Recombinant Parachlorella kessleri Elongation factor 2, partial

What is Parachlorella kessleri and why is it used as an expression system for recombinant proteins?

Parachlorella kessleri is a microalgal species known for its high biomass and lipid production capabilities, making it a promising candidate for biotechnological applications. As demonstrated in recent studies, P. kessleri-I produces high biomass and lipid content that could be suitable for economically viable biofuel production at a commercial scale . Beyond biofuel applications, P. kessleri has garnered attention as a recombinant protein expression system due to several key advantages:

The complete chloroplast genome sequence (cpDNA) of P. kessleri-I has been assembled and annotated, providing crucial information for developing species-specific transformation vectors . This genomic characterization enables targeted genetic modification approaches for expressing foreign proteins. The chloroplast genome exhibits a quadripartite structure with two reverse repeat regions (IRA and IRB), a long single copy (LSC), and a small single copy (SSC) region, encoding 116 genes including 80 protein-coding genes .

The robust growth characteristics of P. kessleri under various environmental conditions make it adaptable to different cultivation parameters. Unlike some expression systems that require strictly controlled environments, P. kessleri can be cultivated using relatively simple methods while maintaining acceptable growth rates and protein expression levels.

What is Elongation Factor 2 and what functions does it serve in cellular protein synthesis?

Elongation Factor 2 (EF2) is a highly conserved protein essential for protein biosynthesis across multiple species. While specific information about P. kessleri EF2 is limited in the literature, we can understand its general function based on studies of EF2 in other organisms:

EF2 plays a crucial role in the translocation step of protein synthesis, facilitating the movement of the ribosome along the mRNA molecule. This process is critical for maintaining efficient protein synthesis rates in the cell. The high degree of conservation of EF2 across species is evidenced by studies showing remarkable sequence similarity in EF2 proteins from different organisms. For instance, research on Eimeria species demonstrated an amino acid sequence similarity of 99% among three different Eimeria species .

Due to its crucial role in cellular function and high degree of conservation, EF2 has been investigated as a target for immunological studies. Research demonstrated that EF2 from Eimeria maxima (EmEF2) was effectively recognized by antibodies and could induce immune responses , suggesting the immunogenic potential of this protein.

How does the chloroplast transformation of Parachlorella kessleri work, and what are its key advantages?

Chloroplast transformation in P. kessleri involves integrating foreign DNA into the chloroplast genome through homologous recombination. This approach offers several key advantages for recombinant protein production:

The chloroplast genome of P. kessleri-I (109,642 bp) has been fully sequenced and annotated, providing essential information such as codons, UTRs, and flank sequences for homologous recombination to create species-specific transformation vectors . This genomic information facilitates more precise genetic engineering approaches.

For selection of transformants, antibiotic resistance markers have been successfully employed. Studies have tested two antibiotic resistance markers: aminoglycoside adenine transferase (aadA) gene and Sh-ble gene, conferring resistance to spectinomycin and zeocin, respectively . Spectinomycin resistance at 400 mg.L-1 has proven effective for selecting transformed colonies .

Confirmation of successful transformation involves multiple techniques. Transgene integration and homoplasty (the complete replacement of all wild-type chloroplast genomes with the transformed version) can be confirmed using PCR, Southern blot analysis, and Droplet Digital PCR . These methods ensure the stable integration of the transgene into the chloroplast genome.

What cultivation conditions are optimal for maximizing recombinant protein expression in P. kessleri?

Optimizing cultivation conditions is critical for achieving high-level expression of recombinant proteins in P. kessleri. Based on studies of this microalga's growth parameters:

Culture Medium and Nutrients: Guillard's F/2 media supplemented with MgSO4 and CaCl2 at final concentrations of 0.3 mM and 0.17 mM, respectively, has been successfully used for P. kessleri cultivation . Maintaining pH between 7.4 and 7.8 is recommended for optimal growth .

Temperature and Aeration: Batch cultures maintained at 23°C and continuously aerated with ambient air have shown good growth characteristics . This moderate temperature appears to balance growth rate with stress minimization.

