TFF1 Human, His

What is the molecular structure of human TFF1 and how does His-tagging affect it?

Human TFF1 is a small secreted protein comprising 60 amino acids with a characteristic trefoil domain extending from residues 6-47. The domain is stabilized by three intramolecular disulfide bonds formed between Cys7-Cys33, Cys17-Cys32, and Cys27-Cys44 . A unique feature of TFF1 is the presence of a C-terminal 7th cysteine residue (Cys58) located outside the TFF domain, flanked by four glutamic acid residues (E55EEC58E59) .

When producing recombinant TFF1 with a histidine tag (TFF1-His), researchers should consider tag positioning carefully. N-terminal His-tags are generally preferred as the C-terminal region contains the unpaired Cys58 that is functionally significant. The addition of a His-tag facilitates protein purification through immobilized metal affinity chromatography (IMAC) but may potentially influence protein folding or activity, necessitating validation studies comparing tagged and untagged versions.

How stable is the TFF1 domain under reducing conditions and what implications does this have for experimental handling?

The TFF1 domain exhibits remarkable stability against reducing conditions. Experimental evidence shows that full reduction of TFF1's disulfide bonds only occurs with a large excess of reducing agent (150-fold TCEP), with no partially reduced intermediates observed . This unusual stability is supported by molecular dynamics simulations revealing that the domain substantially retains its compactness and solvent exposure even when one or two disulfide bonds are removed .

For researchers, this has significant implications:

• Standard reducing buffers may be insufficient for complete denaturation during SDS-PAGE analysis

• Experimental protocols requiring reduction should use higher concentrations of reducing agents than typically employed for other proteins

• The protein maintains structural integrity in mildly reducing environments, which may be relevant for studies in different cellular compartments

What are the optimal expression systems for producing functional human TFF1-His protein?

The selection of an expression system for TFF1-His production should consider the protein's disulfide bonding requirements and post-translational modifications.

Recommended expression systems comparison:

When using bacterial systems, co-expression with disulfide isomerases or directing TFF1-His to the periplasmic space can improve correct disulfide bond formation. For mammalian systems, optimizing secretion signal sequences can enhance yields.

What purification strategy ensures high purity TFF1-His while preserving its functional properties?

A multi-step purification strategy is recommended for TFF1-His:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins (pH 7.4-8.0, 20-50 mM imidazole in binding buffer to reduce non-specific binding)

• Intermediate purification: Ion-exchange chromatography (IEX) - given TFF1's acidic nature, anion exchange chromatography at pH 8.0 is effective for separating different forms (monomers, dimers)

• Polishing step: Size exclusion chromatography (SEC) on a high-resolution column to separate monomeric, dimeric, and heterodimeric forms

• Quality control: Verification of purity by SDS-PAGE under non-reducing and reducing conditions to confirm the presence of different oligomeric states

This approach allows separation of different TFF1 forms, which is crucial since human TFF1 exists in multiple states including monomers with free thiol groups, homodimers, and heterodimers with partners like GKN2 .

How can researchers effectively distinguish between different oligomeric forms of TFF1?

Distinguishing between TFF1 oligomeric forms requires a combination of techniques:

• Non-reducing vs. reducing SDS-PAGE: The different forms (monomer, homodimer, heterodimers) will show distinct migration patterns under non-reducing conditions but convert to monomers under reducing conditions

• Size exclusion chromatography: Use high-resolution columns (e.g., Sephacryl S-500) to separate forms based on molecular size

• Anion-exchange chromatography: Different TFF1 forms show distinct elution patterns, with the TFF1-GKN2 heterodimer and TFF1-FCGBP complexes eluting at different positions than monomers

• Mass spectrometry: To precisely identify the components in each oligomeric form, particularly for novel heterodimers

• Thiol-specific labeling: PEG-maleimide can be used to detect the presence of free thiol groups, characteristic of monomeric TFF1 with unpaired Cys58

Research indicates that human gastric TFF1 exists predominantly in monomeric forms with free thiols, which contradicts the expectation that secretory proteins typically have all cysteines engaged in disulfide bonds .

