Recombinant Xenopus laevis Peptidyl-prolyl cis-trans isomerase FKBP1A (fkbp1a)

What is Xenopus laevis FKBP1A and what is its fundamental structure?

FKBP1A (FK506 Binding Protein 1A, 12kDa) is a peptidyl-prolyl cis-trans isomerase found in Xenopus laevis (African clawed frog) . The protein consists of 108 amino acids, with recombinant forms typically expressing amino acids 2-108 . Its sequence is: GVQVETITE GDGRTFPKKG QTVVVHYVGS LENGKKFDSS RDRNKPFKFI IGRCEVIRGW EEGVAQMSVG QRARLTCSPD FAYGATGHPG IIPPNATLTF DVELLRLE . This protein belongs to the immunophilin family and possesses peptidyl-prolyl isomerase activity, which catalyzes the cis-trans isomerization of proline residues in peptide bonds, a rate-limiting step in protein folding.

What expression systems are most effective for recombinant Xenopus laevis FKBP1A production?

Various expression systems have been employed for recombinant FKBP1A production, each with distinct advantages for different research applications:

The choice of expression system should be determined by experimental requirements, as each system produces protein with slightly different characteristics that may influence function or activity in specific assays .

How can researchers verify the identity and purity of recombinant Xenopus laevis FKBP1A?

Verification of recombinant FKBP1A identity and purity typically involves multiple complementary techniques:

• SDS-PAGE analysis is routinely used to assess purity, with most commercial preparations achieving >90-95% purity .

• Western blotting with anti-His antibodies confirms the presence of the His-tagged recombinant protein.

• Mass spectrometry provides precise molecular weight verification and can identify post-translational modifications.

• Enzymatic activity assays measure peptidyl-prolyl isomerase function, confirming not just presence but functional integrity.

• Circular dichroism spectroscopy can verify proper protein folding by analyzing secondary structure elements.

For experiments requiring exceptionally high purity, researchers should consider additional purification steps beyond the initial IMAC (immobilized metal affinity chromatography) used for His-tagged proteins .

What is the developmental role of FKBP1A in Xenopus laevis embryogenesis?

FKBP1A plays a critical role in Xenopus embryonic development, particularly in axis formation. Experimental evidence demonstrates that xFKBP1A induces an ectopic dorsal axis when injected into ventral blastomeres of 4-cell stage Xenopus embryos . This axis-inducing activity appears to operate through a pathway that can be antagonized by BMP4 and Smad1, suggesting interaction with key developmental signaling pathways .

The specific molecular mechanisms involve:

• Regulation of protein folding through peptidyl-prolyl isomerase activity

• Potential modulation of TGF-β/BMP signaling pathways

• Possible interaction with calcium release channels during early development

Methodologically, these functions are typically studied through microinjection of mRNA into specific blastomeres, followed by phenotypic analysis and molecular marker expression assessment .

How do FKBP1A and FKBP1B differ in their developmental functions in Xenopus?

While FKBP1A and FKBP1B are paralogs sharing significant sequence homology, they demonstrate distinct functional roles during Xenopus development:

When co-injected, FKBP1A and FKBP1B demonstrate a multiplier effect on secondary axis induction, suggesting they operate through complementary but distinct pathways . This synergy provides an excellent experimental model for studying cooperative protein functions in developmental contexts.

What are the optimal approaches for studying FKBP1A function through loss-of-function experiments?

Loss-of-function studies of FKBP1A in Xenopus laevis typically employ morpholino oligonucleotides for targeted knockdown. Based on published methodologies, researchers should consider:

• Morpholino design: Target the 5' UTR or start codon region of xFKBP1A mRNA for maximum efficacy.

• Injection parameters: Typically 10-25 ng morpholino per embryo, injected into specific blastomeres at the 2-4 cell stage depending on the developmental process being studied .

• Controls: Include both standard control morpholinos and rescue experiments with morpholino-resistant mRNA to confirm specificity.

• Phenotypic analysis: Assess developmental outcomes through both morphological observation and molecular marker analysis.

• Timing considerations: Effects of xFKBP1A knockdown may vary depending on developmental stage, requiring time-course analyses .

It's worth noting that morpholinos do not elicit an innate immune response during early Xenopus embryogenesis, making them suitable for developmental studies without confounding inflammatory effects .

How can researchers effectively evaluate the impact of molecular crowding on FKBP1A stability and function?

Molecular crowding significantly impacts protein stability and function in cellular environments. For studying FKBP1A under crowding conditions:

• In vitro crowding models: Use polymers of different molecular weights (PEG, dextran, Ficoll) at 20-30% w/v to mimic cellular crowding .

• Heterogeneous crowding: Combine crowders of different sizes to better simulate physiological conditions, as this creates synergistic effects beyond simple volume exclusion .

• Comparative stability assays: Measure thermal stability (Tm) and folding cooperativity under different crowding conditions using techniques such as differential scanning calorimetry or circular dichroism .

• Functional assays: Assess peptidyl-prolyl isomerase activity using standard substrates under various crowding conditions.

• Cell extract systems: For more physiologically relevant conditions, consider using Xenopus egg extracts as they provide a complex biological environment that can be experimentally manipulated .