Stress Management: It's important to recognize that while certain stressors can be used to induce specific cellular responses, excessive stress can compromise recombinant protein expression. For instance, high turbulence (300 rpm) has been shown to significantly decrease growth while increasing superoxide production , which could potentially impact protein synthesis efficiency.

What are the most effective transformation methods for introducing recombinant EF2 into P. kessleri?

Successful transformation of P. kessleri for recombinant EF2 expression requires careful consideration of several methodological factors:

Vector Design: The creation of a species-specific vector that includes elements from the P. kessleri chloroplast genome is crucial for successful transformation. Research has shown that incorporating essential information like codons, UTRs, and flank sequences for homologous recombination from the chloroplast genome facilitates transformation efficiency .

Antibiotic Selection: Optimized antibiotic concentrations are critical for selecting transformed cells while minimizing false positives. Studies have determined that spectinomycin at 400 mg.L-1 completely inhibits the growth of wild-type P. kessleri cells, making it an effective selective agent . While zeocin at 20 mg.L-1 has also been tested, research indicates that spectinomycin may be more reliable for selection of chloroplast transformants in this species .

Transformation Protocol: Following transformation, plating approximately 1.5 × 106 cells onto solid media containing the appropriate antibiotic and incubating under 16:8 h light (irradiance of 2000 lux) and dark conditions at 25±1°C has proven effective . Colonies typically appear after 4-6 weeks of incubation.

Verification Methods: Screening putative transformants using colony PCR with gene-specific primers is a practical first step. For the verification of transformants, a PCR reaction mixture containing 1X Taq Reaction buffer, 0.1μg DNA template, 200 μM of each dNTPs, 1 Unit Taq polymerase, and 0.5 μM of each primer has been used successfully .

How can researchers effectively verify the integration and expression of recombinant EF2 in P. kessleri?

Verification of successful transformation and expression requires a multi-faceted approach:

PCR Verification: Initial screening using gene-specific primers targeting the inserted EF2 gene can rapidly identify potential transformants. For example, designing primers that span the junction between the chloroplast genome and the inserted gene provides strong evidence of proper integration .

Southern Blot Analysis: This technique provides definitive evidence of transgene integration into the chloroplast genome via homologous recombination. It can confirm both the presence of the transgene and its correct location within the genome .

Droplet Digital PCR: This advanced technique can precisely determine the chloroplast genome copy number in both wildtype and transgenic P. kessleri, allowing researchers to assess whether complete homoplasty has been achieved . Homoplasty is critical for stable and high-level expression of the recombinant protein.

Western Blot Analysis: To verify expression of the recombinant EF2 protein, Western blotting using antibodies specific to the expressed protein or to an added tag (such as a His-tag) is essential. For example, in studies with recombinant EF2 from other organisms, the protein was effectively recognized by His-tag monoclonal antibody .

Functional Assays: Depending on the intended application of the recombinant EF2, functional assays may be necessary to confirm that the expressed protein retains its biological activity.

How do environmental stressors affect recombinant protein expression in P. kessleri, and can these be leveraged to enhance production?

Environmental stressors can significantly impact cellular physiology in P. kessleri, with potential implications for recombinant protein expression:

Turbulence Stress: High levels of culture turbulence (300 rpm) have been shown to significantly decrease growth while increasing superoxide production and flocculation efficiency in P. kessleri . While this stress response might initially seem detrimental to protein production, strategic application of moderate turbulence could potentially be used to trigger cellular responses that enhance certain aspects of protein expression or facilitate harvesting through increased cellular aggregation.

Photoperiod Manipulation: Variations in day length have been shown to affect extracellular polysaccharide (EPS) production in P. kessleri, with significant increases observed under both shorter (8 and 12-hour) and longer (24-hour) day lengths compared to the standard 16:8 light:dark cycle . This physiological response could potentially be leveraged to enhance secretion of recombinant proteins designed for extracellular accumulation.

