BMP 7 Human, His

What is BMP-7 Human His-tagged protein and how is it produced?

BMP-7 Human His-tagged protein is a recombinant form of human Bone Morphogenetic Protein 7 engineered with a histidine tag to facilitate purification and experimental applications. The mature human BMP-7 protein consists of 139 amino acids derived from a larger 431 amino acid pro-protein form . When produced with a His-tag in E. coli expression systems, the protein typically contains 148 amino acids (spanning positions 293-431 of the native sequence) with a molecular weight of approximately 16.5kDa .

The production methodology typically involves:

• Cloning the coding sequence for mature BMP-7 into a bacterial expression vector

• Adding a 6x histidine tag (His-tag) sequence, usually at the C-terminus

• Transforming the construct into E. coli

• Inducing protein expression under optimized conditions

• Purifying the protein through chelating chromatography that captures the His-tag

This recombinant form is non-glycosylated due to E. coli's inability to perform post-translational glycosylation, which distinguishes it from native BMP-7 and from versions produced in eukaryotic expression systems .

What are the key differences between E. coli-produced and mammalian cell-produced BMP-7?

The expression system fundamentally alters the properties of recombinant BMP-7:

The choice between these forms depends on your experimental needs. E. coli-produced His-tagged BMP-7 is advantageous for structural studies, high-yield applications, and experiments where glycosylation is not critical . Conversely, HEK cell-produced BMP-7 more closely resembles the native protein configuration and is preferred for in vivo studies and cellular assays where physiological activity is paramount .

How should BMP-7 His-tagged protein be stored and handled for optimal stability?

Based on established protocols for recombinant BMP proteins, proper handling of BMP-7 His-tagged protein requires:

• Storage at -80°C for long-term stability or -20°C for short-term use

• Avoiding repeated freeze-thaw cycles (aliquot upon initial thawing)

• Working with the protein in buffers containing stabilizing agents:

  • Typically phosphate or HEPES-based buffers at pH 7.4

  • Often containing 0.15M NaCl

  • May include low concentrations of carrier proteins (0.1-0.5% BSA)

  • Sometimes with 1-5% glycerol to prevent freeze damage

When conducting experiments, His-tagged BMP-7 is typically used at concentrations between 1-1000 nM depending on the assay type . Surface Plasmon Resonance (SPR) experiments with BMP-7 commonly employ HBS-EP buffer (0.01M HEPES, pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% surfactant P20) .

How does BMP-7 function in bone and cartilage development?

BMP-7 plays critical roles in the differentiation of mesenchymal cells into bone and cartilage through several key mechanisms:

• Receptor Binding: BMP-7 binds to type I and type II serine/threonine kinase receptors on the cell surface

• SMAD Pathway Activation: Upon binding, BMP-7 induces phosphorylation of SMAD1 and SMAD5 transcription factors

• Gene Transcription: Phosphorylated SMADs translocate to the nucleus and activate transcription of numerous osteogenic genes

• Differentiation Markers: BMP-7 treatment induces the expression of all genetic markers associated with osteoblast differentiation in multiple cell types

• Cartilage Maintenance: BMP-7 helps maintain chondrocyte phenotype and counteracts pathological osteoarthritic changes in cartilage

For experimental verification of BMP-7 activity in osteogenesis, researchers commonly assess:

• Alkaline phosphatase (ALP) activity

• Expression of osteogenic markers including RUNX2, COL1A1, and osteocalcin

• Mineral deposition using Alizarin Red staining

• SMAD phosphorylation by Western blotting

For chondrogenesis, key assessment parameters include:

• SOX9, COL2A1, and NKX3-2 expression (chondrocyte markers)

• Suppression of COL10A1, RUNX2, and ALPL (hypertrophic markers)

How does BMP-7 heterodimerization with other BMPs affect its signaling properties?

Recent research has revealed that BMP-7 functions predominantly as a heterodimer with BMP-2 or BMP-4 during mammalian development, which significantly impacts its signaling properties:

• Formation of Heterodimers: BMP-7 readily forms heterodimers with BMP-2 and BMP-4 in vivo, and these heterodimers appear to be the predominant active forms

• Developmental Importance: Studies using a cleavage mutant mouse (Bmp7 R-GFlag) that prevents proteolytic activation of BMP-7 demonstrated that:

  • While Bmp7-null homozygotes are live born, Bmp7 R-GFlag homozygotes are embryonic lethal

  • Compound heterozygotes carrying the R-GFlag Bmp7 allele with null alleles of Bmp2 or Bmp4 die during embryogenesis with defects in ventral body wall closure and/or heart development

• Signaling Potency: BMP heterodimers typically exhibit enhanced signaling potency compared to homodimers, with:

  • Increased binding affinity to receptors

  • More efficient induction of downstream SMAD phosphorylation

  • Different threshold concentrations for biological effects

To experimentally investigate BMP-7 heterodimers, researchers employ:

• Co-immunoprecipitation assays to confirm the existence of endogenous BMP heterodimers

• Recombinant production of defined heterodimers through co-expression systems

• Cleavage-resistant mutant forms to disrupt heterodimer formation in vivo

What experimental approaches can be used to study BMP-7 interactions with its receptors?

