MIF Human His N

What is Human MIF protein with N-His tag?

Human MIF with N-terminal His tag is a recombinantly expressed version of the human cytokine MIF. It consists of the full 1-115 amino acid sequence of human MIF with a 6x Histidine tag added at the N-terminus. This 12 kDa protein is typically produced in E. coli expression systems and purified via Ni-NTA affinity chromatography . The N-His tag facilitates protein purification while maintaining the biological activities of MIF.

What are the structural characteristics of Human MIF?

Human MIF exists as a homotrimer in its active form. The crystal structure reveals a trimeric assembly with an inner pore created by β-stranded sheets from each subunit . MIF possesses a unique tautomerase enzyme activity that depends on a free N-terminal proline residue . Crystal structure analysis of MIF homologues shows remarkable conservation across species despite considerable phylogenetic distances, indicating essential structural features for its biological functions .

How is recombinant Human MIF with N-His tag produced and purified?

Recombinant Human MIF with N-His tag is commonly produced using E. coli expression systems. After expression, the protein undergoes purification via Ni-NTA affinity chromatography, taking advantage of the N-terminal His tag's affinity for nickel. The purified protein is typically filtered through a 0.2μm filter and formulated in Tris-HCl buffer (pH 8.0) before lyophilization . Alternative expression systems include insect cells, which may provide different post-translational modifications .

What are the optimal storage conditions for MIF Human His N protein?

According to product specifications, lyophilized Human MIF with N-His tag maintains stability when stored at -20°C for 2-3 years . After reconstitution with distilled PBS, the protein should be handled according to standard protein stability protocols. For experimental work requiring extended periods, it's advisable to prepare small aliquots to minimize freeze-thaw cycles that could compromise protein integrity and activity.

How can I assess MIF's tautomerase activity and the impact of the N-terminal His tag?

The tautomerase activity of MIF depends on a free N-terminal proline residue , making assessment of N-His tagged MIF's enzymatic function critical. Established methodologies include:

• Spectrophotometric assays using D-dopachrome or L-dopachrome methyl ester as substrates

• Monitoring absorbance decrease at 475 nm as substrate converts to product

• Comparative enzyme kinetics between His-tagged and untagged MIF

• Site-directed mutagenesis of the N-terminal proline to alanine as a negative control

Researchers should consider that the N-terminal His tag could potentially interfere with tautomerase activity. If enzymatic function is crucial to your research, enzymatic characterization comparing tagged and untagged versions is recommended.

What methods are most effective for studying MIF-inhibitor interactions?

Multiple complementary approaches provide comprehensive insight into MIF-inhibitor interactions:

• Virtual screening: Computational docking of potential inhibitors into MIF's active site using platforms like Glide. This approach has successfully identified inhibitors from libraries of over 2.1 million compounds .

• Enzyme kinetics assays: Determining inhibition constants (Ki or IC50) and mode of inhibition through:

  • Tautomerase activity assays with D-dopachrome or L-dopachrome methyl ester substrates

  • Lineweaver-Burk plots to visualize competitive vs. non-competitive inhibition

• Reversibility assessment: Activity recovery experiments where MIF is pre-incubated with saturating inhibitor concentrations (e.g., 25 μM) followed by rapid dilution (20-fold) to monitor enzyme activity recovery over time .

• Receptor binding assays: Coating 96-well plates with recombinant MIF receptor ectodomain (sCD74), incubating with biotinylated human MIF and potential inhibitors, then detecting binding with streptavidin-conjugated alkaline phosphatase .

How can I investigate MIF's role in cell death pathways?

The research literature identifies MIF's involvement in parthanatos, a PARP-1-dependent programmed cell death pathway. To investigate this role:

• MNNG-induced parthanatos model: Treat cells with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) to induce PARP-1 overactivation and subsequent cell death .

• Cell viability assessment: Use flow cytometry with dual staining (Hoechst 33342 and propidium iodide) to distinguish between live and dead cells following treatment with MIF inhibitors .

• Comparative inhibitor studies: Test different classes of MIF inhibitors (e.g., allosteric inhibitors like compound 6y versus competitive tautomerase inhibitors like ZP143) to distinguish between enzymatic and protein-protein interaction roles .

• Cellular morphology analysis: Observe changes in cell morphology to determine if MIF inhibition restores normal phenotype in cells undergoing parthanatos .

What techniques are available for studying MIF's interaction with CD74?

