Recombinant Rat FK506-binding protein-like (Fkbpl)

What is the structural organization of recombinant rat Fkbpl and how does it differ from other immunophilins?

Recombinant rat Fkbpl belongs to the immunophilin family but represents a divergent member with unique structural features. Based on homology with human FKBPL, rat Fkbpl contains:

• A C-terminal tetratricopeptide repeat (TPR) domain that shares homology with other FKBP family members (particularly FKBP52/51 and cyclophilin 40)

• A weakly homologous peptidyl-prolyl isomerase (PPIase) domain that lacks crucial residues required for enzymatic activity

• Unlike classical FKBPs, Fkbpl has limited binding affinity for immunosuppressive drugs like FK506 and rapamycin due to its divergent structure

This structural organization suggests rat Fkbpl functions primarily through protein-protein interactions rather than enzymatic activity, particularly via its TPR domain which enables interaction with heat shock proteins and steroid receptor complexes .

What expression systems are most effective for producing recombinant rat Fkbpl?

The optimal expression system depends on your research requirements. Based on approaches used for similar proteins:

Bacterial Expression Systems:

• E. coli BL21(DE3) strains with pET vector systems typically yield high quantities

• Expression is generally induced with IPTG (0.1-1.0 mM)

• Lower temperatures (16-25°C) post-induction often improve solubility

• Addition of glycerol (5-10%) to lysis buffer enhances stability during purification

Mammalian Expression Systems:

• HEK293 or CHO cells are preferable when post-translational modifications are critical

• Strong expression can be achieved with CMV promoter-containing vectors

Purification Strategy:

• Affinity chromatography using His-tag or GST-tag for initial capture

• Size exclusion chromatography for further purification

• Expected yields: 2-5 mg/L in bacterial systems; 0.5-2 mg/L in mammalian systems

How should recombinant rat Fkbpl be stored to maintain maximum activity?

Storage conditions significantly impact the stability and activity of recombinant Fkbpl:

Recommended Storage Buffer:

• PBS (pH 7.4) with 10% glycerol

• Addition of 1 mM DTT may improve long-term stability

• Protease inhibitors are advisable for extended storage

Best Practices:

• Aliquot protein to minimize freeze-thaw cycles

• Use rapid thawing at room temperature followed by immediate transfer to ice

• For experimental use, maintain on ice and use within 8 hours after thawing

How does recombinant rat Fkbpl interact with steroid receptor complexes and what are the functional implications?

Recombinant rat Fkbpl, like human FKBPL, functions as a co-chaperone within steroid hormone receptor complexes:

Interaction Mechanism:

• Fkbpl associates with Hsp90 through its TPR domain

• This interaction enables Fkbpl to participate in steroid receptor signaling complexes

• Unlike some FKBPs with PPIase activity, Fkbpl's role appears to be primarily structural rather than enzymatic

Functional Implications:

• Fkbpl is involved in regulating steroid receptor maturation, trafficking, and signaling

• Its presence in receptor complexes can affect hormone binding affinity and downstream responses

• These interactions suggest Fkbpl may influence hormone-dependent processes in various tissues

When designing experiments to study these interactions:

• Co-immunoprecipitation assays can demonstrate physical interaction between rat Fkbpl and Hsp90

• Cell-based reporter assays can assess Fkbpl's ability to modulate steroid receptor transcriptional activity

• Confocal microscopy can reveal co-localization patterns with steroid receptors

What is the current understanding of recombinant rat Fkbpl's role in angiogenesis?

