Recombinant Buchnera aphidicola subsp. Acyrthosiphon pisum Probable protease sohB (sohB)

What is Buchnera aphidicola and why is its protease SohB of particular research interest?

Buchnera aphidicola is an obligate endosymbiont of aphids that cannot be cultured outside of hosts. It exists as diverse strains in different aphid species, and phylogenetic reconstructions show that it has been maternally transmitted in aphids for over 100 million years . The SohB protein is classified as a probable protease, and while its exact function remains under investigation, proteases play critical roles in symbiotic relationships between aphids and their bacterial symbionts.

The Buchnera-aphid symbiosis represents one of the best-studied insect obligate symbioses, particularly in the pea aphid (Acyrthosiphon pisum) with Buchnera aphidicola APS . This symbiosis is primarily nutritional - aphids feed on plant phloem sap deficient in essential amino acids, and Buchnera supplements this diet through its biosynthetic capabilities . Proteases like SohB may be involved in protein processing crucial for this nutritional symbiosis.

What are the optimal expression systems and conditions for producing recombinant SohB protein?

Based on established protocols for similar bacterial proteases, the optimal expression system for recombinant SohB is E. coli, particularly the BL21(DE3) pLysS strain which allows tight control of protein expression . This strain has the following advantages for expression of potential proteases:

• The T7 expression system allows controlled induction

• The pLysS plasmid produces T7 lysozyme that inhibits T7 RNA polymerase, reducing leaky expression

• The addition of glucose can further repress the lac UV5 promoter

Table 1: Comparison of Expression Systems for Recombinant SohB Production

The expression protocols typically involve induction with IPTG followed by cell harvest, lysis, and purification. The purity can range from 80-95% depending on the amount of soluble expressed recombinant protein .

What purification methods yield the highest purity and activity for recombinant SohB?

Immobilized metal affinity chromatography (IMAC) is the recommended purification method for His-tagged recombinant SohB protein . The purification process should include:

• Preparation: Brief centrifugation of the vial prior to opening to bring contents to the bottom

• Reconstitution: Dissolving in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Stabilization: Addition of glycerol (5-50% final concentration) for long-term storage

• Aliquoting: Division into working volumes to avoid freeze-thaw cycles

For optimal purity (>90% as determined by SDS-PAGE), batch purification using a 2 mL 96-well filter block has been effective for similar proteases . For constructs with alternative fusion tags such as GST, glutathione agarose resin purification is recommended instead of IMAC .

What are the optimal storage conditions for maintaining SohB stability and activity?

The recommended storage conditions for recombinant SohB are:

• Store at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use

• Avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

The protein should be stored in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . The addition of glycerol (recommended final concentration of 50%) helps maintain stability during long-term storage at -20°C/-80°C .

How does SohB protease function in the context of the aphid-Buchnera symbiotic relationship?

While the exact function of SohB in the aphid-Buchnera symbiosis remains under investigation, research on similar proteases in symbiotic systems provides insights. The aphid-Buchnera symbiosis is primarily nutritional, with Buchnera supplying essential amino acids that are deficient in the aphid's phloem sap diet .

Proteases may play several critical roles in this relationship:

• Metabolism regulation: Proteases could be involved in regulating metabolic pathways crucial for amino acid biosynthesis

• Protein turnover: SohB might participate in protein quality control within Buchnera

• Signaling: Proteolytic processing may be involved in molecular communication between symbiont and host

• Adaptation: Differential expression or activity of proteases like SohB might contribute to biotype differentiation in aphids

Recent research has shown that reduction of Buchnera abundance through antibiotic treatment can alter the virulence of aphid biotypes, suggesting that Buchnera proteins, potentially including proteases like SohB, might influence aphid-plant interactions .

How does SohB compare with proteases identified in aphid saliva and other endosymbionts?