MIF initiates signal transduction by binding to the transmembrane protein CD74 . This interaction can be studied using:

• ELISA-based binding assays: Coat plates with recombinant MIF receptor ectodomain (sCD74 = CD74 73–232), add biotinylated human MIF with or without test compounds, and detect binding using streptavidin-conjugated alkaline phosphatase and p-nitrophenyl phosphate substrate .

• Cell-based receptor studies: Analyze signaling pathways downstream of MIF-CD74 binding, including MAPK activation .

• Inhibition analysis: Test compounds over 10-12 concentrations spanning a ~500-fold concentration range to generate accurate inhibition curves for the MIF-CD74 interaction .

How do MIF homologues from different species compare structurally and functionally?

Human MIF shares remarkable functional conservation with homologues from phylogenetically distant organisms, despite relatively low sequence identity:

• Parasite homologues: B. malayi MIF homologues (Bm-MIF-1 and Bm-MIF-2) share only 26-27% sequence identity with human MIF but maintain parallel functions .

• Conserved functions across species include:

  • Chemotactic activity for human monocytes

  • Activation of monocytes to produce IL-8, TNF-α, and endogenous MIF

  • Tautomerase enzyme activity dependent on the N-terminal proline residue

• Structural conservation: Crystal structure of Bm-MIF-2 at 1.8-Å resolution reveals a trimeric assembly similar to human MIF, with an inner pore created by β-stranded sheets from each subunit .

• Invariant residues: Six amino acid residues are completely conserved across all 19 known MIF homologues, indicating their critical importance for structure or function .

This evolutionary conservation suggests that MIF-mediated activities are essential biological processes conserved across diverse organisms, potentially informing therapeutic targeting strategies.

What is known about MIF expression and function in human skin?

MIF is expressed in human skin with specific localization patterns:

• Epidermal expression: RT-PCR analysis confirms MIF mRNA expression in both surgically obtained normal human epidermis and primary cultured human keratinocytes .

• Protein verification: Western blot analysis using polyclonal antibodies against human recombinant MIF demonstrates a single band at approximately 12.5 kDa in skin samples .

• Histological localization: Immunohistochemical studies reveal that MIF is present throughout human epidermis, with particularly high expression in the basal layer .

• Potential functions: While the precise pathophysiological role remains under investigation, research suggests MIF may play important roles in cutaneous immunity, inflammatory responses, and cellular differentiation of epidermal cells .

What types of MIF inhibitors exist and how do they differ in mechanism and effect?

Two primary classes of MIF inhibitors have been characterized with distinct mechanisms and effects:

• Competitive tautomerase inhibitors:

  • Target MIF's enzymatic activity by competing for the tautomerase active site

  • Examples include ISO-1, ISO-17, and ZP143

  • Generally do not protect against MNNG-induced parthanatos

• Allosteric inhibitors:

  • Modulate MIF's protein-protein interactions rather than enzymatic activity

  • Example: compound 6y shows reversible binding to MIF

  • Provide protection against MNNG-induced parthanatos in a dose-dependent manner with an EC50 of 7.7 ± 2.1 μM

  • Reduce cell death as measured by propidium iodide staining (20% in MNNG and 6y co-treated cells versus 50% in MNNG-only treated cells)

These different inhibitor classes help distinguish between MIF's enzymatic functions and its protein-protein interaction roles in various biological processes, providing valuable tools for mechanistic studies and potential therapeutic development.

What biological processes does MIF influence that make it a potential therapeutic target?

MIF influences numerous biological processes with implications for disease pathogenesis:

• Inflammation regulation: MIF counter-regulates glucocorticoid expression, which normally suppresses pro-inflammatory molecules .

• Angiogenesis promotion: MIF stimulates tumor cells to produce angiogenic factors such as IL-8 and VEGF .

• Tumor suppression inhibition: MIF directly downregulates the expression and function of the tumor-suppressor protein p53 .

• MAPK pathway activation: MIF activates MAPKs, enhancing cellular responses to various stimuli .

• Cell death modulation: MIF plays a role in parthanatos, a form of programmed cell death .

• Receptor-mediated signaling: MIF initiates signal transduction by binding to CD74, with inhibition of this interaction shown to attenuate tumor growth and angiogenesis .

These diverse biological activities position MIF as a potential therapeutic target for inflammatory conditions, cancer, and cell-death related disorders.