Recombinant Bombyx mori Antichymotrypsin-2

What is Bombyx mori Antichymotrypsin-2 and how does it relate to the serpin superfamily?

Bombyx mori Antichymotrypsin (sw-Achy) is a serine protease inhibitor (serpin) expressed in the silkworm. The mature sw-Achy protein begins with Phe1 and ends with Phe384, with a preceding 16-amino-acid signal peptide. Sequence analysis reveals similarity with other serpins: 29.6% with silkworm antitrypsin, 30.3% with tobacco hornworm alaserpin, 26.1% with human α-1-antitrypsin, and 25.0% with human α-1-antichymotrypsin . Antichymotrypsin-2 represents a specific isoform within this protein family, sharing the characteristic serpin fold and inhibitory mechanism. Like other serpins, it functions as a suicide substrate that undergoes a significant conformational change upon binding to its target protease.

What are the optimal conditions for cloning and expressing recombinant Bombyx mori Antichymotrypsin-2?

For successful cloning and expression of recombinant Bombyx mori Antichymotrypsin-2, researchers typically isolate mRNA from larval fat body tissue, which is a primary site of serpin expression in silkworms . The cDNA can be synthesized using reverse transcription and then amplified using PCR with specific primers designed based on the published sequence. For expression, systems similar to those used for other silkworm proteins can be employed, such as the Pichia pastoris expression system used for BmAChE II .

A typical expression protocol involves:

• Vector selection: pPICZα for secreted expression in P. pastoris

• Transformation: Electroporation (1.5 kV, 200 Ω, 25 μF)

• Selection: Using zeocin resistance (100 μg/mL)

• Induction: Methanol addition (0.5-1.0% final concentration) every 24 hours for 72-96 hours

• Purification: Affinity chromatography using a His-tag or specialized serpin-targeted methods

How can one confirm the identity and purity of the recombinant protein?

Verification of recombinant Bombyx mori Antichymotrypsin-2 identity and purity should involve multiple complementary approaches:

• SDS-PAGE analysis: Expect a band at approximately 45-50 kDa for the mature protein

• Western blotting: Using anti-serpin antibodies or epitope tag-specific antibodies

• Mass spectrometry: For precise molecular weight determination and peptide mapping

• N-terminal sequencing: To confirm the start with Phe1

• Enzyme inhibition assay: Measuring inhibitory activity against α-chymotrypsin with the reactive site identified at Thr343-Ser344

• Circular dichroism: To verify proper folding through secondary structure analysis

Purity assessment should achieve >95% homogeneity as verified by densitometry analysis of stained gels and chromatographic profiles.

What methods are suitable for analyzing the inhibitory activity of recombinant Bombyx mori Antichymotrypsin-2?

The inhibitory activity of recombinant Bombyx mori Antichymotrypsin-2 can be determined using several methods:

• Enzyme kinetic assays: Measure α-chymotrypsin activity with chromogenic or fluorogenic substrates (e.g., N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) in the presence of increasing concentrations of the inhibitor. Calculate IC50 and Ki values.

• Complex formation assays: Analyze the formation of covalent complexes between the serpin and protease using SDS-PAGE under non-reducing conditions.

• Progress curve analysis: Monitor the time-dependent inhibition to distinguish between different inhibition mechanisms.

• Reactive center loop cleavage assays: Assess the cleavage at the Thr343-Ser344 reactive site using mass spectrometry or N-terminal sequencing of cleavage products.

These methods can be calibrated using commercial serpins like α-1-antichymotrypsin as positive controls.

How does the structure of Bombyx mori Antichymotrypsin-2 compare to other insect serpins, and what implications does this have for its function?

Comparative structural analysis of Bombyx mori Antichymotrypsin with other insect serpins reveals important evolutionary and functional insights. The conserved serpin fold consists of three β-sheets, 8-9 α-helices, and the exposed reactive center loop (RCL). The RCL contains the protease recognition and cleavage site (Thr343-Ser344) .

Phylogenetic analysis using multiple alignment of sw-Achy with 23 other serpins shows that insect serpins form a distinct branch in the serpin evolutionary tree , suggesting specialized adaptation to insect-specific proteolytic systems. Key structural differences from human serpins include variations in the hinge region preceding the reactive center and in surface-exposed loops.

When compared with other insect immunity proteins like PGRP, which exist in multiple forms with distinct functions (BmPGRP2-1 as transmembrane and BmPGRP2-2 as intracellular) , Antichymotrypsin-2 likely represents an adaptation to specific proteolytic challenges in the silkworm immune system.

These structural distinctions have important functional implications:

• Specific protease targeting

• Altered inhibition kinetics

• Potential interactions with unique insect signaling pathways

• Possible participation in silkworm-specific immune responses against pathogens

What is the role of Bombyx mori Antichymotrypsin-2 in immune response, particularly against viral infections?

While direct evidence for Antichymotrypsin-2's role in viral immunity is limited, insights can be drawn from studies of related proteins in the silkworm immune system. The serpin family members, including antichymotrypsin, primarily regulate proteolytic cascades involved in various physiological processes including immunity.

Research on other silkworm immune proteins like BmPGRP2-2 shows that they can be induced by viral infection (BmNPV) and manipulate host signaling pathways like PTEN-PI3K/Akt to inhibit apoptosis . Similar mechanisms might apply to Antichymotrypsin-2.

Studies of transcriptome responses to viral infection in Bombyx mori have revealed complex interactions between host immunity proteins and viral pathogens. For example, BmTex261 overexpression inhibited AcMNPV infection in BmN cells, indicating its antiviral properties .