Based on studies with human FKBPL, recombinant rat Fkbpl likely exhibits anti-angiogenic properties:

Anti-angiogenic Mechanisms:

• Inhibition of endothelial cell migration and proliferation

• Disruption of vessel formation in angiogenesis models

• Potential interference with VEGF signaling pathways

Experimental Evidence:

• Human recombinant FKBPL and its peptide derivatives have been assessed in human microvascular endothelial cell (HMEC-1) assays

• These studies demonstrated inhibition of proliferation and migration

• The anti-angiogenic activity appears distinct from other known anti-angiogenic agents

Research Applications:

• Rat models using recombinant rat Fkbpl can provide valuable insights into angiogenic regulation

• Comparative studies between rat and human Fkbpl can help identify conserved mechanisms

• Understanding species differences may inform translation of Fkbpl-based therapeutics

To investigate this property, researchers typically employ:

• Endothelial cell proliferation assays (BrdU incorporation or MTT)

• Migration assays (Boyden chamber or scratch wound)

• Tube formation assays on Matrigel

• In vivo Matrigel plug assays

How does recombinant rat Fkbpl compare functionally with other members of the immunophilin family?

Recombinant rat Fkbpl exhibits several key functional differences from classical immunophilins:

Key Functional Distinctions:

• Fkbpl lacks the enzymatic PPIase activity characteristic of other FKBPs

• Fkbpl functions primarily through protein-protein interactions rather than enzymatic activity

• Fkbpl has emerging roles in cancer biology that may not be shared by other FKBPs

• Unlike FKBP35 found in Plasmodium, Fkbpl does not appear to be inhibited by FK506

These functional differences suggest Fkbpl has evolved distinct biological roles despite sharing structural elements with other immunophilins.

What is the potential of recombinant rat Fkbpl in therapeutic applications?

While most therapeutic development has focused on human FKBPL, recombinant rat Fkbpl provides important insights for preclinical studies:

Potential Therapeutic Applications:

• Anti-cancer agent, particularly for targeting tumor angiogenesis

• Modulator of steroid receptor signaling in hormone-dependent conditions

• Regulator of DNA damage response in cancer

Preclinical Evidence:

• Studies with human FKBPL have shown promising anti-tumor activity

• Anti-angiogenic peptide derivatives of FKBPL have approached clinical trials

• Rat models using recombinant rat Fkbpl provide crucial proof-of-concept data

Mechanism of Action:

• Inhibition of angiogenesis appears to be a key mechanism for anti-tumor effects

• Modulation of steroid receptor signaling may contribute to efficacy in hormone-dependent cancers

• FKBPL has been described as "a key player in the DNA damage response, steroid receptor signalling and control of tumour growth"

When developing therapeutic applications:

• Consider species-specific differences between rat and human Fkbpl

• Use recombinant rat Fkbpl for preclinical validation in rodent models

• Address potential immunogenicity concerns when transitioning from rat to human applications

What are the most reliable assays for evaluating the biological activity of recombinant rat Fkbpl?

Several assays can be employed to assess the biological activity of recombinant rat Fkbpl:

Angiogenesis-related Assays:

• Endothelial Cell Proliferation Assay:

  • Measure BrdU incorporation or MTT conversion in rat endothelial cells

  • Typical concentration range: 10-100 nM recombinant Fkbpl

  • Include bevacizumab as positive control

  • Expected result: Dose-dependent inhibition of proliferation

• Migration Assay:

  • Quantify migration of endothelial cells using Boyden chamber or scratch wound

  • Time course: 0-24 hours

  • Analysis: Image-based quantification of migration distance or area

  • Expected result: Significant reduction in migration distance/rate

• Tube Formation Assay:

  • Assess formation of capillary-like structures on Matrigel

  • Duration: 4-16 hours

  • Quantification: Total tube length, branch points, loop formation

  • Expected result: Disruption of network formation

Protein-Protein Interaction Assays:

• Co-immunoprecipitation:

  • Pull-down assays with Hsp90 or steroid receptors

  • Detection by Western blot with anti-Fkbpl antibodies

  • Controls: Include interaction inhibitors as negative controls

• Reporter Gene Assays:

  • Transfect cells with steroid-responsive reporter constructs

  • Measure transcriptional activity with/without Fkbpl

  • Hormone concentrations: 1-10 nM (estrogen, progesterone, etc.)

