Recombinant Bovine Peptidyl-prolyl cis-trans isomerase FKBP11 (FKBP11)

What is FKBP11 and what is its primary function in bovine cells?

Bovine FKBP11 belongs to the FK506-binding protein family of peptidyl-prolyl cis/trans isomerases. These enzymes catalyze the folding of proline-containing polypeptides, which is often a rate-limiting step in protein folding processes. FKBP11 likely plays a crucial role within the endoplasmic reticulum (ER), particularly for the proper folding of secretory proteins. The peptidyl-prolyl isomerase activity of FKBP proteins, including FKBP11, is inhibited by immunosuppressant compounds such as FK506 (tacrolimus) and rapamycin (sirolimus) .

The primary function of bovine FKBP11 involves facilitating proper protein folding through the isomerization of peptide bonds preceding proline residues. This catalytic activity helps overcome energy barriers in protein folding, ensuring efficient processing of newly synthesized proteins. Based on human FKBP11 characteristics, the bovine variant likely participates in quality control mechanisms within the ER and may be upregulated during ER stress as part of the unfolded protein response . The enzyme's activity is especially important for proteins containing proline residues in structurally critical positions where isomerization is required for proper folding.

What are the structural characteristics of bovine FKBP11?

While specific structural data for bovine FKBP11 is limited, important features can be inferred from human FKBP11 studies and the general conservation of FKBP family proteins. Human FKBP11 is a 201-amino acid protein with a calculated molecular mass of approximately 22 kDa . The bovine ortholog likely shares similar structural organization with several key domains:

• An N-terminal signal sequence directing the protein to the endoplasmic reticulum

• A peptidyl-prolyl isomerase (PPIase) domain responsible for catalytic activity

• A transmembrane region anchoring the protein to ER membranes

• A lysine-rich C-terminal tail containing ER retention motifs

The PPIase domain would contain the binding pocket for substrates as well as inhibitors like FK506 and rapamycin. This domain exhibits the characteristic FKBP fold with a hydrophobic pocket that accommodates the proline-containing substrate. The tertiary structure of this domain is highly conserved across most FKBPs, making them structurally similar despite functional differences . The C-terminal region likely contains "a variant of the dilysine motif found in endoplasmic reticulum membrane proteins," which facilitates retention in the ER .

Where is FKBP11 expressed in bovine tissues?

While the search results don't provide specific information about FKBP11 tissue distribution in cattle, expression patterns can be reasonably inferred from human data. In humans, FKBP11 shows "ubiquitous expression, with highest levels in pancreas and other secretory tissues, such as stomach, pituitary, salivary gland, and lymph node" .

Given the conservation of FKBP expression patterns across species, bovine FKBP11 would likely show similar tissue distribution, with highest expression in secretory organs. This pattern is consistent with FKBP11's presumed role in protein folding within the ER, as tissues with high secretory activity require robust protein folding mechanisms. The following table summarizes the expected tissue expression profile of bovine FKBP11 based on human data:

Researchers studying bovine FKBP11 should verify this predicted expression pattern through quantitative PCR, Western blotting, or analysis of bovine transcriptome datasets.

What are the recommended approaches for measuring the peptidyl-prolyl isomerase activity of recombinant bovine FKBP11?

Measuring the peptidyl-prolyl isomerase (PPIase) activity of recombinant bovine FKBP11 requires specialized assays that detect the cis-trans isomerization of peptide bonds. Several established methods are suitable for bovine FKBP11 characterization:

• Protease-coupled spectrophotometric assay: This classical approach uses a proline-containing peptide substrate (typically Suc-Ala-Leu-Pro-Phe-pNa) where only the trans isomer is susceptible to chymotrypsin cleavage. The rate of p-nitroaniline release, monitored at 390-400 nm, reflects PPIase activity. This assay offers good sensitivity and is widely used for FKBP family proteins.

