Recombinant Bovine Peptidyl-prolyl cis-trans isomerase B (PPIB)

What is the primary function of bovine Peptidyl-prolyl cis-trans isomerase B (PPIB)?

Bovine Peptidyl-prolyl cis-trans isomerase B (PPIB) belongs to the family of peptidyl-prolyl isomerases that catalyze the cis/trans isomerization of proline residues in peptide bonds. This isomerization represents a rate-limiting step in protein folding for many polypeptides. Similar to other prolyl-cis/trans isomerases, PPIB facilitates the correct folding of newly synthesized proteins by accelerating the otherwise slow isomerization of peptide bonds preceding proline residues . The enzyme functions as a molecular chaperone, particularly important for proteins with multiple proline residues where proper conformational changes are essential for achieving their native structure.

How does PPIB differ from other peptidyl-prolyl isomerases like PPIA or Pin1?

While all peptidyl-prolyl isomerases catalyze proline isomerization, they differ in structure, substrate specificity, and cellular localization:

Unlike PPIA which is primarily cytoplasmic, PPIB localizes to the endoplasmic reticulum where it assists in the folding of nascent polypeptides . In contrast to Pin1, which specifically recognizes and isomerizes phosphorylated serine/threonine-proline motifs (pS/TP) as seen in its interaction with hepatitis B virus core particles , PPIB has broader substrate specificity for proline-containing sequences regardless of phosphorylation status.

What expression systems are most suitable for producing recombinant bovine PPIB?

Multiple expression systems can be used for recombinant bovine PPIB production, with E. coli being the most common due to its simplicity and high yield potential. For optimal expression in E. coli, consider these methodological approaches:

• Vector selection: pET expression systems with T7 promoter offer high-level inducible expression

• Strain selection: BL21(DE3) or derivatives like Rosetta 2 for enhanced expression of eukaryotic proteins

• Fusion partners: Consider fusion with solubility-enhancing proteins like MBP, thioredoxin, or GST

• Expression conditions: Lower temperatures (16-25°C) and reduced inducer concentrations often improve soluble protein yield

The choice of expression system should be based on the specific research needs, including required yield, post-translational modifications, and downstream applications .

What are the optimal conditions for high-yield expression of soluble recombinant bovine PPIB in E. coli?

For maximizing soluble PPIB expression, a multivariate experimental design approach is recommended. Based on statistical optimization studies of recombinant protein expression, the following conditions typically yield optimal results:

The experimental design methodology allows for evaluation of multiple variables simultaneously, accounting for interactions between them—which is more thorough than traditional univariant approaches . For PPIB specifically, co-expression with folding modulators like DsbC may increase yields of correctly folded protein .

How can I troubleshoot poor solubility of recombinant bovine PPIB?

Poor solubility of recombinant PPIB can be addressed through several methodological approaches:

• Adjust expression conditions:

  • Reduce temperature to 16°C during induction

  • Lower IPTG concentration to 0.1 mM

  • Shorten induction time to 4 hours

• Use solubility-enhancing fusion partners:

  • MBP (maltose-binding protein) fusion

  • Thioredoxin fusion

  • SUMO fusion

• Co-express with folding chaperones:

  • GroEL/GroES system

  • DnaK/DnaJ/GrpE system

  • Specific PPIases like FkpA or SurA

• Modify buffer conditions during purification:

  • Include 5-10% glycerol

  • Add low concentrations (0.1-0.5%) of non-ionic detergents

  • Test various salt concentrations (150-500 mM NaCl)

It should be noted that fusion partners may sometimes result in soluble but incorrectly folded target proteins, as observed with some antibody fragments fused to MBP . Therefore, functional assays should be performed to confirm proper folding.

What assays are available for measuring the isomerase activity of recombinant bovine PPIB?

Several robust assays can quantify the peptidyl-prolyl isomerase activity of recombinant PPIB:

• Spectrophotometric coupled assay:

  • Principle: Measures the rate of conformational change in proline-containing peptide substrates

  • Substrate: Typically N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide

  • Detection: Chymotrypsin cleaves only the trans isomer, releasing p-nitroaniline that is measured at 390 nm

  • Advantages: Continuous monitoring, quantitative results

• Protease-coupled fluorescence assay:

  • Principle: Similar to spectrophotometric assay but with fluorescent substrates

  • Substrate: Peptides labeled with fluorophore/quencher pairs

  • Detection: Increased fluorescence upon protease cleavage

  • Advantages: Higher sensitivity than spectrophotometric methods

• NMR-based exchange spectroscopy:

  • Principle: Directly measures cis/trans exchange rates of proline peptide bonds

  • Detection: NOESY or ROESY spectra reveal exchange cross-peaks between cis and trans isomers

  • Advantages: Provides direct evidence of isomerization as demonstrated with similar PPIases

For accurate assessment of PPIB activity, temperature control and buffer optimization are critical. The appearance of exchange cross-peaks between cis and trans proline conformers in NOESY or ROESY spectra, similar to those observed with PPIA, provides definitive evidence of increased isomerization rates .