  • Expected result: Modulation of hormone-induced transcription

These assays should include appropriate controls and standardized conditions to ensure reproducibility across experiments.

What strategies can be used to optimize expression and purification of recombinant rat Fkbpl?

Optimizing expression and purification requires addressing several key factors:

Expression Optimization:

• Codon Optimization:

  • Adapt codons to E. coli preference if using bacterial system

  • Focus on rare codons that may limit expression (especially arginine and leucine codons)

• Expression Conditions:

  • Test induction at different OD600 values (0.4-0.8)

  • Vary IPTG concentration (0.1-1.0 mM)

  • Optimize temperature (15-37°C) and duration (3-24 hours)

  • Example optimization matrix:

  Temperature	IPTG Concentration	OD600 at Induction	Yield/Solubility
  37°C	1.0 mM	0.6	High yield/Low solubility
  25°C	0.5 mM	0.6	Medium yield/Medium solubility
  16°C	0.1 mM	0.8	Lower yield/High solubility

• Fusion Tags:

  • His6-tag for IMAC purification

  • MBP or SUMO tags to enhance solubility

  • Cleavable tags with TEV protease recognition site

Purification Strategy:

• Initial Capture:

  • IMAC for His-tagged proteins (50 mM imidazole wash, 250-300 mM elution)

  • GST affinity for GST-fusion proteins

  • Typical binding capacity: 5-10 mg protein per mL resin

• Intermediate Purification:

  • Ion exchange chromatography based on theoretical pI

  • Tag cleavage and reverse IMAC to remove tags

• Polishing:

  • Size exclusion chromatography (Superdex 75/200)

  • Expected elution volume corresponding to ~35 kDa

Quality Assessment:

• SDS-PAGE: >90% purity

• Western blot: Confirmation of identity

• Mass spectrometry: Verification of intact mass

• Endotoxin testing: <1 EU/mg for cell-based applications

What are common troubleshooting strategies when working with recombinant rat Fkbpl?

Researchers may encounter several challenges when working with recombinant rat Fkbpl:

Expression and Purification Issues:

• Low Expression Yield:

  • Problem: Poor expression in bacterial systems

  • Solution: Optimize codon usage, reduce expression temperature (16-18°C), use specialized strains (e.g., Rosetta)

  • Alternative: Switch to mammalian expression if complex folding is required

• Protein Insolubility:

  • Problem: Formation of inclusion bodies

  • Solution: Add solubility tags (SUMO, MBP), reduce induction temperature, co-express with chaperones

  • Alternative: Develop refolding protocol from solubilized inclusion bodies

• Degradation During Purification:

  • Problem: Multiple bands on SDS-PAGE

  • Solution: Add protease inhibitors, reduce purification time, maintain cold temperature

  • Verification: N-terminal sequencing to identify cleavage sites

Stability and Storage Issues:

• Activity Loss During Storage:

  • Problem: Decreased functional activity over time

  • Solution: Add stabilizers (glycerol, trehalose), store at -80°C in small aliquots

  • Monitoring: Implement regular activity testing

• Aggregation After Thawing:

  • Problem: Visible precipitates or high molecular weight bands

  • Solution: Centrifuge after thawing, add reducing agents, optimize buffer

  • Prevention: Avoid freeze-thaw cycles

Experimental Challenges:

• Inconsistent Results in Cell-Based Assays:

  • Problem: Variable cellular responses

  • Solution: Standardize cell passage number, use serum-free media when possible

  • Control: Include positive controls (e.g., known anti-angiogenic agents for angiogenesis assays)

• Weak or No Activity in Functional Assays:

  • Problem: Protein appears pure but shows limited activity

  • Solution: Verify proper folding, check for inhibitory contaminants

  • Alternative: Compare with commercially available standards

A systematic troubleshooting approach will help overcome these challenges and ensure successful experiments with recombinant rat Fkbpl.