Several methodological approaches have proven effective for investigating BMP-7 interactions with its receptors:

• Surface Plasmon Resonance (SPR):

  • BMP-7 growth factor (GF) is covalently coupled to sensor chips (~500 resonance units)

  • Potential binding partners are flowed over in appropriate buffers

  • For receptor competition experiments, BMP-7 is immobilized and soluble BMPRII extracellular domain is injected at varying concentrations (0-500 nM)

  • Detection of bound growth factor can be enhanced using monoclonal anti-BMP-7 antibodies

• Co-immunoprecipitation:

  • Cells expressing BMP receptors are treated with His-tagged BMP-7

  • Complexes are immunoprecipitated using anti-receptor or anti-His antibodies

  • Western blotting confirms the presence of receptor-ligand complexes

• Reporter Gene Assays:

  • Cells transfected with BMP-responsive elements driving luciferase expression

  • Dose-dependent activation of reporter gene expression by BMP-7

  • Competition with soluble receptors or inhibitors quantifies binding affinities

• SMAD Phosphorylation Assays:

  • Western blotting for phosphorylated SMAD1/5/8 following BMP-7 treatment

  • Time-course and dose-response analyses provide kinetic binding information

SPR experiments with BMP-7 have shown that pH significantly affects binding interactions, with studies conducted at both physiological pH 7.4 and acidic pH 4.5 revealing different binding characteristics .

How can bioactive peptides derived from BMP-7 be identified and characterized?

Recent research has successfully identified bioactive peptides derived from BMP-7 that retain specific biological activities, particularly for applications in osteoarthritis treatment. The methodological approach includes:

• Peptide Library Design:

  • Create overlapping sequential peptides covering the complete mature human BMP-7 sequence

  • For optimal screening, use 20-mer peptides with 2 amino acid intervals (18 amino acid overlap)

  • Substitute cysteine residues with serine to avoid uncontrolled oxidation of cysteine groups

  • This approach yielded 61 individual peptides spanning the complete mature BMP-7 sequence

• Screening Methodology:

  • Test peptides at multiple concentrations (1, 10, 100, 1000 nM)

  • Use relevant cell types (e.g., primary human osteoarthritic chondrocytes)

  • Assess changes in expression of disease-relevant genes compared to full-length BMP-7

• Validation of Candidate Peptides:

  • Confirm activity in disease-relevant environments (e.g., OA synovial fluid)

  • Analyze dose-response relationships

  • Compare with full-length BMP-7

Using this approach, researchers identified two specific regions within BMP-7 that yield bioactive peptides capable of attenuating the pathological osteoarthritic chondrocyte phenotype: peptides p[63-82] and p[113-132] . These peptides counteracted OA-associated changes in gene expression, including:

• Increasing SOX9, COL2A1, and NKX3-2 expression

• Decreasing RUNX2, COL10A1, ALPL, MMP13, ADAMTS5, COX-2, and IL6 expression

What methodological approaches are used to assess BMP-7's anti-inflammatory properties?

BMP-7 is recognized as a potent anti-inflammatory growth factor. To experimentally assess its anti-inflammatory properties:

• Gene Expression Analysis:

  • qRT-PCR for inflammatory cytokines (IL-1β, IL-6, TNF-α)

  • Assessment of anti-inflammatory markers (IL-10, TGF-β)

  • Analysis of inflammatory enzymes (COX-2, iNOS)

• Protein Secretion Measurements:

  • ELISA for inflammatory mediators

  • Multiplex cytokine arrays

  • Measurement of prostaglandin E2 (PGE2) secretion

• Signaling Pathway Analysis:

  • Western blotting for NFκB pathway components

  • Phosphorylation status of inflammatory signal transducers

  • Nuclear translocation of transcription factors

• Functional Assays:

  • Migration and invasion assays of inflammatory cells

  • Inflammatory cell adhesion to endothelial cells

  • Macrophage polarization (M1/M2 ratio)

In cellular models of osteoarthritis, BMP-7 and its derived peptides have been shown to counteract inflammation by:

• Reducing the expression of pro-inflammatory mediators (COX-2, IL-6)

• Decreasing PGE2 secretion in chondrocytes exposed to osteoarthritic synovial fluid

• Modulating the expression of matrix-degrading enzymes (MMP13, ADAMTS5)

What are the key considerations for designing experiments with BMP-7 in kidney research models?