• NMR spectroscopy: This technique allows direct observation of cis and trans isomers and their interconversion kinetics. While providing detailed mechanistic insights, it requires specialized equipment and larger quantities of purified protein.

• Fluorescence-based assays: Modified peptide substrates with environmentally sensitive fluorophores exhibit altered fluorescence properties depending on cis/trans conformation, enabling real-time monitoring of isomerization.

A typical protocol for the protease-coupled assay would include:

• Expression and purification of recombinant bovine FKBP11

• Pre-equilibration of the tetrapeptide substrate at low temperature (0-4°C) to enrich for the cis conformation

• Rapid mixing with FKBP11 and chymotrypsin at room temperature

• Spectrophotometric monitoring of p-nitroaniline release at 390 nm

• Calculation of isomerization rate constants from the progress curves

• Validation with known FKBP inhibitors (FK506, rapamycin) as controls

When performing these assays, researchers should consider buffer conditions that mimic the ER environment, test activity across a range of substrate concentrations, and include appropriate controls to confirm specificity.

What analytical techniques provide the most reliable quantification of bovine FKBP11 expression levels?

Reliable quantification of bovine FKBP11 expression requires selecting appropriate techniques based on specific research questions. Both RNA-level and protein-level approaches offer complementary information:

RNA-level Quantification:

• Quantitative RT-PCR (qRT-PCR): The gold standard for mRNA quantification requires designing primers specific to bovine FKBP11 that avoid cross-reactivity with other FKBP family members. Reference genes like GAPDH, β-actin, or 18S rRNA should be carefully selected based on their stability in the specific tissues being studied.

• RNA-Seq: Provides comprehensive transcriptome analysis and enables detection of splice variants and novel isoforms. This approach is particularly valuable for discovering tissue-specific expression patterns and regulatory mechanisms.

Protein-level Quantification:

• Western blotting: Requires antibodies specific to bovine FKBP11. Based on available antibody information, polyclonal antibodies with human and mouse reactivity might cross-react with bovine FKBP11 due to sequence conservation . Validation for bovine specificity is essential.

• ELISA: Offers higher throughput than Western blotting but similarly requires validated antibodies. Commercial ELISA kits for FKBP11 quantification might be adaptable to bovine samples after proper validation .

• Mass spectrometry-based proteomics: Provides antibody-independent quantification through techniques like multiple reaction monitoring (MRM) targeting peptides unique to bovine FKBP11.

For optimal results, researchers should:

• Include appropriate controls to account for technical variations

• Validate antibodies specifically against bovine FKBP11

• Consider potential post-translational modifications

• Account for subcellular localization during sample preparation

• Use multiple techniques for cross-validation of important findings

The combination of qRT-PCR and Western blotting provides a practical approach for most research applications, while more specialized techniques offer advantages for specific research questions.

What are the recommended protocols for expressing and purifying recombinant bovine FKBP11?

Expressing and purifying recombinant bovine FKBP11 with optimal enzymatic activity requires careful consideration of expression systems and purification strategies. While specific protocols for bovine FKBP11 are not provided in the search results, the following recommendations are based on general principles for recombinant protein production and FKBP family characteristics:

Expression System Selection:

Expression Construct Design:

• Include an appropriate affinity tag (His₆, GST, or MBP) for purification

• Consider expressing just the PPIase domain (more soluble) or full-length protein

• Optimize codon usage for the expression system

• Include TEV or PreScission protease sites for tag removal

Purification Strategy:

• Affinity chromatography (Ni-NTA for His-tagged proteins) as initial capture

• Ion exchange chromatography as intermediate purification

• Size exclusion chromatography as final polishing step

A comprehensive protocol would include:

• Cloning bovine FKBP11 cDNA into an expression vector with an N-terminal His₆ tag

