Recombinant Macaca fascicularis T-cell immunomodulatory protein (ITFG1)

What is the relationship between Macaca fascicularis ITFG1 and human ITFG1?

ITFG1, also known as T-cell immunomodulatory protein (TIP), is highly conserved across species. Human ITFG1 shares 89% amino acid identity with mouse ITFG1 and 88% with rat ITFG1 . The Macaca fascicularis genome sequencing has enabled identification of 17,387 orthologs of human protein-coding genes, including ITFG1 . When comparing sequences, researchers should perform detailed analysis using the UniProt accession numbers Q95KC8 (M. fascicularis) and Q8TB96 (human) . To properly characterize homology:

• Use multiple sequence alignment tools (MUSCLE, Clustal Omega)

• Perform phylogenetic analysis focusing on functional domains

• Compare post-translational modification sites across species

What are the key structural domains and features of M. fascicularis ITFG1?

M. fascicularis ITFG1, like its human counterpart, is a transmembrane glycoprotein with distinct structural elements:

• Contains an N-terminal signal peptide and C-terminal transmembrane domain

• Features integrin-alpha FG-GAP repeats (giving the protein its name)

• Has multiple potential N-linked glycosylation sites (human ITFG1 contains twelve)

• In full-length form, it's a 98 kDa glycoprotein

The amino acid sequence available (from search result ) allows researchers to predict structural domains using bioinformatics tools like SMART, Pfam, or PROSITE.

What expression systems are optimal for producing functional recombinant M. fascicularis ITFG1?

The choice of expression system depends on research needs and which domains require study:

For studies requiring immunomodulatory function, mammalian expression systems are strongly recommended to maintain post-translational modifications essential for activity .

What purification strategies yield the highest purity and activity for recombinant ITFG1?

A multi-step purification strategy yields the highest purity while maintaining protein activity:

• Initial capture: Affinity chromatography using appropriate tags (His-tag, GST, etc.)

• Intermediate purification: Ion exchange chromatography to remove contaminants with similar affinity but different charge properties

• Polishing step: Size exclusion chromatography to achieve final purity

Products described in the literature achieve >95% purity using SDS-PAGE analysis . For optimal activity maintenance:

• Perform all purification steps at 4°C

• Include protease inhibitors in buffers

• Minimize exposure to harsh pH conditions

• Validate functional activity after each purification step

How should recombinant M. fascicularis ITFG1 be stored to maintain stability?

Proper storage conditions are critical for maintaining ITFG1 stability and activity:

• Lyophilized protein: Store at -20°C for up to 12 months

• Reconstituted protein: Store at 2-8°C for up to 1 month under sterile conditions

• For reconstitution:

  • Centrifuge vial at 10,000 rpm for 1 minute

  • Reconstitute at 200 μg/mL in sterile distilled water

  • Mix by gentle pipetting (2-3 times), avoid vortexing

• For long-term storage of reconstituted protein, use buffer containing trehalose or glycerol (e.g., PBS with 5% trehalose, pH 7.4)

What assays effectively measure the immunomodulatory activity of recombinant ITFG1?

Based on known functions of ITFG1, these assays provide comprehensive functional characterization:

• Cytokine secretion profiles:

  • Measure IFN-γ, TNF-α, and IL-10 production by T cells using ELISA or multiplex assays

  • Compare cytokine ratios to determine pro/anti-inflammatory balance

• T cell functional assays:

  • Proliferation assays (CFSE dilution, tritiated thymidine incorporation)

  • Activation marker expression (CD25, CD69) by flow cytometry

• Advanced functional models:

  • In vitro mixed lymphocyte reactions

  • Protection in graft-versus-host disease models

  • Analysis of signaling pathway activation by phospho-flow cytometry

How does M. fascicularis ITFG1 affect specific cell types beyond T cells?

While ITFG1 was initially characterized for its effects on T cells, emerging research indicates broader functions:

• Hepatocytes: ITFG1 knockdown accelerates hepatocyte proliferation and wound healing in vitro, suggesting a role in regulating liver regeneration

• Membrane localization studies: ITFG1 primarily localizes to the plasma membrane , suggesting potential interactions with cell surface receptors on multiple cell types

• Protein interaction networks: Proteomic analysis identified 180 proteins that specifically associate with ITFG1, involving multiple cellular networks including cell cycle, mitochondrial translation initiation, and regulation of DNA repair

For comprehensive cellular effect characterization, researchers should:

• Perform RNA-seq on multiple cell types after ITFG1 treatment

• Use quantitative proteomics to identify changes in protein expression

• Employ tissue-specific conditional knockout models

What signaling pathways are modulated by ITFG1, and how can they be investigated?

