitors:

  • Include a cocktail of protease inhibitors (PMSF, aprotinin, leupeptin, pepstatin A)

  • Consider using EDTA-free protease inhibitor formulations to preserve metal-dependent conformations

• Gentle elution conditions:

  • For affinity chromatography, use competitive elution rather than harsh pH changes

  • If using immobilized metal affinity chromatography (IMAC), use an imidazole gradient

  • For antibody-based purification, consider mild acid elution with immediate neutralization

• Monitoring methods:

  • Use circular dichroism spectroscopy to assess secondary structure

  • Employ dynamic light scattering to check for aggregation

  • Verify activity through binding assays after purification

How can I validate the functionality of purified Recombinant Bovine ITGA3?

Validating the functionality of purified Recombinant Bovine ITGA3 requires multiple complementary approaches:

• Binding assays:

  • Solid-phase binding to known ligands (laminin, fibronectin, collagen)

  • Competition with function-blocking antibodies to confirm specificity

  • Analysis of binding in the presence of RGD-containing peptides, which have been shown to partially inhibit α3β1 integrin interactions

• Cell adhesion assays:

  • Coat surfaces with purified ITGA3 (complexed with β1 if available)

  • Assess adhesion of cells known to express ITGA3 ligands

  • Include controls with function-blocking antibodies against α3 and β1 subunits

• Receptor signaling assays:

  • Monitor downstream signaling events when cells expressing the recombinant protein are exposed to ligands

  • Assess phosphorylation of focal adhesion kinase (FAK) and other pathway components

• Heterodimer formation:

  • Co-immunoprecipitation with β1 integrin subunit

  • Size exclusion chromatography to confirm complex formation

  • Native PAGE to visualize heterodimer formation

How does the binding mechanism of bovine ITGA3 compare with its human homolog?

The binding mechanism of bovine ITGA3 shares similarities with its human homolog but may exhibit species-specific differences:

Similarities:

• Both bovine and human ITGA3 primarily form heterodimers with β1 integrin

• Both recognize similar extracellular matrix ligands, including laminin, fibronectin, and collagen

• Both participate in RGD-dependent and RGD-independent interactions

Differences:

• While the exact sequence homology between bovine and human ITGA3 is not specified in the available data, information from related integrins suggests potential variations:

  • Bovine and human integrin αv subunits show 98.8% amino acid sequence similarity in ligand-binding domains

  • The β3 subunits show only 93% similarity between the two species

• These differences can result in functional variations, as demonstrated in studies of other integrins where the bovine version showed increased efficiency as a viral receptor compared to its human counterpart

Research approaches to compare binding mechanisms include generating binding affinity profiles for both bovine and human ITGA3 against a panel of ligands, performing structural studies, and creating chimeric proteins to identify regions responsible for binding differences.

What role does ITGA3 play in pathogen recognition and infection processes?

Research suggests ITGA3 may play significant roles in pathogen recognition and infection processes:

• Viral interactions:

  • Integrin α3β1 has been identified as an alternative cellular receptor for adenovirus in human systems

  • Studies have shown that adenovirus penton base protein can bind to integrin α3β1 in vitro

  • Virus-mediated transduction can be inhibited by anti-α3 and anti-β1 function-blocking antibodies

• Binding mechanisms:

  • The RGD (Arg-Gly-Asp) motif, a well-known integrin binding motif, is only part of the binding interface with α3β1

  • Multiple additional contact sites are involved in the interaction between pathogens and α3β1

  • Competition experiments have shown that the GRGDSP peptide can inhibit penton base binding to α3β1, but this competition is substantially weaker than that observed with other integrin receptors like αvβ3

• Pathogen entry:

  • α3β1 binding to pathogens may facilitate cellular entry and infection

  • Gene transduction mediated by adenovirus has been shown to be inhibited by anti-α3 antibodies, suggesting a functional role in infection

Understanding these interactions could lead to novel strategies for preventing or treating infectious diseases that involve ITGA3 as a receptor.

How can structure-function relationship studies of bovine ITGA3 inform therapeutic development?

Structure-function relationship studies of bovine ITGA3 can provide valuable insights for therapeutic development:

• Identification of critical binding domains:

  • Peptide library screenings and binding experiments can reveal the importance of specific motifs beyond the canonical RGD sequence

  • Research on penton base protein binding to α3β1 has shown that the RGD motif is only part of multiple binding sites in the complete interaction interface

• Species-specific therapeutic targeting:

  • Understanding structural differences between bovine and human ITGA3 can guide the development of species-specific therapeutics

  • Studies of other integrins show that the C-terminal one-third of the β3 subunit ectodomain, containing a highly structured cysteine-rich repeat region, can affect receptor efficiency

• Binding interface complexity:

  • Research indicates that even well-established binding motifs like RGD may play only partial roles in complete protein-protein interactions

  • For example, mutations in the RGD motif (R340E) only partially impaired penton base-α3β1 interaction, suggesting additional binding determinants

  • These findings suggest that effective therapeutics targeting ITGA3 interactions may need to address multiple binding determinants simultaneously

• Therapeutic design approaches:

  • Structure-guided design of peptide inhibitors targeting specific ITGA3 interactions

  • Development of function-blocking antibodies that recognize critical domains

  • Identification of small molecule binding sites for synthetic drug development

What are the most effective approaches for detecting and quantifying bovine ITGA3 expression in tissue samples?