• Transforming into E. coli BL21(DE3) or other suitable strain

• Culturing at 37°C until OD₆₀₀ reaches 0.6-0.8

• Inducing with 0.1-0.5 mM IPTG at reduced temperature (16-18°C) overnight

• Harvesting cells and lysing by sonication or French press

• Clarifying lysate by centrifugation (20,000 × g, 30 min)

• Purifying by Ni-NTA affinity chromatography

• Further purification by ion exchange and size exclusion chromatography

• Assessing purity by SDS-PAGE and activity using PPIase assay

• Storing in small aliquots at -80°C in buffer with 20% glycerol

For optimal enzymatic activity, the purification buffers should include:

• Reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol)

• Protease inhibitors during initial extraction

• Stabilizing agents (10-20% glycerol) in final storage buffer

• Physiological pH (7.0-7.5) and salt concentration (100-150 mM NaCl)

How can researchers effectively distinguish between FKBP11 and other FKBP family members in bovine samples?

• Immunological approaches:

  • Immunodepletion using validated FKBP11-specific antibodies

  • Sequential immunoprecipitation to remove specific FKBPs from complex samples

  • Western blotting with antibodies recognizing unique regions outside the conserved catalytic domain

• Biochemical approaches:

  • Subcellular fractionation to separate ER-localized FKBP11 from cytosolic FKBPs

  • Separation by chromatographic techniques (ion exchange, hydrophobic interaction)

  • Proteomic identification of purified fractions

• Genetic approaches:

  • CRISPR/Cas9 knockout or RNAi-mediated knockdown of FKBP11

  • Overexpression of tagged FKBP11 for activity measurement above endogenous background

  • Mutation of key catalytic residues to create dominant-negative variants

• Substrate and inhibitor profiling:

  • Screening substrate peptides with varying sequences surrounding the proline residue

  • Testing selective FKBP inhibitors that exploit minor differences in binding pockets

  • Determining kinetic parameters (Km, kcat) for different substrates

For analysis of complex biological samples, a combination of approaches is typically required:

• Begin with subcellular fractionation to enrich for ER components containing FKBP11

• Perform immunodepletion with specific antibodies

• Compare PPIase activity profiles before and after depletion

• Confirm results using genetic approaches in cell culture systems

Researchers should be aware that complete separation of FKBP activities may not be possible due to functional redundancy, and interpretations should consider potential contributions from multiple family members.

How does the inhibition profile of bovine FKBP11 by FK506 and rapamycin compare to that of other species?

The peptidyl-prolyl isomerase activity of FKBP proteins, including FKBP11, is generally inhibited by the immunosuppressant compounds FK506 and rapamycin . The binding pocket that accommodates these inhibitors is highly conserved across FKBP family members, though subtle differences can affect binding affinities and selectivity.

To characterize the inhibition profile of bovine FKBP11:

• Perform in vitro PPIase assays with purified recombinant bovine FKBP11 in the presence of varying concentrations of FK506 and rapamycin

• Determine IC₅₀ values and compare with published data for human FKBP11

• Evaluate binding affinities using techniques such as isothermal titration calorimetry or surface plasmon resonance

• Test newer, selective FKBP ligands that have been developed, such as GPI-1485, V10367, and ElteN378

The table below summarizes expected inhibition characteristics based on FKBP family properties:

Understanding species-specific differences in inhibition profiles could be relevant for veterinary applications and for using bovine models in drug development studies. Researchers should note that while the FK506-binding domain is conserved, species-specific differences in the binding pocket microenvironment could result in altered binding kinetics or inhibition profiles.

How can computational modeling be used to predict potential selective inhibitors of bovine FKBP11?

Computational modeling offers powerful approaches for predicting selective inhibitors of bovine FKBP11, particularly important given the challenge of achieving selectivity among highly similar FKBP family members. As noted in the search results, "high-affinity ligands with selectivity between close homologs are scarce" , making computational methods essential for rational inhibitor design.

