Recombinant Pseudoalteromonas haloplanktis ATP synthase subunit c (atpE)

What makes Pseudoalteromonas haloplanktis TAC125 suitable for recombinant protein production?

Pseudoalteromonas haloplanktis TAC125 (PhTAC125) offers unique advantages as a recombinant protein expression host due to its psychrophilic nature and adaptability to extreme environments. As a cold-adapted marine bacterium, it can grow efficiently at temperatures as low as 4-15°C, with 15°C being its optimal growth temperature . This cold-adapted metabolism provides significant advantages for expressing proteins that tend to form inclusion bodies or become non-functional when produced in conventional mesophilic hosts.

The bacterium possesses a fully sequenced and annotated multipartite genome, consisting of two chromosomes and the cryptic pMtBL plasmid, which has facilitated the development of various genetic tools for recombinant protein expression . PhTAC125 has demonstrated exceptional capabilities in preventing insoluble protein aggregates, making it particularly valuable for the production of "difficult proteins" that are challenging to express in functional forms using traditional systems .

How does the growth of PhTAC125 in biofilm conditions compare to planktonic culture for recombinant protein production?

Biofilm cultivation of PhTAC125 offers distinct advantages for recombinant protein production compared to traditional planktonic culture, though with some trade-offs. Research demonstrates that biofilm-based production systems require lower concentrations of carbon sources and can eliminate the need for antibiotic supplementation during cultivation . This represents a significant cost reduction for long-term protein production processes.

While biofilm-based production requires a longer cultivation period than planktonic approaches, it has been demonstrated to have comparable production potential for some proteins. In the specific case of the fluorescent protein mScarlet, biofilm-based expression actually outperformed planktonic systems in terms of recombinant product quality . This suggests that the microenvironment within biofilms may provide conditions that favor proper folding and maturation of certain recombinant proteins.

For optimal recombinant protein production in biofilm conditions, the induction timing is critical. Adding the inducer (IPTG) at the beginning of biofilm formation resulted in the best ratio between fluorescent cells and matrix biomass, while adding IPTG after biofilm establishment was ineffective . This is likely due to limitations in molecule diffusion within established biofilms and/or lack of oxygen required for proper maturation of fluorescent proteins.

What genetic tools and vectors are available for expressing recombinant proteins in PhTAC125?

Researchers have developed several expression vectors and genetic tools specifically for PhTAC125:

• Expression vectors based on pMtBL replication signals with both constitutive and inducible promoters are available for recombinant protein production in both planktonic and biofilm cultures .

• Techniques for construction of insertion/deletion genomic mutants using homologous recombination have been established, allowing for allelic exchange and/or gene inactivation by in-frame deletion using counter-selection markers with non-replicative plasmids .

• A conditional gene silencing system based on PTasRNA technology has been developed to effectively down-regulate chromosomal gene expression in PhTAC125, achieving high levels of gene silencing .

• Intergeneric conjugation methods have been established as an effective means of transforming PhTAC125 cells with appropriate vectors .

These tools allow for complex genetic manipulations and precise control over gene expression, providing researchers with multiple approaches to optimize recombinant protein production, including potential ATP synthase subunit c expression.

What are the recommended induction conditions for optimal expression of recombinant proteins in PhTAC125?

The optimal induction conditions for recombinant protein expression in PhTAC125 depend significantly on whether the cells are grown in planktonic or biofilm conditions. For biofilm-based expression systems:

• Adding the inducer (IPTG) at time zero (the beginning of biofilm formation) results in the best ratio between fluorescent cells and matrix biomass .

• Attempting to induce expression after the biofilm is already structured results in ineffective production, likely due to limitations in molecule diffusion within established biofilms .

This timing consideration is particularly important for proteins requiring post-translational modifications, such as the oxygen-dependent maturation needed for fluorescent proteins. The oxygen gradient present in biofilms must be considered when designing expression protocols for proteins with similar requirements .

How can the extracellular proteolytic activity of PhTAC125 be reduced to improve recombinant protein yields?

Extracellular proteases can significantly reduce the quality and yield of secreted recombinant proteins. PhTAC125 naturally secretes multiple extracellular proteases, which can degrade recombinant products. A successful strategy to address this issue has been the functional inactivation of the Type II Secretion System (T2SS) through targeted mutation of the gspE gene .

The gspE gene encodes a specialized ATP-synthase essential for T2SS functionality. Researchers have developed a gene insertion strategy to knock out this gene, resulting in a mutant strain with remarkably reduced extracellular protease secretion . Importantly, this genetic modification does not affect the strain's ability to secrete the psychrophilic α-amylase, which serves as a secretion carrier in many recombinant protein expression systems .