Detecting and quantifying bovine ITGA3 expression in tissue samples requires selecting appropriate methods based on research objectives:

• Protein-level detection:

  • Immunohistochemistry (IHC): Optimal for spatial localization in tissue sections

    • Use validated antibodies specific to bovine ITGA3

    • Consider antigen retrieval methods to expose epitopes

  • Western blotting: Effective for semi-quantitative analysis

    • Use appropriate detergents for membrane protein extraction

    • Include both reducing and non-reducing conditions to assess protein integrity

    • Commercial antibodies are available that have been validated for bovine ITGA3 detection

  • Flow cytometry: Ideal for quantitative analysis in cell suspensions

    • Use gentle enzymatic dissociation methods to preserve surface integrins

    • Consider dual staining for both α3 and β1 subunits

• mRNA-level detection:

  • RT-qPCR: Offers high sensitivity and specificity

    • Design primers specific to bovine ITGA3 sequences

    • Validate primer efficiency using standard curves

    • Use multiple reference genes for accurate normalization

  • In situ hybridization: Provides spatial information on mRNA expression

    • Design probes specific to bovine ITGA3 mRNA

    • Include appropriate controls to confirm specificity

• Data analysis considerations:

  • Normalize protein expression to appropriate loading controls

  • Consider the ratio of ITGA3 to potential beta partners (particularly β1)

  • Account for tissue-specific expression patterns when comparing across samples

What strategies can improve the yield and purity of Recombinant Bovine ITGA3 expression?

Optimizing yield and purity of Recombinant Bovine ITGA3 requires strategic approaches tailored to the chosen expression system:

• E. coli expression optimization:

  • Use codon-optimized sequences for bovine ITGA3

  • Consider expressing functional domains rather than full-length protein

  • Employ fusion partners to enhance solubility

  • Optimize induction conditions (temperature reduction, lower inducer concentrations)

• Insect cell/Baculovirus system:

  • Optimize multiplicity of infection and harvest time

  • Consider using secretion signal sequences for easier purification

  • Implement fed-batch culture techniques to increase cell density

  • Monitor protein expression with time-course analysis

• Mammalian cell expression:

  • Select high-expressing stable cell lines through antibiotic selection

  • Consider using systems with inducible promoters for controlled expression

  • Optimize media composition and feeding strategies

• Purification strategy enhancement:

  • Design multi-step purification protocols combining affinity, ion exchange, and size exclusion chromatography

  • Include specific elution conditions to maintain protein integrity

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

• Quality control methods:

  • SDS-PAGE and Western blotting to assess purity and integrity

  • Mass spectrometry to confirm protein identity

  • Functional binding assays to confirm activity

Multiple commercial sources offer recombinant bovine ITGA3 produced using various expression systems, providing options that may be suitable for different research applications .

How can I develop reliable binding assays for studying bovine ITGA3 interactions with various ligands?

Developing reliable binding assays for bovine ITGA3 requires careful consideration of assay format, detection methods, and controls:

• Solid-phase binding assays:

  • ELISA-based approaches:

    • Coat plates with purified ligands or recombinant ITGA3

    • Block thoroughly to reduce non-specific binding

    • Use detection antibodies with minimal cross-reactivity

    • Previous studies have used this approach to demonstrate penton base protein binding to immobilized α3β1

  • Optimization parameters:

    • Buffer composition (include divalent cations essential for integrin function)

    • Incubation times and temperatures

    • Washing stringency

    • Detection system sensitivity

• Solution-phase binding assays:

  • Surface Plasmon Resonance (SPR):

    • Immobilize either ITGA3 or ligand using appropriate chemistry

    • Optimize surface density to avoid mass transport limitations

    • Include reference surfaces for background subtraction

• Cell-based binding assays:

  • Cell adhesion models:

    • Express bovine ITGA3 in appropriate cell lines (COS cells have been used successfully for integrin expression studies)

    • Quantify adhesion to surfaces coated with potential ligands

    • Use function-blocking antibodies as controls

    • Studies have shown that cells expressing recombinant integrin α3β1 can be used to study virus-receptor interactions

• Critical controls:

  • Positive controls: Known ITGA3 binding partners (laminin, fibronectin)

  • Negative controls: Non-binding proteins of similar size and charge

  • Specificity controls: Competition with function-blocking antibodies

  • System validation: Comparison with published binding constants

• Competition approaches:

  • Include potential inhibitory peptides (such as GRGDSP)

  • Test function-blocking antibodies against specific integrin subunits

  • Previous research has used anti-α3 antibodies as specific competitors for binding to α3β1

Why might recombinant bovine ITGA3 show reduced binding activity compared to native protein?