Proteomic analysis of aphid saliva has identified several proteases that may play roles in plant-aphid interactions. In the pea aphid Acyrthosiphon pisum, nine proteins secreted in saliva were identified, including:

• A homolog of angiotensin-converting enzyme (an M2 metalloprotease)

• An M1 zinc-dependent metalloprotease

• A glucose-methanol-choline (GMC)-oxidoreductase

• A homolog to regucalcin

These salivary proteases are predicted to inactivate plant protein defenses and inhibit calcium-mediated occlusion of phloem sieve elements, thereby facilitating sustained feeding . In contrast, SohB from Buchnera is an intracellular protease that may serve different functions within the symbiont or in the bacteriocyte (specialized aphid cells housing Buchnera colonies).

In other symbiotic systems, proteases from Serratia symbiotica (another aphid symbiont) have been identified, including DegQ, HtpX, YfgC, SohB, and PepA, which may be important for tritrophic interactions between symbionts, insects, and plants . Quantitative PCR analysis has shown that S. symbiotica can be transmitted to host plants by aphids and persist for up to 10 days post-feeding .

What is the genomic context of the sohB gene in Buchnera aphidicola and how has it evolved?

The genomic context of sohB in Buchnera aphidicola reflects the evolutionary history of this obligate endosymbiont. Buchnera genomes are highly reduced and show conserved gene order and no gene acquisition, but encoded proteins undergo rapid evolution .

The Buchnera genome has undergone extensive reduction during its >100 million years of symbiosis with aphids. Despite this reduction, comparative genomics of different Buchnera strains reveals that certain genes, including those involved in essential functions, have been retained . The sohB gene is one such conserved gene, suggesting its importance for Buchnera survival or for the symbiotic relationship.

Buchnera aphidicola genomes from four aphid species have been sequenced:

• B. aphidicola BAp from Acyrthosiphon pisum

• B. aphidicola BSg from Schizaphis graminum

• B. aphidicola BBp from Baizongia pistaciae

• B. aphidicola BCc from Cinara cedri

These genomes exhibit "extreme evolutionary stasis with nearly perfect gene order conservation" , suggesting that the genomic context of sohB is likely similar across different Buchnera strains.

What are effective experimental designs for studying SohB protease activity and specificity?

To effectively study SohB protease activity and specificity, researchers can employ several methodological approaches:

• Zymography: This technique allows visualization of proteolytic activity in gels containing protein substrates. It has been successfully used to detect novel proteolytic activities in recombinant proteases .

• Fluorescence-based assays: These assays use fluorogenic substrates to measure protease activity. For recombinant proteases, this approach has confirmed proteolytic activity in approximately 46% of purified proteases and 40% of hypothetical proteins predicted to be proteases .

• Comparative activity assays: Testing SohB against various substrates can help determine its specificity. The relative activity can be calculated as a percentage of the maximum activity observed.

• Inhibitor profiling: Testing different protease inhibitors can help classify SohB and understand its catalytic mechanism.

Table 2: Recommended Experimental Design for SohB Activity Characterization

How can researchers investigate SohB's role in the aphid-Buchnera-plant interaction system?

Investigating SohB's role in the complex aphid-Buchnera-plant interaction system requires a multifaceted approach:

• Localization studies: Determine where SohB is expressed and active within the system using immunohistochemistry or fluorescent protein fusions.

• Quantitative gene expression analysis: Measure sohB expression levels in different aphid biotypes, under different conditions, or when fed on different host plants using qRT-PCR . For example, in a study of Serratia symbiotica genes in aphids, expression of proteases including SohB was evaluated by qRT-PCR after normalization to the expression level of the rpl32 reference gene .

• Antibiotic treatment: Reduce Buchnera abundance using antibiotics like rifampicin and observe effects on aphid virulence and fitness . This approach has shown that reduction in Buchnera abundance can alter the virulence of aphid biotypes.

• Artificial diet experiments: Supplement aphid diets with purified recombinant SohB or with specific amino acids to determine effects on aphid development and fecundity. Research has shown that leucine and tryptophan deficiencies significantly impact nymph development duration and aphid fecundity .

• Plant response analysis: Examine plant responses to aphid feeding with normal or reduced Buchnera levels to assess potential roles of Buchnera proteins in suppressing plant defenses.