Salt Stress Response: P. kessleri significantly increases cell size and lipid droplet (LD) content when exposed to salt stress, suggesting major physiological adaptations to this stressor . Research indicates that lipid droplets play an important role in salt-stress tolerance mechanisms . This stress response pathway could potentially be manipulated to enhance production of recombinant proteins associated with stress response or lipid metabolism.

Combined Stress Strategies: Research indicates that stress responses in P. kessleri vary according to stress type and magnitude . This suggests the possibility of developing sophisticated stress protocols that combine multiple stressors at carefully calibrated levels to optimize recombinant protein production while maintaining adequate growth rates.

What challenges are associated with achieving homoplasty in P. kessleri chloroplast transformation, and how can they be overcome?

Achieving homoplasty (complete replacement of all wild-type chloroplast genomes with the transformed version) presents several challenges:

Multiple Chloroplast Genome Copies: Each chloroplast contains multiple copies of the genome, and each cell may contain multiple chloroplasts, resulting in numerous genome copies that must all be transformed to achieve homoplasty. Droplet Digital PCR has been used to determine chloroplast genome copy number in both wildtype and transgenic P. kessleri , providing valuable information for developing strategies to achieve complete homoplasty.

Multiple Rounds of Selection: Often, achieving homoplasty requires multiple rounds of selection on increasing antibiotic concentrations to eliminate any remaining wild-type chloroplast genomes. This process can be time-consuming but is essential for stable, high-level expression of the recombinant protein.

Verification Techniques: Confirming true homoplasty requires sensitive detection methods that can identify even small populations of wild-type chloroplast genomes. Southern blot analysis combined with Droplet Digital PCR provides a powerful approach for assessing homoplasty status .

What are the comparative advantages of chloroplast versus nuclear transformation for expressing recombinant EF2 in P. kessleri?

The choice between chloroplast and nuclear transformation presents important considerations for researchers:

Expression Levels: Chloroplast transformation typically allows for higher expression levels of recombinant proteins compared to nuclear transformation. This is due to the high copy number of chloroplast genomes within each cell and the prokaryotic-like nature of chloroplast gene expression machinery.

Post-Translational Modifications: Chloroplasts lack many of the post-translational modification capabilities present in the eukaryotic cytoplasm. If the recombinant EF2 requires specific modifications for functionality, nuclear transformation may be preferable despite potentially lower expression levels.

Gene Containment: Chloroplast genes are typically maternally inherited in most plant species, which provides an additional level of transgene containment that may be relevant for certain research or commercial applications.

Integration Stability: Homologous recombination in chloroplast transformation offers precise integration of the transgene into predetermined sites in the genome, while nuclear transformation often results in random integration, which can lead to position effects and variable expression levels.

Polycistronic Expression: The chloroplast genome allows for polycistronic expression of multiple genes from a single promoter, which can be advantageous for expressing EF2 along with other proteins of interest or with proteins that enhance expression or stability.

How does recombinant EF2 expression in P. kessleri compare with other microalgal expression systems?

When comparing P. kessleri with other microalgal expression systems for recombinant EF2 production:

Growth Characteristics: P. kessleri demonstrates robust growth under various environmental conditions, with stock cultures maintainable in relatively simple media such as Guillard's F/2 supplemented with MgSO4 and CaCl2 . This adaptability may provide advantages over more fastidious microalgal species.

Transformation Efficiency: The chloroplast genome of P. kessleri has been fully sequenced and annotated (109,642 bp) , providing essential information for developing efficient transformation vectors. This genomic characterization may offer advantages over less well-characterized microalgal species.

Stress Tolerance: P. kessleri exhibits notable stress tolerance mechanisms, including accumulation of lipid droplets under salt stress and production of extracellular polysaccharides in response to photoperiod variations . This stress resilience could potentially be advantageous for maintaining protein expression under suboptimal conditions.

Scalability: P. kessleri cultures can be maintained in simple batch systems with basic aeration and illumination requirements , potentially facilitating scaling of recombinant protein production compared to more demanding microalgal species.