A comprehensive computational strategy should include:

• Structure-based approaches:

  • Homology modeling of bovine FKBP11 based on crystal structures of human FKBPs

  • Refinement using molecular dynamics simulations

  • Comparative binding site analysis to identify unique features of bovine FKBP11

  • Molecular docking of compound libraries against the model

• Ligand-based approaches:

  • Pharmacophore modeling based on known FKBP inhibitors

  • Quantitative structure-activity relationship (QSAR) analysis

  • Fragment-based design targeting specific regions of the binding pocket

• Advanced computational methods:

  • Molecular dynamics simulations to study protein flexibility

  • Machine learning models trained on existing FKBP inhibitor data

  • Free energy calculations to estimate binding affinities

Implementation workflow:

• Develop a high-quality homology model of bovine FKBP11 using multiple templates

• Validate the model through energy minimization and Ramachandran plot analysis

• Perform comparative analysis of binding sites across the FKBP family

• Identify unique features of the bovine FKBP11 binding pocket

• Screen virtual libraries targeting these unique features

• Prioritize compounds based on predicted selectivity and affinity

• Validate top candidates experimentally

Special considerations for FKBP11:

• Focus on exploiting the differences in non-conserved residues near the binding pocket

• Consider targeting the region where the PPIase domain interfaces with other domains

• Design compounds that extend beyond the conserved FK506 binding site

• Incorporate knowledge of species-specific structural features

This computational workflow should be iterative, with experimental validation informing refinement of the models and subsequent rounds of virtual screening or de novo design. The goal is to identify compounds that maintain high affinity for FKBP11 while discriminating against other FKBP family members.

How can researchers assess the impact of FKBP11 inhibition on cellular stress responses in bovine models?

Assessing the impact of FKBP11 inhibition on cellular stress responses in bovine models requires a multi-faceted approach focusing on endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), given FKBP11's presumed role in ER protein folding.

Experimental Approaches for FKBP11 Inhibition:

• Pharmacological inhibition: Using FK506, rapamycin, or newer selective inhibitors

• Genetic approaches: CRISPR/Cas9 knockout, shRNA knockdown, or dominant-negative mutants

• Antibody-mediated inhibition in cell-permeable formats if available

Key ER Stress Markers to Evaluate:

Comprehensive Assessment Protocol:

• Establish baseline ER stress markers in normal conditions

• Apply FKBP11 inhibition (pharmacological or genetic)

• Challenge with escalating doses of ER stressors:

  • Tunicamycin (N-glycosylation inhibitor)

  • Thapsigargin (SERCA inhibitor)

  • DTT (reducing agent disrupting disulfide bonds)

• Monitor time-dependent changes in stress responses

• Assess recovery capacity after stress removal

• Compare results with inhibition of other FKBPs to determine specificity

This approach would provide insights into whether FKBP11 plays a critical role in managing ER stress in bovine cells or if functional redundancy with other FKBPs prevents major stress response disruptions. Given FKBP11's expression in secretory tissues , researchers should consider using relevant bovine cell types such as pancreatic cells, mammary epithelial cells, or immune cells for these studies.

What are the known protein interactions of bovine FKBP11 and how do they compare to those of human FKBP11?

As an ER-resident peptidyl-prolyl isomerase, FKBP11 likely interacts with:

• Nascent polypeptide chains undergoing folding

• Components of the ER quality control machinery

• Other ER chaperones and folding enzymes (BiP/GRP78, PDI family members)

• ER stress response factors (PERK, IRE1α, ATF6)

Recommended Approaches for Identifying FKBP11 Interactors:

When comparing bovine and human FKBP11 interactions, researchers should consider:

• Core interactions with the protein folding machinery are likely conserved across species

• Species-specific interactions may exist, particularly with proteins that have diverged significantly

• Tissue-specific interaction networks may differ based on the expression patterns of partners

• Post-translational modifications may influence interaction profiles

Understanding FKBP11's protein interactions would provide insights into its roles beyond simple peptidyl-prolyl isomerase activity, potentially revealing functions in stress response pathways, protein quality control, or tissue-specific processes in cattle. These interactions could also highlight potential therapeutic targets for diseases affecting bovine secretory tissues where FKBP11 is highly expressed.