Recombinant bovine ITGA3 may show reduced binding activity compared to the native protein due to several factors:

• Structural considerations:

  • Incomplete folding: Recombinant expression may not fully recapitulate the native folding pathway

  • Missing post-translational modifications: Glycosylation patterns may differ depending on the expression system

  • Improper disulfide bond formation: Especially in bacterial expression systems

  • Lack of heterodimer formation: Native ITGA3 functions as a heterodimer with β1, which may be absent in recombinant preparations

• Expression system limitations:

  • E. coli: Lacks machinery for proper folding and post-translational modifications of complex eukaryotic proteins

  • Yeast: May introduce hypermannosylation not present in bovine cells

  • Insect cells: Closer to mammalian systems but still have differences in glycosylation

• Methodological factors:

  • Denaturation during purification: Harsh elution conditions or pH changes

  • Loss of cofactors: Divalent cations (Ca²⁺, Mg²⁺) are essential for integrin function

  • Protein truncation: "Partial" recombinant ITGA3 may lack crucial binding regions

• Solutions and workarounds:

  • Co-express with bovine β1 integrin to form functional heterodimers

  • Include metal ions in all buffers

  • Use mild purification conditions and avoid harsh elution methods

  • Consider expressing only the extracellular domain with appropriate tags

How can I address specificity concerns when working with anti-bovine ITGA3 antibodies?

Addressing specificity concerns with anti-bovine ITGA3 antibodies requires systematic validation and appropriate controls:

• Sources of non-specificity:

  • Cross-reactivity with other integrin alpha subunits (particularly α5, α6, α7)

  • Recognition of denatured epitopes not present in native conformation

  • Species cross-reactivity when using commercial antibodies

  • Multiple commercial sources offer anti-ITGA3 antibodies with different applications and species reactivity profiles

• Validation strategies:

  • Western blotting:

    • Compare patterns in ITGA3-expressing vs. knockout/knockdown samples

    • Look for bands of the expected molecular weight

    • Include recombinant ITGA3 as positive control

  • Immunoprecipitation followed by mass spectrometry:

    • Confirm identity of precipitated proteins

    • Identify potential cross-reactive proteins

  • Flow cytometry:

    • Compare staining patterns on cells with known ITGA3 expression levels

    • Use appropriate controls to assess background and non-specific binding

• Methodological considerations:

  • Use multiple antibodies targeting different epitopes when possible

  • Consider monoclonal antibodies for higher specificity

  • Optimize antibody concentration through titration experiments

  • Commercial antibodies are available in various formats including unconjugated, HRP-conjugated, and FITC-conjugated

• Controls to include:

  • Positive controls: Tissues/cells known to express ITGA3 (epithelial cells)

  • Negative controls: Tissues/cells with minimal ITGA3 expression

  • Technical controls: Isotype controls, secondary-only controls

  • Validation controls: siRNA knockdown or CRISPR knockout of ITGA3

What are the potential pitfalls in interpreting ITGA3 binding data from in vitro versus in vivo studies?

Interpreting ITGA3 binding data requires careful consideration of differences between in vitro and in vivo contexts:

• Structural and conformational differences:

  • In vitro simplification: Purified recombinant ITGA3 may lack native conformational states

  • Activation state: Integrins exist in multiple conformational states that may not be fully recapitulated in vitro

  • Heterodimer formation: ITGA3 functions as a heterodimer with β1, which may be incomplete in some in vitro systems

• Environmental factors:

  • Matrix complexity: In vivo environments contain multiple overlapping ligands and binding partners

  • Mechanical forces: Cell-generated tension affects integrin activation and binding, often absent in vitro

  • Local concentrations: Spatial organization and clustering effects in cellular membranes

• Cellular context:

  • Membrane composition: Lipid rafts and membrane microdomains affect integrin function

  • Cytoskeletal interactions: Intracellular integrin domains interact with cytoskeletal components

  • Signaling crosstalk: Other receptors modulate integrin activity through inside-out signaling

• Methodological considerations:

  • Binding kinetics: In vitro measurements may not capture the dynamic nature of in vivo interactions

  • Detection limitations: Some assays may not distinguish between specific and non-specific binding

  • Avidity effects: Multivalent interactions in cellular contexts versus monovalent binding in solution

• Bridging the gap:

  • Studies of adenovirus interactions with integrins demonstrate that while the RGD motif is important in simple in vitro assays, the actual virus-integrin interaction involves multiple contact sites

  • This illustrates how simplified in vitro binding studies may not capture the full complexity of biological interactions