What are the key differences between using bacterial versus microalgal systems for recombinant EF2 production?

Bacterial and microalgal expression systems offer distinct advantages and limitations for recombinant EF2 production:

Codon Usage: When expressing eukaryotic proteins, microalgal systems may offer more compatible codon usage patterns compared to bacterial systems, potentially reducing the need for extensive codon optimization that might be required for efficient expression in bacteria.

Scalability and Economics: While bacterial fermentation is well-established at industrial scales, microalgal cultivation can be performed using photobioreactors that utilize light energy and CO2, potentially offering economic and sustainability advantages for large-scale production.

Protein Solubility and Folding: Complex eukaryotic proteins like EF2 may fold more correctly in a microalgal system compared to bacterial cytoplasm, potentially reducing issues with inclusion body formation that often plague bacterial expression systems.

What are common challenges in achieving stable expression of recombinant EF2 in P. kessleri, and how can they be addressed?

Researchers may encounter several challenges when expressing recombinant EF2 in P. kessleri:

Unstable Expression: Incomplete homoplasty can lead to unstable expression as wild-type chloroplast genomes persist and potentially outcompete transformed genomes in the absence of selection pressure. To address this:

• Maintain selective pressure with appropriate antibiotic concentrations (e.g., 400 mg.L-1 spectinomycin)

• Perform multiple rounds of selection to drive the population toward complete homoplasty

• Verify homoplasty status using sensitive methods like Droplet Digital PCR

Low Expression Levels: If expression levels are lower than expected, consider:

• Optimizing the promoter and 5' UTR elements used to drive expression

• Adjusting cultivation conditions, particularly light intensity and photoperiod, as these factors significantly affect chloroplast gene expression

• Evaluating codon optimization strategies based on the codon usage preferences of the P. kessleri chloroplast genome

Protein Degradation: Recombinant proteins may be susceptible to degradation by chloroplast proteases. Strategies to address this include:

• Co-expressing chaperones or protease inhibitors

• Including stabilizing fusion partners or tags that can later be removed if necessary

• Optimizing harvest timing to capture peak expression before degradation occurs

Purification Challenges: Extracting and purifying the recombinant protein from the chloroplast environment can present difficulties. Consider:

• Including affinity tags such as His-tags for simplified purification, which have been successfully used with recombinant EF2 from other organisms

• Optimizing cell disruption methods that effectively break chloroplast membranes while minimizing protein degradation

• Developing fractionation protocols that enrich for chloroplast proteins

How can researchers optimize codon usage for maximum expression of recombinant EF2 in P. kessleri chloroplasts?

Codon optimization is a critical consideration for efficient expression of recombinant proteins:

Chloroplast-Specific Codon Usage: The chloroplast genome has distinct codon usage preferences that differ from those of the nuclear genome. Analysis of the P. kessleri chloroplast genome sequence (which encodes 80 protein-coding genes) can provide valuable information about preferred codons.

GC Content Adjustment: Adjusting the GC content of the recombinant gene to match that of highly expressed chloroplast genes can improve expression efficiency. This adjustment should be made while preserving the amino acid sequence of the protein.

Avoiding Rare Codons: Identifying and eliminating rare codons in the recombinant EF2 sequence is crucial. Rare codons can cause ribosomal pausing during translation, leading to reduced expression levels or truncated proteins.

Removal of Unfavorable Sequence Elements: Certain sequence elements can negatively impact expression, including:

• Internal Shine-Dalgarno-like sequences that may cause translational pausing

• Sequences that form stable RNA secondary structures, particularly near the 5' end of the transcript

• Cryptic splice sites that could lead to incorrect RNA processing

Codon Harmonization: Rather than simply using the most common codon for each amino acid, codon harmonization aims to mimic the codon usage pattern of the native gene, preserving the translational rhythm while avoiding rare codons. This approach may be particularly relevant for a complex protein like EF2.

