Recombinant Coelogyne cristata Protein psbN (psbN), partial

What is the psbN protein in Coelogyne cristata and what is its function?

The psbN protein in Coelogyne cristata is a chloroplast-encoded protein found in the photosystem II complex. It functions as a small subunit involved in photosynthetic electron transport and plays a role in the assembly and stability of photosystem II complexes. The psbN gene is located in the large single-copy (LSC) region of the chloroplast genome, which in Coelogyne species typically spans approximately 87,600-87,760 bp . The protein is considered "partial" when the recombinant form represents a fragment rather than the complete protein sequence, which is often used for specific research applications focusing on particular functional domains.

How is the chloroplast genome of Coelogyne cristata structured and where is the psbN gene located?

The chloroplast genome of Coelogyne species, including C. cristata, exhibits a typical quadripartite structure consisting of a pair of inverted repeats (IRs) separated by large single-copy (LSC) and small single-copy (SSC) regions. Based on closely related Coelogyne species like C. fimbriata and C. ovalis, the complete chloroplast genome is approximately 159,795-160,040 bp in length . The psbN gene is typically located in the LSC region, which contains the majority of protein-coding genes (61 protein-coding genes in related Coelogyne species) . The chloroplast genome demonstrates a relatively high conservation in structure across Coelogyne species, with GC content around 37.3-37.4% .

What expression systems are commonly used for producing recombinant orchid proteins?

For recombinant orchid proteins like psbN from Coelogyne cristata, several expression systems can be employed, with yeast-based systems such as Saccharomyces cerevisiae and Pichia pastoris being particularly effective for plant proteins . These systems allow for post-translational modifications which may be necessary for proper protein folding and function. P. pastoris has gained popularity for recombinant protein expression due to its ability to achieve high cell densities (up to 120 g DCW per liter) in chemically defined media and its eukaryotic protein processing capabilities . Plant-based expression systems are also viable alternatives, offering advantages such as proper glycosylation patterns, low production costs, and reduced risk of contamination with animal pathogens .

What are the challenges in expressing and purifying functional recombinant psbN protein from Coelogyne cristata?

Expressing and purifying functional recombinant psbN protein from Coelogyne cristata presents several significant challenges. As a chloroplast membrane protein, psbN is inherently hydrophobic and may form inclusion bodies when overexpressed, particularly in bacterial systems. Expression in eukaryotic systems like P. pastoris may improve solubility, but optimization of expression conditions is critical . The small size of psbN also presents purification challenges, requiring careful selection of affinity tags that minimize interference with protein function while facilitating efficient purification. Additionally, maintaining the protein's native conformation during extraction and purification is essential, often requiring specialized detergents or membrane-mimetic systems. The limited genomic information available for Coelogyne cristata compared to model organisms further complicates the design of appropriate expression constructs .

How can CRISPR/Cas9 technology be applied to enhance the production of recombinant Coelogyne cristata proteins?

CRISPR/Cas9 technology can be strategically applied to optimize expression systems for recombinant Coelogyne cristata proteins. In yeast expression systems like P. pastoris, CRISPR/Cas9 enables precise genetic modifications including gene integration, knockout of unwanted genes, and multiplexed gene deletions without marker genes . Specifically, researchers can use CRISPR/Cas9 to integrate the psbN gene at specific genomic loci known to enhance expression, with integration efficiencies approaching 100% in ku70 deletion strains . The technology also allows for one-step multiloci gene integration without selective markers, which is valuable for complex expression strategies . Additionally, CRISPR/Cas9 can be used to knock out proteases that might degrade the recombinant protein or modify glycosylation pathways to produce proteins with more human-like glycosylation patterns, potentially enhancing stability and functionality .

What comparative genomic approaches can reveal evolutionary insights about psbN conservation across Orchidaceae?