How can I distinguish between the chaperone and isomerase activities of PPIB?

Differentiating between the catalytic isomerase activity and general chaperone function of PPIB requires specific experimental approaches:

• Site-directed mutagenesis of catalytic residues:

  • Create active site mutants that retain structure but lack isomerase activity

  • Compare the effects of wild-type and mutant PPIB on substrate folding

  • Residual activity in catalytic mutants indicates chaperone function

• Selective inhibition:

  • Use cyclosporin A to specifically inhibit the isomerase activity

  • Measure remaining protein folding assistance in the presence of inhibitor

  • Chaperone function typically persists despite isomerase inhibition

• Substrate specificity tests:

  • Use substrates with and without proline residues

  • Assistance in folding of proline-free substrates indicates chaperone activity

  • Comparison with quadruple cysteine mutants (as done with PDI) can separate isomerase and chaperone functions

• Aggregation prevention assays:

  • Monitor prevention of thermal aggregation of model substrates

  • This activity is typically independent of catalytic isomerase function

  • May persist under conditions where isomerase activity is inhibited

These approaches allow for mechanistic distinction between the enzymatic and chaperone functions of PPIB in protein folding.

How does PPIB interact with other components of the protein folding machinery in the endoplasmic reticulum?

PPIB functions as part of the complex protein folding machinery in the endoplasmic reticulum, interacting with multiple components:

• Interactions with other chaperones:

  • Forms functional complexes with BiP (Hsp70 family)

  • Collaborates with protein disulfide isomerases (PDIs)

  • May interact with calnexin/calreticulin in glycoprotein folding

• Role in disulfide bond formation:

  • Coordinates with DsbA, DsbB, DsbC, and DsbD homologs

  • Proline isomerization often precedes or follows disulfide bond formation

  • The sequential or simultaneous action with disulfide isomerases enhances folding efficiency

• Substrate handoff mechanisms:

  • Evidence suggests sequential processing of substrates between PPIB and other chaperones

  • Proline isomerization may expose hydrophobic patches that recruit other chaperones

  • Temporal coordination of different folding activities is critical for complex protein structures

Similar to findings with DsbA and DsbC, which showed improved yields of properly folded proteins when co-expressed , PPIB likely participates in a coordinated network of folding assistants rather than working in isolation.

What role might PPIB play in liquid-liquid phase separation (LLPS) of proline-rich proteins?

Recent research with related peptidyl-prolyl isomerases suggests potential roles for PPIB in modulating liquid-liquid phase separation (LLPS) of proline-rich proteins:

• Potential mechanism of action:

  • PPIB may concentrate inside liquid-like droplets of proline-rich proteins

  • Isomerization of prolines could disrupt intermolecular interactions maintaining phase separation

  • This may lead to dissolution of droplets and return to a single mixed phase

• Biological significance:

  • Could represent a regulatory mechanism for proline-rich protein assemblies

  • May prevent pathological aggregation of proline-rich proteins

  • Could be particularly relevant for intrinsically disordered proteins with high proline content

• Experimental evidence from related PPIases:

  • PPIA has been shown to concentrate in liquid droplets formed by tau protein

  • PPIA recruitment triggers dissolution of these droplets

  • NMR studies confirmed that this effect occurs through proline isomerization

While direct evidence for PPIB's role in LLPS regulation is still emerging, the concentration-dependent modulation of phase separation observed with PPIA provides a compelling model for potential PPIB functions with appropriate substrates .

How can molecular dynamics simulations enhance our understanding of PPIB catalytic mechanism?

Molecular dynamics (MD) simulations offer powerful insights into PPIB's catalytic mechanism:

• Catalytic site dynamics:

  • Simulations can reveal conformational changes during substrate binding

  • Water molecule positioning and hydrogen bond networks can be visualized

  • Energy barriers for cis/trans isomerization can be calculated

• Substrate recognition mechanism:

  • MD reveals induced fit mechanisms upon substrate binding

  • Helps identify residues involved in substrate specificity

  • Can predict effects of mutations on catalytic efficiency

• Methodological approach:

  • Begin with crystal structure coordinates or homology model

  • Perform equilibration in explicit solvent

  • Run production simulations of 100-500 ns

  • Apply enhanced sampling techniques for capturing rare events

• Comparative analysis with other PPIases:

  • MD can reveal mechanistic differences between PPIB and related enzymes like Pin1

  • Explains substrate specificity differences between family members

  • Identifies potential allosteric regulation sites

These computational approaches complement experimental methods and can guide the design of mutants with altered specificity or enhanced catalytic efficiency.