Potential immune functions of Antichymotrypsin-2 may include:

• Regulation of proteolytic cascades activated during viral infection

• Protection of host tissues from excessive proteolysis during immune response

• Modulation of apoptotic pathways, similar to BmPGRP2-2's anti-apoptotic function

• Direct interaction with viral proteins containing protease domains

What site-directed mutagenesis strategies would be most effective for enhancing the stability and activity of recombinant Bombyx mori Antichymotrypsin-2?

Based on the mutagenesis approaches applied to other Bombyx mori proteins like AChE II , several strategies can be proposed for Antichymotrypsin-2 enhancement:

• Reactive site loop engineering: Mutations near the Thr343-Ser344 reactive site could alter specificity and inhibition kinetics. Consider conservative substitutions (Thr→Ser, Ser→Ala) to fine-tune protease recognition.

• Stabilizing the serpin fold: Introduce disulfide bridges at strategic locations to enhance thermostability without compromising flexibility needed for the inhibitory mechanism.

• Surface charge optimization: Modify surface residues to improve solubility and reduce aggregation propensity.

• Glycosylation site engineering: Add or remove N-glycosylation sites to influence stability and half-life.

Drawing from the Y398 mutation studies in BmAChE II , a systematic approach to mutagenesis would involve:

Each mutation should be analyzed for structural integrity using circular dichroism and functional activity through enzyme inhibition assays.

What are the challenges and solutions for large-scale production of functionally active recombinant Bombyx mori Antichymotrypsin-2?

Scaling up production of recombinant Bombyx mori Antichymotrypsin-2 presents several challenges:

• Proper folding: Serpins have a metastable structure critical for their inhibitory function. At large scales, misfolding and polymerization can occur.

  Solution: Optimize expression conditions including temperature (typically lowered to 16-20°C during induction), use chaperone co-expression systems, and consider fusion partners that enhance solubility.

• Proteolytic degradation: Serpins are susceptible to cleavage by host proteases.

  Solution: Use protease-deficient expression hosts, add protease inhibitors during purification, and optimize purification protocols for speed.

• Maintaining inhibitory activity: Loss of activity during purification and storage.

  Solution: Validate activity at each purification step, determine optimal buffer conditions using differential scanning fluorimetry, and formulate with stabilizing agents like glycerol or specific salts.

• Expression system selection: Different systems have varying glycosylation patterns and folding machinery.

  Solution: Compare insect cell lines (Sf9, High Five), yeast (P. pastoris), and mammalian cells to identify optimal expression hosts. For methodology similar to that used for BmAChE II , P. pastoris often provides a good balance of yield and proper folding.

• Purification strategy: Obtaining homogeneous preparations at scale.

  Solution: Develop a multi-step purification protocol typically involving:

  • Initial capture with affinity chromatography (if tagged)

  • Intermediate purification using ion exchange chromatography

  • Polishing step using size exclusion chromatography

  • Activity-based separation to isolate only functional protein

How does Bombyx mori Antichymotrypsin-2 interact with signaling pathways involved in immune response and programmed cell death?

Understanding how Bombyx mori Antichymotrypsin-2 interacts with immune signaling pathways requires investigation of serpin-protease-signaling axis in silkworms. While specific data for Antichymotrypsin-2 is limited, insights can be drawn from other Bombyx mori immune proteins like PGRP.

BmPGRP2-2 negatively regulates PTEN, suppressing PTEN-PI3K/Akt signaling to inhibit cell apoptosis during viral infection . By analogy, Antichymotrypsin-2 might:

• Regulate proteolytic cascades that activate immune signaling pathways

• Inhibit specific proteases involved in programmed cell death pathways

• Protect signaling pathway components from proteolytic degradation

• Modulate the balance between pro-survival and pro-apoptotic signals

Possible experimental approaches to investigate these interactions include:

• Co-immunoprecipitation with components of immune signaling pathways

• RNAi-mediated knockdown followed by pathway analysis

• Overexpression studies to identify affected downstream targets

• Phosphoproteomic analysis to detect changes in signaling pathway activation

A proposed model for Antichymotrypsin-2 in immune modulation might involve inhibition of proteases that activate pro-apoptotic signals, thereby promoting cell survival during immune challenge, similar to how BmPGRP2-2 functions during viral infection .

What are the most effective approaches for studying structure-function relationships in recombinant Bombyx mori Antichymotrypsin-2?

Elucidating structure-function relationships in Bombyx mori Antichymotrypsin-2 requires an integrated approach combining structural biology, biochemistry, and functional assays:

• Structural determination:

  • X-ray crystallography of both native and cleaved forms

  • Cryo-EM for visualizing serpin-protease complexes

  • NMR for analyzing dynamics of the reactive center loop

  • Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

• Computational modeling:

  • Homology modeling based on known serpin structures

  • Molecular dynamics simulations to study conformational changes

  • Docking studies with target proteases

  • Similar to the approach used for BmAChE II , steered molecular dynamics can reveal binding mechanisms

• Systematic mutagenesis:

  • Alanine scanning of the reactive center loop

  • Domain swapping with other serpins

  • Introduction of reporter groups at strategic positions

  • Conservative and non-conservative substitutions at the reactive site (Thr343-Ser344)

• Functional correlation:

  • Enzyme kinetics with various target proteases

  • Stability measurements using thermal shift assays

  • Binding affinity determination via surface plasmon resonance

  • Cellular assays to monitor effects on proteolytic cascades

The Y398 mutation studies in BmAChE II provide a valuable template, showing how single residue changes can dramatically alter both activity and substrate interactions . Similar critical residues in Antichymotrypsin-2 can be identified through computational analysis and verified experimentally.