BMP-7 plays critical roles in kidney development and protection from fibrosis, requiring specialized experimental approaches:

• Developmental Studies:

  • BMP-7 induces mesenchymal-epithelial transition (MET) of the metanephrogenic blastema, critical for nephron formation

  • Ex vivo kidney explant cultures can be used to assess BMP-7's role in tubular and glomerular development

  • Lineage tracing with reporter genes helps track MET progression

• Fibrosis Models:

  • BMP-7 inhibits epithelial-mesenchymal transition (EMT) which can lead to kidney fibrosis

  • Unilateral ureteral obstruction (UUO) models

  • Ischemia-reperfusion injury models

  • Monitoring of fibrotic markers (α-SMA, collagen deposition, fibronectin)

  • Histological assessment using Masson's Trichrome or Sirius Red staining

• Dosing Considerations:

  • Recombinant BMP-7 is typically administered at 50-300 μg/kg in animal models

  • For in vitro studies with kidney cell lines (HK-2, MDCK), concentrations of 50-200 ng/ml are commonly used

  • Treatment duration varies from acute (24-72h) to chronic (1-4 weeks) depending on the model

• Delivery Methods:

  • Systemic administration (intravenous or intraperitoneal injection)

  • Local administration (subcapsular injection)

  • Sustained release systems (critical for maintaining therapeutic levels)

BMP-7 expression is known to be attenuated when the kidney is under inflammatory or ischemic stress, which contributes to EMT and fibrosis development . Therefore, experimental timing is crucial when studying BMP-7's protective effects.

How can CRISPR/Cas9 technology be applied to study BMP-7 function?

CRISPR/Cas9 technology offers powerful approaches for investigating BMP-7 function:

• Gene Knockout Strategies:

  • Complete BMP-7 gene knockout to study null phenotypes

  • Conditional knockout using Cre-loxP systems for tissue-specific deletion

  • Targeting of specific domains to create truncated proteins

• Knock-in Approaches:

  • Introduction of point mutations to study specific amino acid functions

  • Creation of cleavage-resistant mutants (e.g., Bmp7 R-GFlag) to prevent proteolytic activation

  • Addition of epitope tags (Flag, HA) for tracking endogenous BMP-7

• Regulatory Element Modification:

  • Targeting enhancers or promoters to modulate expression levels

  • Creating reporter knock-ins to monitor BMP-7 expression patterns

• Experimental Design Considerations:

  • Off-target effects must be carefully controlled

  • Multiple guide RNAs should be used to confirm phenotypes

  • Rescue experiments with wild-type BMP-7 confirm specificity

  • Heterozygous models may be more informative than complete knockouts in some cases

A particularly informative approach used in recent research involved generating knock-in mice carrying a mutation (Bmp7 R-GFlag) that prevents proteolytic activation of the BMP-7 precursor protein, which revealed that BMP-7 functions predominantly as a heterodimer with BMP-2 or BMP-4 during mammalian development .

What quality control methods should be used to verify the integrity of BMP-7 His-tagged protein?

Rigorous quality control is essential to ensure experimental reproducibility with BMP-7 His-tagged protein:

• SDS-PAGE Analysis:

  • Confirms proper molecular weight (16.5 kDa for E. coli-produced BMP-7-His)

  • Assess purity (typically >95% for research applications)

  • Under reducing conditions to evaluate monomer size

  • Under non-reducing conditions to assess potential dimer formation

• Western Blotting:

  • Anti-BMP-7 antibodies confirm identity

  • Anti-His antibodies verify tag presence and integrity

• Mass Spectrometry:

  • Precise molecular weight determination

  • Sequence coverage analysis

  • Post-translational modification assessment

• Endotoxin Testing:

  • Critical for E. coli-produced proteins

  • Limulus Amebocyte Lysate (LAL) assay

  • Acceptable levels typically <1 EU/μg protein

• Bioactivity Assays:

  • SMAD phosphorylation in responsive cell lines

  • ALP induction in pre-osteoblasts

  • Reporter gene assays with BMP-responsive elements

• Structure Verification:

  • Circular dichroism to assess secondary structure

  • Dynamic light scattering for aggregation detection

A common issue with recombinant BMP-7 is the potential presence of misfolded or inactive protein. Functional assays are therefore essential to confirm biological activity before experimental use.