What methodological approaches can verify the tumor suppressor function of TFF1 in experimental models?

To investigate TFF1's tumor suppressor function, researchers can employ multiple complementary approaches:

• In vitro cellular assays:

  • Gain-of-function: Expressing TFF1 in TFF1-negative cell lines (e.g., MCF10A, MDA-MB-231) to assess changes in migration, invasion, and anchorage-independent growth

  • Loss-of-function: Knockdown TFF1 in TFF1-positive cell lines (e.g., MCF7, ZR75.1) using siRNA or CRISPR-Cas9 to evaluate oncogenic potential

• In vivo xenograft models:

  • Implant TFF1-expressing vs. control cells in nude mice to assess tumor formation and growth rates

  • TFF1-knockdown cells show enhanced tumorigenicity compared to parental cells

• Transgenic mouse models:

  • TFF1-deficient (TFF1-KO) mice exhibit higher incidence of mammary tumors and larger tumor sizes compared to wild-type mice when exposed to chemical carcinogens

  • Similar enhanced tumor development is observed in TFF1-KO ovary and lung tissues

• Molecular mechanism studies:

  • Analyze signaling pathway activation/inhibition (e.g., MAPK, PI3K/AKT)

  • Investigate TFF1 binding partners using co-immunoprecipitation followed by mass spectrometry

  • Examine effects on cell cycle progression and apoptosis markers

Research consistently shows that TFF1 reduces tumor development rather than exhibiting oncogenic properties, aligning with clinical observations that patients with TFF1-positive breast primary tumors have better outcomes .

How does the unique free cysteine residue (Cys58) in TFF1 contribute to its antioxidant properties, and how can this be experimentally validated?

The free cysteine residue (Cys58) in TFF1, flanked by acidic residues (PPEEEC58EF), is thought to function as a scavenger for extracellular reactive oxygen/nitrogen species (ROS/RNS), potentially protecting the gastric mucosa from oxidative damage .

Experimental approaches to validate this function:

• In vitro ROS/RNS scavenging assays:

  • Compare wild-type TFF1 vs. C58S mutant in H₂O₂ or peroxynitrite neutralization assays

  • Measure thiol oxidation rates using fluorescent probes like dibromobimane

• Cellular protection assays:

  • Expose gastric epithelial cells to oxidative stressors with/without TFF1 supplementation

  • Assess cell viability, membrane integrity, and intracellular ROS levels

• Redox state analysis:

  • Use mass spectrometry to characterize post-translational modifications of Cys58 in proteins isolated from gastric mucosa

  • Identify S-nitrosylation, S-glutathionylation, or other oxidative modifications

• Structure-function studies:

  • Investigate how copper ions, which bind to glutamic acid residues flanking Cys58, might catalyze S-nitrosylation reactions

  • Perform site-directed mutagenesis of flanking acidic residues to assess their contribution to the reactivity of Cys58

The unique position of Cys58 outside the TFF domain and its flanking by acidic residues makes it particularly suited for redox reactions, especially in the acidic environment of the stomach where nitrogen oxide chemistry is active .

What are the molecular mechanisms behind TFF1's selective binding to MUC6 but not MUC5AC, and how does this inform gastric mucosal protection studies?

Despite TFF1 being synthesized in surface mucous cells that produce MUC5AC, binding studies show that TFF1 preferentially interacts with MUC6, which is secreted by mucous neck and antral gland cells . This unexpected finding has significant implications for understanding gastric mucosal protection.