Comparative genomic analysis of the psbN gene across Orchidaceae can provide valuable evolutionary insights through several methodological approaches. Researchers should begin by extracting and sequencing the chloroplast genomes from multiple Coelogyne species and other orchid genera, following protocols similar to those used for C. fimbriata and C. ovalis, which employed Illumina MiSeq sequencing with reference-guided assembly . Sequence alignment of the psbN coding regions can identify conserved domains which likely represent functionally critical regions of the protein. dN/dS ratio analysis can detect signatures of selection pressure, indicating whether psbN evolution has been primarily driven by purifying, neutral, or positive selection. Phylogenetic tree construction based on psbN sequences should be compared with trees derived from other chloroplast genes and the complete plastome to identify potential incongruence that might suggest horizontal gene transfer events or lineage-specific selection pressure . The close relationship between Coelogyne and Pleione genera identified in previous phylogenetic analyses provides context for interpreting psbN evolution patterns .

What are the optimal extraction and amplification protocols for isolating the psbN gene from Coelogyne cristata?

For optimal extraction and amplification of the psbN gene from Coelogyne cristata, a comprehensive protocol should be followed. Begin with fresh, young leaf tissue (approximately 100 mg) and employ a CTAB-based extraction method with modifications for plants rich in secondary metabolites. Include 2% polyvinylpyrrolidone (PVP) and 2% β-mercaptoethanol in the extraction buffer to neutralize phenolic compounds and prevent oxidation. After DNA extraction, verify quality using spectrophotometry (A260/A280 ratio between 1.8-2.0) and gel electrophoresis.

For PCR amplification, design primers based on conserved regions flanking the psbN gene identified from the chloroplast genome sequences of related Coelogyne species . A nested PCR approach may increase specificity, with initial amplification of a larger chloroplast region followed by psbN-specific amplification. Optimize PCR conditions with a touchdown protocol starting at 65°C and decreasing to 55°C over 10 cycles, followed by 25 cycles at 55°C. Purify the amplified product using silica column-based methods and verify through sequencing before proceeding to cloning steps.

For challenging samples, whole chloroplast genome sequencing following the methods used for C. fimbriata and C. ovalis might be necessary, which yielded approximately 3 million clean reads with an average read length of 300 bp .

What expression vector designs maximize recombinant psbN protein yield and functionality?

Designing optimal expression vectors for recombinant psbN protein requires careful consideration of multiple elements. For P. pastoris expression, construct a vector incorporating the strong, inducible AOX1 promoter for controlled expression, paired with a terminator sequence that ensures efficient transcription termination . Include a secretion signal (such as the α-mating factor from S. cerevisiae) to direct protein secretion, potentially improving folding and reducing toxicity. For membrane proteins like psbN, consider fusion tags that enhance solubility such as MBP (maltose-binding protein) or SUMO, positioned at the N-terminus with a precision protease cleavage site for tag removal.

Vector construction should include centromeric DNA sequences as found in newly developed P. pastoris plasmids, which exhibit high stability for plasmid retention without integration, facilitating genetic manipulation and high-throughput screening . Optimize codon usage for the expression host to enhance translation efficiency, particularly for rare codons in the psbN sequence. For complex experiments, implement the newly developed CRISPR/Cas9-compatible vectors that enable marker-less genome integration at efficiencies approaching 100% .

The vector should contain multiple restriction sites for flexible cloning strategies and must be verified through sequencing before transformation. This comprehensive design approach has shown success in expressing complex membrane proteins in yeast systems.

How can researchers optimize purification strategies for recombinant psbN protein while maintaining structural integrity?

Optimizing purification of recombinant psbN protein requires a multi-step approach that preserves structural integrity. Begin with careful cell lysis using either mechanical disruption (for yeast cells) or gentle enzymatic methods (for plant cells), maintaining low temperature (4°C) throughout all purification steps. For membrane-associated proteins like psbN, incorporate a detergent screen testing multiple detergents (CHAPS, DDM, Triton X-100) at various concentrations to identify optimal solubilization conditions.