The gspE mutant strain even demonstrates improved growth characteristics compared to the wild type, growing faster and achieving higher cellular biomass under standard conditions . SDS-PAGE analysis of concentrated culture supernatants confirmed that the mutant strain secretes significantly fewer proteins compared to the wild type, indicating reduced proteolytic activity in the extracellular environment .

This improved host strain offers enhanced biotechnological potential for recombinant protein secretion at low temperatures, making it particularly valuable for expressing sensitive proteins that might otherwise be degraded by host proteases .

What is the impact of pMEGA plasmid curing on the performance of PhTAC125 as a recombinant host?

The endogenous megaplasmid (pMEGA) in PhTAC125 has been successfully removed to streamline the host genetic background. Researchers employed a sequential genetic approach combining homologous recombination with PTasRNA gene-silencing technology that interferes with the pMEGA replication machinery .

• Enhanced resistance to oxidative stress, suggesting that some elements on the pMEGA plasmid may negatively regulate stress response mechanisms .

• Reduced capacity for biofilm formation, indicating that pMEGA carries genetic elements that contribute to the development of biofilm structures .

These findings have important implications for recombinant protein production. The enhanced oxidative stress resistance may benefit the expression of proteins sensitive to oxidative damage, while the reduced biofilm formation capacity must be considered when designing biofilm-based expression systems with this strain. The cured strain potentially offers a cleaner genetic background for recombinant protein expression, with fewer competing endogenous processes .

What are the key optimization parameters for expressing membrane proteins like ATP synthase subunit c in PhTAC125?

Expressing membrane proteins such as ATP synthase subunit c presents unique challenges due to their hydrophobic nature and complex integration into lipid bilayers. When using PhTAC125 as an expression host, several key parameters must be optimized:

• Media composition: The growth medium can significantly impact recombinant protein production in PhTAC125. For biofilm-based systems, optimizing media composition is essential for both biofilm formation and protein expression . For membrane proteins, the availability of specific lipids may affect proper folding and insertion into membranes.

• Induction conditions: As demonstrated with fluorescent proteins, the timing of induction is critical, especially in biofilm conditions where inducer diffusion may be limited . For membrane proteins, gradual induction at lower concentrations of inducer may help prevent overwhelming the membrane insertion machinery.

• Expression vector design: Constructing specialized expression vectors suitable for the intended growth conditions (planktonic vs. biofilm) can significantly improve yields . For membrane proteins like ATP synthase subunit c, incorporating appropriate signal sequences or fusion partners may facilitate proper membrane targeting.

• Temperature optimization: As a psychrophilic organism, PhTAC125 offers the advantage of low-temperature expression, which can slow down protein synthesis and allow proper folding and membrane insertion of complex proteins. The optimal temperature range of 4-15°C should be carefully evaluated for ATP synthase subunit c expression .

How does the performance of PhTAC125 compare to other expression systems for producing ATP synthase components?

PhTAC125 offers several distinct advantages for expressing ATP synthase components compared to conventional expression systems:

• Cold-adapted expression: The psychrophilic nature of PhTAC125 allows protein expression at low temperatures (4-15°C), which can be particularly beneficial for complex membrane proteins like ATP synthase subunit c that may aggregate or misfold at higher temperatures .

• Reduced proteolytic degradation: The engineered gspE mutant strain with reduced extracellular proteolytic activity provides a more stable environment for secreted recombinant proteins . This can be particularly valuable for exposed portions of membrane proteins or during purification processes.

• Prevention of insoluble aggregates: PhTAC125 has demonstrated exceptional capabilities in preventing insoluble protein aggregates, making it valuable for expressing "difficult proteins" that are challenging to produce in functional forms using traditional systems .

• Versatile growth conditions: The ability to grow PhTAC125 in either planktonic or biofilm conditions offers flexibility in expression strategies . For membrane proteins like ATP synthase subunit c, the structured environment of a biofilm might provide advantages for proper membrane insertion and assembly.

• Advanced genetic tools: The availability of various expression vectors, gene knockout techniques, and conditional gene silencing systems allows for sophisticated genetic manipulations to optimize expression conditions .

What are the recommended approaches for purifying recombinant membrane proteins from PhTAC125?

Purifying recombinant membrane proteins like ATP synthase subunit c from PhTAC125 requires specialized approaches to maintain protein integrity and functionality:

• Cell disruption at low temperatures: Given the psychrophilic nature of PhTAC125, all purification steps should be performed at low temperatures (preferably 4°C) to maintain protein stability. Mechanical disruption methods like French press or sonication at controlled temperatures are recommended to avoid protein denaturation.