The choice between simplified polymer crowders and complex biological extracts depends on whether you need a controlled, defined environment or a more physiologically relevant but complex system .

What spectroscopic techniques are most informative for studying FKBP1A structural dynamics in different environments?

Multiple complementary spectroscopic approaches provide insights into FKBP1A structural dynamics:

For in-cell studies, a combination of fluorescence-based and NMR experiments provides complementary data on both structure and stability . Fast Relaxation Imaging combined with other methods enables analysis of kinetic and thermodynamic parameters with high spatial resolution, particularly valuable for studying crowding effects .

How do FKBP1A and FKBP1B coordinate to regulate eye development in Xenopus embryos?

FKBP1B plays a crucial role in eye formation during Xenopus embryogenesis, with expression in the dorsal side of the embryo including the eye region throughout development . Research has revealed:

• Spatial regulation: xFKBP1B is expressed in the dorsal side of the embryo including the eye during embryogenesis at least until stage 46 .

• Morphological impact: Injection of xFKBP1B mRNA in dorsal blastomeres leads to eye malformation .

• Gene expression effects: xFKBP1B injection suppresses the expression of key eye development genes including Rx1, Mitf, and Vax2 mRNAs .

• Loss-of-function phenotypes: Morpholino knockdown of xFKBP1B induces additional retina or failure to close tapetum nigrum in the ventral side within the optic cap .

• Regulatory targets: xFKBP1B knockdown reduces expression of Xotx2 and Rx1 mRNAs in the eye, suggesting regulatory functions .

These findings indicate that xFKBP1B is a key factor regulating gene expression during eye formation, with implications for understanding developmental eye disorders and potential therapeutic approaches.

What experimental approaches can differentiate between the signaling pathways affected by FKBP1A versus FKBP1B during axis formation?

Distinguishing between FKBP1A and FKBP1B signaling pathways requires several specialized experimental approaches:

• Co-injection studies: When BMP4 and Smad1 mRNAs are co-injected with either FKBP1A or FKBP1B in ventral blastomeres, they suppress the axis-inducing ability of FKBP1A but not FKBP1B, providing clear evidence for distinct pathways .

• Pathway inhibition assays: Selective inhibitors of different developmental pathways (Wnt, BMP, Nodal) can be used to identify the specific cascade affected by each protein.

• Protein interaction studies: Co-immunoprecipitation and proximity ligation assays can identify different binding partners for each protein.

• Transcriptional profiling: RNA-seq analysis of embryos overexpressing either FKBP1A or FKBP1B can reveal distinct transcriptional signatures.

• Rescue experiments: Testing whether one protein can rescue defects caused by knocking down the other can reveal functional redundancy or independence.

The multiplier effect observed when both proteins are co-injected suggests that they operate through different but potentially complementary mechanisms to induce ectopic dorsal axis formation .

What are the critical parameters for successful microinjection experiments with recombinant FKBP1A in Xenopus embryos?

Successful microinjection of FKBP1A mRNA or protein into Xenopus embryos requires careful optimization of multiple parameters:

• Preparation quality: Use highly purified, capped mRNA (for expression) or recombinant protein (for direct activity). For mRNA, in vitro transcription should yield a single band on gel electrophoresis.

• Injection volume: Typically 5-10 nl per blastomere, with careful calibration to ensure consistent delivery.

• Concentration optimization: Perform dose-response experiments (typically 50-500 pg of mRNA) to determine the minimal effective dose that avoids non-specific toxicity.

• Targeting precision: For axis induction studies, ventral blastomere injection at the 4-cell stage is critical; for eye development studies, dorsal blastomere targeting is essential .

• Co-injection markers: Include lineage tracers (β-galactosidase mRNA or fluorescent dextran) to verify accurate targeting and cell fate.

• Timing considerations: Inject at consistent developmental stages (typically 2-4 cell) and maintain embryos at standardized temperature to ensure reproducible developmental timing.

• Controls: Include both uninjected and control-injected (e.g., GFP mRNA) embryos in each experiment to distinguish specific effects from injection trauma.

How can researchers optimize in-cell studies of FKBP1A function and stability?

In-cell studies of FKBP1A require specialized approaches to obtain meaningful data from complex cellular environments:

• Expression systems: For Xenopus studies, consider microinjection of mRNA encoding fluorescently tagged FKBP1A into embryos or oocytes.

• Stability measurements: Employ in-cell NMR spectroscopy or fluorescence-based sensors (e.g., FRET-labeled constructs) to monitor protein stability under physiological conditions .

• Crowding effects: Compare in vitro results using artificial crowders with in-cell measurements to distinguish specific interactions from general crowding effects .

• Environmental variables: Systematically alter cellular conditions through osmotic perturbations to change crowding density or induce cell stress to observe effects on FKBP1A stability .

• Localization studies: Use fluorescent tagging combined with high-resolution microscopy to determine subcellular distribution and potential compartment-specific functions.

• Interaction networks: Employ proximity labeling approaches (BioID, APEX) to identify interaction partners specific to cellular compartments.

Given the complexity of cellular environments, researchers should validate in-cell findings through a workflow that rigorously compares different contributions of crowding, cosolutes, and biomolecular interactions to provide comprehensive interpretation of results .