What are the best practices for designing CRISPR/Cas9 knockouts of FKBP11 in bovine cell lines?

Designing effective CRISPR/Cas9 knockouts of FKBP11 in bovine cell lines requires careful consideration of several factors specific to both the FKBP11 gene and the characteristics of bovine cells. Although the search results don't provide specific guidelines for CRISPR-based editing of bovine FKBP11, the following best practices can be derived from general CRISPR principles and FKBP biology.

Target Selection Strategies:

• Identify conserved, functionally critical exons by aligning bovine FKBP11 with human and mouse orthologs

• Target early exons to maximize the likelihood of complete loss of function

• Focus on regions encoding the catalytic peptidyl-prolyl isomerase domain

• Avoid regions with high homology to other FKBP family members to minimize off-target effects

Guide RNA Design Considerations:

Delivery and Screening Protocols:

• Optimize transfection conditions specifically for bovine cell lines

• Consider ribonucleoprotein (RNP) delivery for reduced off-target effects

• Design PCR primers flanking the target site for mutation detection

• Use T7 Endonuclease I assay or Sanger sequencing for initial screening

• Perform Western blot analysis to confirm protein loss

• Validate functional knockout with PPIase activity assays

Special Considerations for Bovine Cells:

• Account for potential polyploidy in certain bovine cell types

• Consider the lower transfection efficiency often observed in bovine cells

• Be aware of potential embryonic lethality if working with bovine embryos

• Establish appropriate screening protocols for mixed cell populations before single-cell cloning

A well-designed experiment would include thorough validation of the knockout through multiple methods, including genomic analysis, protein expression assessment, and functional assays to confirm the loss of FKBP11-specific PPIase activity. Considering FKBP11's role in protein folding, researchers should also monitor for potential compensatory upregulation of other FKBP family members.

What are the recommended approaches for studying post-translational modifications of bovine FKBP11?

Studying post-translational modifications (PTMs) of bovine FKBP11 requires specialized techniques to identify, localize, and quantify these modifications. Based on what is known about FKBP proteins, potential PTMs of interest might include phosphorylation, glycosylation, and possibly SUMOylation or ubiquitination that could regulate its activity or localization.

Identification and Mapping of PTMs:

Validation and Functional Analysis:

• Site-directed mutagenesis:

  • Mutate modified residues to non-modifiable amino acids (e.g., Ser→Ala for phosphosites)

  • Create phosphomimetic mutations (e.g., Ser→Asp) to simulate constitutive phosphorylation

  • Express mutants in bovine cells and assess effects on localization and function

• Functional assays:

  • Compare PPIase activity of wild-type and PTM-modified FKBP11

  • Assess protein-protein interactions with and without specific modifications

  • Examine subcellular localization changes in response to stimuli that affect PTMs

PTM Dynamics Studies:

• Apply ER stress inducers and monitor changes in FKBP11 modifications

• Use phosphatase or kinase inhibitors to manipulate modification states

• Perform time-course experiments after stimulation to track dynamic changes

• Employ quantitative proteomics (SILAC, TMT) for relative quantification across conditions

A comprehensive workflow for studying bovine FKBP11 PTMs might involve:

• Expressing tagged bovine FKBP11 in a suitable bovine cell system

• Purifying the protein under conditions that preserve PTMs

• Processing parallel samples with and without ER stress induction

• Analyzing by LC-MS/MS with PTM-specific search parameters

• Validating key sites by site-directed mutagenesis

• Performing functional studies comparing wild-type and mutant proteins

This approach would provide insights into how PTMs regulate FKBP11 function in bovine cells and potentially reveal species-specific regulatory mechanisms not present in human or mouse orthologs.