What statistical approaches should be used when optimizing conditions for PPIB expression?

Robust statistical design is critical for efficiently optimizing PPIB expression:

• Fractional factorial designs:

  • Allow investigation of multiple variables with fewer experiments

  • Maintain statistical orthogonality for independent parameter estimation

  • Particularly valuable when evaluating more than four variables

• Response surface methodology (RSM):

  • After initial screening, RSM can fine-tune optimal conditions

  • Generates mathematical models predicting protein expression

  • Identifies interactions between variables that univariate approaches would miss

• Practical implementation:

  • Select 6-8 key variables (temperature, IPTG concentration, media composition, etc.)

  • Design a fractional factorial experiment (e.g., 2^8-4 design with center points)

  • Measure multiple responses (cell growth, biological activity, productivity)

  • Use statistical software to analyze results and identify significant factors

• Data analysis considerations:

  • Normalize data to allow comparison between effect sizes

  • Include center point replicates to estimate experimental error

  • Validate model predictions with confirmation runs

This multivariate approach is superior to traditional one-variable-at-a-time methods and can characterize experimental error while gathering high-quality information with minimal experiments .

How can I resolve conflicting data regarding PPIB substrate specificity?

When faced with conflicting data about PPIB substrate specificity, apply these methodological approaches:

• Standardize experimental conditions:

  • Use identical buffer components, pH, and temperature across experiments

  • Ensure protein concentrations are accurately determined by multiple methods

  • Control for potential confounding variables (contaminants, cofactors)

• Compare direct versus indirect assays:

  • Direct methods (NMR, CD spectroscopy) measure isomerization directly

  • Indirect methods (protease-coupled assays) measure consequences of isomerization

  • Discrepancies often arise from different detection principles

• Control for non-catalytic effects:

  • Include heat-inactivated PPIB controls

  • Use catalytically inactive mutants to control for binding effects

  • Consider substrate sequestration by PPIB independent of catalysis

• Meta-analysis approach:

  • Systematically compare methodologies across conflicting studies

  • Weight evidence based on experimental rigor

  • Consider developing a consensus assay protocol

Data conflicts may reflect genuine biological complexity rather than experimental error, as PPIB might exhibit context-dependent substrate preferences depending on local environment, post-translational modifications, or interaction partners.

How might PPIB be involved in disease processes, and what are the implications for therapeutic development?

PPIB has emerging connections to multiple disease processes with potential therapeutic implications:

• Viral infections:

  • Related PPIases like Pin1 interact with viral proteins, including hepatitis B virus core particles

  • Pin1 binds to HBV core particles through phosphorylated serine/threonine-proline motifs

  • Overexpression of Pin1 increased viral DNA synthesis and virion secretion

  • PPIB may play analogous roles with other viruses that use the ER for assembly

• Protein misfolding disorders:

  • PPIB's role in protein folding suggests potential involvement in diseases characterized by misfolding

  • Similar to PDI's protective effects against tau aggregation, PPIB may inhibit pathological aggregation

  • May influence liquid-liquid phase separation processes implicated in neurodegenerative diseases

• Therapeutic strategies:

  • Small molecule modulators of PPIB activity could alter disease progression

  • Gene therapy approaches might leverage PPIB's chaperone functions

  • Cell-penetrating PPIB variants could address cytoplasmic protein misfolding

Future research should investigate disease-specific substrates of PPIB and develop selective modulators of its activity for potential therapeutic applications.

What novel technological approaches are being developed to study PPIB interactions and dynamics?

Cutting-edge technologies are expanding our ability to study PPIB:

• Single-molecule techniques:

  • Optical tweezers can directly measure forces during PPIB-catalyzed folding

  • FRET-based approaches reveal conformational dynamics during catalysis

  • These techniques overcome limitations of ensemble measurements

• Cryo-electron microscopy:

  • Near-atomic resolution structures of PPIB-substrate complexes

  • Visualization of different catalytic states

  • Insights into substrate binding modes

• Proteomics approaches:

  • Proximity labeling methods (BioID, APEX) identify transient PPIB interactors

  • Crosslinking mass spectrometry maps interaction interfaces

  • Thermal proteome profiling reveals substrate engagement in cells

• Patient and public involvement (PPI) in research:

  • While distinct from the protein PPIB, PPI approaches in research design can provide valuable insights

  • Patient experiences can help identify relevant disease contexts for PPIB research

  • This involvement enhances research relevance and applicability

These technologies are transforming our understanding of PPIB from static snapshots to dynamic views of its cellular functions and interactions.