ITFG1 appears to interact with multiple cellular pathways. To investigate these mechanisms:

• Protein interaction studies:

  • Validated interaction between ITFG1 and RUVBL1 provides a starting point

  • Conduct co-immunoprecipitation with mass spectrometry analysis to identify additional partners

  • Use proximity labeling techniques (BioID, APEX) to identify transient interactions

• Signaling pathway analysis:

  • Perform phosphoproteomic analysis before and after ITFG1 treatment

  • Use pathway inhibitor panels to identify essential signaling components

  • Apply CRISPR/Cas9 screening to identify genes required for ITFG1 response

• Transcriptomic analysis:

  • RNA-seq of cells/tissues after ITFG1 treatment or knockdown

  • The M. fascicularis-specific gene expression microarray mentioned in the literature provides a valuable tool

How does ITFG1 contribute to liver regeneration, and what mechanisms are involved?

Recent research has identified ITFG1 as a promising target for enhanced liver regeneration and chronic liver disease treatment:

• Experimental evidence:

  • Knockdown of ITFG1 in mouse and human hepatocytes accelerates proliferation and wound healing in vitro

  • In vivo knockdown accelerates liver repopulation in FAH-deficient mice

  • ITFG1 knockdown enhances regeneration after partial hepatectomy

• Mechanism investigation methods:

  • Transcriptomic and proteomic analysis of liver tissue after ITFG1 knockdown

  • Analysis of cell cycle regulators and proliferation markers

  • Investigation of inflammatory mediators that may influence regeneration

• Disease models:

  • Thioacetamide (TAA) model of chronic liver disease

  • "Western Diet" model of NAFLD

How can gene editing approaches be used to investigate ITFG1 function?

CRISPR/Cas9 technology offers powerful tools for ITFG1 functional studies:

• Available tools:

  • ITFG1 sgRNA CRISPR/Cas9 All-in-One Lentivector sets are commercially available for human ITFG1

  • These tools include three sgRNA targets designed to cleave exonic gDNA, resulting in frameshift mutations and gene knockout

• Experimental approaches:

  • Complete gene knockout studies to assess loss-of-function phenotypes

  • Domain-specific mutations to determine structure-function relationships

  • Knockin of reporter tags for live cell imaging of ITFG1 localization and trafficking

• Validation methods:

  • Surveyor assay to confirm editing efficiency

  • Sanger sequencing to characterize specific mutations

  • Functional assays to confirm phenotypic changes

How can researchers leverage M. fascicularis ITFG1 studies for human therapeutic applications?

The cynomolgus monkey (M. fascicularis) is one of the most important nonhuman primate models in biomedical research . For translational ITFG1 research:

• Comparative studies:

  • Determine functional conservation between species using recombinant proteins from both sources

  • Test cross-reactivity of therapeutic candidates against both human and M. fascicularis ITFG1

  • Compare signaling responses in cells from both species

• Preclinical model development:

  • The sequenced M. fascicularis genome provides a foundation for genetically precise models

  • M. fascicularis-specific gene expression microarrays enable detailed transcriptomic analysis

• Therapeutic development strategies:

  • Anti-ITFG1 antibodies for blocking studies (commercially available conjugated antibodies can be used as a starting point)

  • Small molecule inhibitors targeting ITFG1-dependent pathways

  • RNA interference approaches similar to those used in liver regeneration studies

What challenges exist in developing ITFG1-targeted therapeutics?

Several technical and biological challenges must be addressed:

• Biological complexity:

  • ITFG1 appears to have multiple functions in different tissues

  • As a transmembrane protein, accessibility for therapeutic targeting may be limited

  • Potential for compensatory mechanisms through related proteins

• Technical considerations:

  • Production of consistent, fully glycosylated recombinant protein for functional studies

  • Development of specific assays to measure on-target activity

  • Species differences that may limit predictive value of animal models

• Therapeutic modality selection:

  • Antibody-based approaches for surface-exposed domains

  • Small molecules for disrupting specific protein interactions

  • RNA therapeutics for tissue-specific knockdown similar to successful liver models

Understanding these challenges and developing strategies to address them will be crucial for successful translation of ITFG1 research from bench to bedside.

What are the optimal methods for detecting ITFG1 expression in tissues and cells?

Multiple complementary approaches provide comprehensive expression analysis:

• Protein detection:

  • Western blotting using validated antibodies

  • Flow cytometry for cell surface expression

  • Immunohistochemistry for tissue localization

• mRNA analysis:

  • qRT-PCR using species-specific primers

  • RNA-seq for global expression analysis

  • In situ hybridization for spatial expression patterns

• Subcellular localization:

  • Subcellular fractionation followed by immunoblotting

  • Immunofluorescence microscopy

When analyzing ITFG1 expression in M. fascicularis samples, species-specific reagents or carefully validated cross-reactive antibodies should be used to ensure accurate results.

What experimental models are most suitable for studying ITFG1 function in specific disease contexts?

Based on current research, these models have proven valuable for ITFG1 functional studies:

For M. fascicularis studies specifically, primary cells isolated from tissues provide the most relevant experimental system, though immortalized cell lines may be used for initial screening.