Methodological approaches to investigate this interaction:

• Binding specificity analysis:

  • Overlay assays using ¹²⁵I-labeled TFF1 homodimer with gastric extracts fractionated by anion-exchange chromatography or SEC

  • Compare binding patterns with lectins specific for MUC5AC vs. MUC6 (e.g., GSA-II for MUC6)

• Glycan interaction studies:

  • Characterize the glycan structures on MUC6 that mediate TFF1 binding

  • Employ glycosidase treatments to identify critical sugar moieties

  • Utilize glycan arrays to define TFF1's carbohydrate recognition profile

• Structural biology approaches:

  • X-ray crystallography or cryo-EM studies of TFF1-MUC6 complexes

  • NMR analysis of labeled TFF1 interacting with MUC6-derived glycopeptides

• Functional analyses:

  • Co-localization studies in gastric tissue sections

  • Investigate how TFF1-MUC6 interaction influences mucus rheological properties

  • Assess barrier function in models with disrupted TFF1-MUC6 binding

Research indicates that TFF1's lectin-like activity may explain its binding to MUC6, similar to how TFF1 interacts with Helicobacter pylori through core oligosaccharide binding . This interaction may contribute to TFF1's role as a tumor suppressor by influencing mucosal organization and integrity.

What strategies can overcome the challenges in detecting and quantifying different TFF1 forms in biological samples?

Accurate detection and quantification of TFF1 forms in biological samples present several challenges due to the protein's complex oligomerization behavior and redox sensitivity.

Recommended analytical approaches:

• Sample preparation optimization:

  • Use TCA/acetone precipitation followed by dissolution in 1% SDS to recover all TFF1 forms, especially the TFF1-GKN2 heterodimer which may be difficult to detect in standard extractions

  • Include protease inhibitors and alkylating agents (e.g., iodoacetamide) immediately upon sample collection to prevent artificial disulfide shuffling

• Immunodetection considerations:

  • Develop antibodies against different epitopes to ensure detection of all forms

  • Validate antibodies against recombinant standards of each TFF1 form

  • Use non-reducing conditions when necessary to preserve oligomeric states

• Mass spectrometry approaches:

  • Targeted multiple reaction monitoring (MRM) assays for specific TFF1 forms

  • Top-down proteomics to characterize intact proteins with post-translational modifications

  • Native MS to preserve non-covalent interactions

• Standardization of quantification:

  • Develop recombinant standards for each TFF1 form

  • Create isotopically labeled internal standards for absolute quantification

  • Establish correction factors for differential extraction efficiency of various forms

Research shows that significant amounts of TFF1 may exist in hard-to-solubilize forms or in complexes with other proteins, requiring specialized extraction methods for complete recovery .

How can researchers effectively distinguish between experimental artifacts and true biological heterogeneity when studying TFF1?

Distinguishing genuine biological heterogeneity from artifacts is critical when studying a protein like TFF1 that exists in multiple forms sensitive to redox conditions.

Methodological safeguards:

• Control for extraction artifacts:

  • Compare multiple extraction methods in parallel

  • Include spike-in controls of recombinant TFF1 forms

  • Process samples with minimal delay and under controlled temperature/pH conditions

• Redox state preservation:

  • Use oxygen-free buffers when appropriate

  • Add thiol-blocking agents at the time of sample collection

  • Compare results with and without reducing agents to identify artifactual disulfide formation

• Validation across techniques:

  • Confirm observations using orthogonal methods (e.g., western blotting, mass spectrometry, functional assays)

  • Compare in vitro observations with in vivo imaging when possible

• Biological replicates and controls:

  • Include appropriate tissue/cell controls where TFF1 is absent

  • Analyze samples from multiple individuals/animals

  • Use TFF1-knockout models as negative controls

• Data interpretation frameworks:

  • Establish clear criteria for distinguishing physiological heterogeneity from artifacts

  • Consider statistical approaches for pattern recognition across multiple samples

  • Document all sample handling conditions meticulously

Research indicates that TFF1 heterogeneity is genuine biological phenomenon, with forms like the TFF1-GKN2 heterodimer being reproducibly detected across multiple studies and species when appropriate extraction methods are used .