• Membrane fraction isolation: Differential centrifugation can be used to separate membrane fractions containing the recombinant ATP synthase subunit c. Typically, low-speed centrifugation (5,000-10,000 × g) removes unbroken cells and debris, followed by high-speed ultracentrifugation (100,000-150,000 × g) to pellet membrane fractions.

• Detergent solubilization: Careful selection of detergents is critical for membrane protein extraction while maintaining native structure. For ATP synthase components, mild non-ionic detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin often provide good results.

• Affinity purification: Incorporating affinity tags (His-tag, Strep-tag) in the recombinant construct enables specific purification. For ATP synthase subunit c, C-terminal tags are often preferable to avoid interfering with membrane insertion. The psychrophilic amylase system can also be used as a secretion carrier if appropriate for the experimental design .

• Size exclusion chromatography: As a final purification step, size exclusion chromatography can separate properly folded protein from aggregates while maintaining the protein in appropriate detergent micelles.

The advantage of using PhTAC125 for membrane protein expression includes potentially better folding at low temperatures and reduced proteolytic degradation in engineered strains, which can significantly improve the quality of the purified protein .

What analytical methods are most effective for characterizing recombinant ATP synthase components from PhTAC125?

Several analytical methods are particularly valuable for characterizing recombinant ATP synthase components expressed in PhTAC125:

• Functional assays: ATP hydrolysis/synthesis assays using reconstituted proteoliposomes can confirm functional activity of ATP synthase components. For subunit c specifically, proton translocation assays using pH-sensitive fluorescent dyes can assess channel functionality.

• Structural analysis: Circular dichroism (CD) spectroscopy is useful for assessing secondary structure content and thermal stability, particularly relevant for proteins expressed in a psychrophilic host. Cryo-electron microscopy may provide structural insights for assembled ATP synthase complexes.

• Mass spectrometry: Mass spectrometry techniques can confirm the identity and integrity of the expressed protein, detect post-translational modifications, and analyze protein-protein interactions through cross-linking approaches.

• Protein-lipid interactions: Fluorescence-based assays or surface plasmon resonance can characterize interactions between the recombinant ATP synthase subunit c and lipid membranes, which is crucial for understanding functional integration.

• Comparative analysis: Comparing the properties of ATP synthase components expressed in PhTAC125 with those from conventional expression systems can highlight differences in folding, stability, and activity. This comparative approach is particularly valuable when optimizing expression conditions in the psychrophilic host.

• In-gel activity stains: Clear native PAGE combined with in-gel ATP hydrolysis activity staining can assess the assembly and functionality of ATP synthase complexes while maintaining native interactions.

When characterizing membrane proteins like ATP synthase subunit c, maintaining appropriate detergent or lipid environments throughout the analytical procedures is essential for obtaining meaningful results that reflect native protein characteristics.

How can low expression yields of recombinant ATP synthase subunit c in PhTAC125 be addressed?

Low expression yields of membrane proteins like ATP synthase subunit c in PhTAC125 can be addressed through several strategic approaches:

• Optimize codon usage: Adapting the coding sequence to the codon bias of PhTAC125 can significantly improve translation efficiency. This is particularly important for heterologous genes that may contain rare codons in the psychrophilic host.

• Adjust induction protocol: For biofilm-based expression, inducing at the beginning of biofilm formation rather than after biofilm establishment has been shown to significantly improve protein production . For planktonic cultures, testing different inducer concentrations and induction durations can help identify optimal conditions.

• Engineer expression vectors: Developing specialized expression vectors with appropriate promoters and ribosome binding sites optimized for the psychrophilic host can enhance expression levels. Consider testing both constitutive and inducible promoter systems available for PhTAC125 .

• Fusion partners approach: Using the psychrophilic α-amylase as a secretion carrier in fusion constructs can improve expression and facilitate purification . For membrane proteins like ATP synthase subunit c, carefully designed fusions that don't interfere with membrane integration are essential.

• Strain optimization: Consider using engineered strains like the gspE mutant with reduced extracellular protease activity to minimize proteolytic degradation of expressed proteins . The pMEGA-cured strain may also provide benefits through enhanced oxidative stress resistance .

• Media optimization: Systematically testing different media compositions can identify conditions that favor recombinant protein production while supporting robust growth of the psychrophilic host .

• Temperature optimization: While PhTAC125 has an optimal growth temperature of 15°C, expression of certain recombinant proteins may benefit from even lower temperatures to slow down protein synthesis and allow proper folding.

What strategies can address protein misfolding or aggregation of recombinant ATP synthase components in PhTAC125?

Membrane proteins like ATP synthase components are particularly prone to misfolding and aggregation. Several strategies can mitigate these challenges when expressing such proteins in PhTAC125:

These strategies can be employed individually or in combination, with systematic optimization to determine the most effective approach for expressing functional ATP synthase components in the PhTAC125 system.