What analytical techniques are most effective for characterizing recombinant EF2 expressed in P. kessleri?

Comprehensive characterization of recombinant EF2 requires multiple analytical approaches:

Expression Verification:

• Western blot analysis using antibodies specific to EF2 or to an added tag (e.g., His-tag)

• Mass spectrometry for definitive protein identification and sequence verification

• Quantitative PCR to measure transcript levels and assess expression at the RNA level

Functional Characterization:

• GTPase activity assays to confirm that the recombinant EF2 retains its enzymatic function

• Ribosome binding assays to assess interaction with translational machinery

• If applicable, immunological assays to evaluate antigenic properties, as demonstrated with recombinant EF2 from other organisms

Structural Analysis:

• Circular dichroism spectroscopy to assess secondary structure content

• Differential scanning calorimetry to evaluate thermal stability

• Limited proteolysis to probe folding and domain organization

• For more detailed structural information, X-ray crystallography or cryo-electron microscopy may be considered

Purity Assessment:

• SDS-PAGE with densitometry analysis to quantify purity

• Size-exclusion chromatography to identify aggregates or truncated forms

• Isoelectric focusing to detect charge variants

These analytical techniques provide complementary information about different aspects of the recombinant protein, enabling comprehensive characterization that is essential for research applications.

What emerging techniques could enhance recombinant EF2 production in P. kessleri?

Several emerging approaches show promise for improving recombinant protein expression in microalgal systems:

CRISPR/Cas9 Technology for Chloroplast Genome Editing: While CRISPR/Cas9 has been widely applied to nuclear genomes, its adaptation for chloroplast genome editing in microalgae is an emerging area. Development of efficient CRISPR systems for P. kessleri chloroplasts could enable more precise and efficient genetic modifications.

Synthetic Biology Approaches: Designing synthetic promoters, ribosome binding sites, and regulatory elements specifically optimized for P. kessleri chloroplasts could significantly enhance expression levels. The detailed characterization of the P. kessleri chloroplast genome (109,642 bp) provides valuable information for such synthetic biology approaches.

Inducible Expression Systems: Development of tightly regulated inducible systems for chloroplast gene expression could allow for controlled production of recombinant proteins. This approach could be particularly valuable for proteins that might impact chloroplast function when expressed at high levels.

Secretion Pathways: Engineering efficient secretion pathways could facilitate recovery of recombinant proteins without cell disruption. Research on extracellular polysaccharide production in P. kessleri under different environmental conditions provides insights into natural secretion mechanisms that could potentially be leveraged for recombinant protein secretion.

Bioprocess Optimization Using Artificial Intelligence: Machine learning approaches could accelerate the optimization of cultivation conditions for maximal protein expression, analyzing complex interactions between parameters such as light intensity, nutrient availability, and stress responses.

How might recombinant EF2 from P. kessleri be applied in research and biotechnology?

Recombinant EF2 from P. kessleri could have various research and biotechnological applications:

Immunological Research Tools: EF2 proteins have demonstrated immunogenic properties in other organisms, with studies showing that recombinant EF2 can be effectively recognized by antibodies . Recombinant P. kessleri EF2 could serve as a valuable research tool for immunological studies.

Protein Synthesis Research: As a key component of the translational machinery, recombinant EF2 could be used to study the mechanisms of protein synthesis in vitro, particularly for investigating species-specific aspects of translation.

Structural Biology: Producing sufficient quantities of pure EF2 would enable detailed structural studies, potentially revealing insights into the evolution and function of this highly conserved protein across different taxonomic groups.

Biomedical Applications: If P. kessleri EF2 exhibits unique properties or activities, it could potentially be developed for biomedical applications. For example, EF2 has been investigated as a potential target for antimicrobial or antiparasitic agents due to structural differences between prokaryotic and eukaryotic elongation factors.

Educational and Training Tools: Well-characterized recombinant proteins like EF2 can serve as valuable tools for education and training in molecular biology, providing students with opportunities to study protein structure-function relationships, expression systems, and purification techniques.