What techniques can be used to study potential lateral gene transfer involving the sohB gene?

The study of lateral gene transfer (LGT) involving the sohB gene requires specialized techniques:

• Comparative genomics: Compare the sequence, GC content, and codon usage of sohB across different Buchnera strains and related bacteria to identify potential LGT events.

• Phylogenetic analysis: Construct phylogenetic trees using sohB sequences from different organisms to detect incongruencies that might indicate LGT. For example, a neighbor-joining phylogenetic tree has been used to indicate genetic differentiation of Buchnera among different aphid biotypes .

• Whole genome analysis: Examine the genomic context of sohB for signatures of LGT, such as proximity to mobile genetic elements or regions with atypical sequence characteristics.

• Molecular clock analyses: Compare evolutionary rates of sohB with other genes to identify accelerated or decelerated evolution that might indicate LGT.

• Experimental evolution: Monitor changes in sohB under selective pressure in laboratory settings to understand its evolutionary dynamics.

What strategies can address poor solubility and yield issues when expressing recombinant SohB protein?

Poor solubility and yield are common challenges when expressing recombinant proteases. Several strategies can address these issues:

• Fusion tags: Different fusion tags can significantly improve solubility and expression levels. For example, fusion to large protein domains such as maltose-binding protein (MBP), SP-MBP (containing signal peptide at the N-terminus of MBP), disulfide oxidoreductase (DsbA), or Glutathione S-transferase (GST) has improved expression and solubility of proteases .

• Expression vectors: Utilizing different expression vectors encoding these fusion proteins can enhance recombinant protein solubility. The following expression vectors have proven effective:

  • pMBP: Contains His-tag upstream of MBP but lacks signal peptide

  • pSP-MBP: Engineered with signal peptide at N-terminal end of MBP

  • pDsbA: Contains signal peptide as part of fusion

  • pGST: Contains N-terminal GST fusion tag

• Expression conditions: Optimizing temperature, induction timing, and inducer concentration can significantly improve yields. Lower temperatures (16-18°C) during induction often increase the proportion of soluble protein.

• Host strain selection: E. coli BL21(DE3) pLysS allows tight control of expression of recombinant proteins, which is particularly important for potentially toxic proteins like proteases .

• Co-expression with chaperones: Co-expressing molecular chaperones can facilitate proper folding and increase solubility.

How can researchers verify SohB protease activity when traditional assays yield ambiguous results?

When traditional protease assays yield ambiguous results for SohB, alternative approaches can be employed:

• Zymography with different substrates: Using various protein substrates in zymography gels can help detect activity that might be substrate-specific.

• In-gel activity assays: After non-denaturing PAGE, incubate the gel with different substrates and detection systems to visualize activity bands.

• Mass spectrometry-based approaches: Incubate SohB with potential protein substrates and analyze the mixture using mass spectrometry to identify cleavage products and sites.

• Thermal shift assays: Monitor protein thermal stability in the presence of potential inhibitors; binding typically increases stability.

• Comparative proteomics: Compare protein profiles between samples with and without SohB treatment to identify potential substrates.

• Live-cell imaging: Use fluorescent reporters to visualize protease activity in living cells when possible.

What controls should be included when studying the effects of recombinant SohB in biological systems?

When studying the effects of recombinant SohB in biological systems, the following controls are essential:

• Inactive enzyme control: Use heat-inactivated SohB or create a catalytically inactive mutant by site-directed mutagenesis of the active site.

• Buffer-only control: Include the storage/reconstitution buffer without SohB to control for buffer effects.

• Non-specific protein control: Include an unrelated protein of similar size and preparation to control for general protein effects.

• Concentration gradient: Test multiple concentrations of SohB to establish dose-response relationships.

• Positive control: Include a well-characterized protease with known activity and effects as a benchmark.

• Time-course analysis: Monitor effects over time to distinguish between immediate and delayed responses.

• System-specific controls: For aphid-Buchnera-plant systems, controls could include antibiotic-treated aphids to reduce Buchnera (as in ), aphids fed on different plant varieties, or artificial diet compositions lacking specific amino acids.