Implement a two-phase purification strategy: initial capture using affinity chromatography based on the fusion tag (His-tag or GST), followed by size exclusion chromatography to isolate monomeric psbN from aggregates. Consider intermediate ion-exchange chromatography if contaminants persist. Throughout purification, verify protein integrity using SDS-PAGE and Western blotting with antibodies specific to psbN or the fusion tag.

For structural studies, maintain protein stability using buffer optimization through differential scanning fluorimetry, testing various pH values (6.0-8.0), salt concentrations (100-500 mM NaCl), and stabilizing additives (glycerol, specific lipids). If expression yields are low, scale up production using bioreactor systems capable of achieving high cell densities in P. pastoris (up to 120 g/L) .

Monitor protein folding through circular dichroism spectroscopy and fluorescence-based thermal shift assays to confirm that purification conditions preserve the native structure, which is critical for functional studies of photosystem proteins.

How should researchers interpret discrepancies between predicted and observed properties of recombinant psbN?

When confronted with discrepancies between predicted and observed properties of recombinant psbN protein, researchers should implement a systematic analytical framework. Begin by comparing the amino acid sequence of the expressed protein with the predicted sequence through mass spectrometry to identify potential post-translational modifications or unexpected truncations. Conduct circular dichroism analysis to compare secondary structure elements with computational predictions, noting that membrane proteins like psbN may adopt different conformations in detergent micelles versus native lipid environments.

For unexpected electrophoretic mobility, consider the effects of post-translational modifications and the highly charged nature of many chloroplast proteins. Native electrophoresis paired with size exclusion chromatography can resolve whether anomalous migration is due to oligomerization or extended protein conformations. For functional discrepancies, examine whether the recombinant protein lacks critical cofactors present in the native chloroplast environment, potentially requiring reconstitution experiments with chlorophyll or other photosystem components.

Additionally, compare expression results across different hosts (bacterial, yeast, plant systems) to identify system-specific artifacts. Document all discrepancies methodically, as they may reveal previously unknown properties of the psbN protein that could provide insights into its native function and folding requirements in Coelogyne cristata.

What bioinformatic tools are most effective for analyzing the structure-function relationship of psbN protein?

For comprehensive analysis of psbN structure-function relationships, researchers should employ a multi-tiered bioinformatic approach. Begin with sequence-based tools including BLAST for homology identification across photosynthetic organisms, and MAFFT or CLUSTAL for multiple sequence alignments to identify conserved residues likely critical for function. Employ specialized transmembrane prediction algorithms such as TMHMM and MEMSAT to accurately identify membrane-spanning regions characteristic of photosystem proteins.

For structural prediction, implement AlphaFold2 or RoseTTAFold which have demonstrated remarkable accuracy for protein structure prediction, including membrane proteins. Validate these predictions against experimentally determined structures of homologous photosystem components when available. Use molecular dynamics simulations in explicit membrane environments to assess stability of the predicted structure and identify potential lipid-protein interactions that may be critical for function.

For functional prediction, employ tools like ConSurf to map evolutionary conservation onto structural models, revealing surface patches likely involved in protein-protein interactions within the photosystem complex. Computational docking studies can predict interactions with other PSII components, generating testable hypotheses about psbN's role in photosystem assembly or function.

Compare predicted structures of wild-type psbN with naturally occurring variants identified in different Coelogyne species to understand how sequence divergence may affect structural features and potentially functional adaptations in different orchid species.

How can recombinant psbN protein be utilized in photosynthesis research and biotechnological applications?

Recombinant psbN protein from Coelogyne cristata offers multiple research and biotechnological applications. In fundamental photosynthesis research, purified psbN can be used in reconstitution experiments to elucidate its precise role in photosystem II assembly and stability. By incorporating recombinant psbN into artificial membrane systems with other PSII components, researchers can study the kinetics of complex formation and identify critical interaction partners.

For biotechnological applications, psbN knowledge can contribute to engineering more efficient photosynthetic organisms. Understanding the structure-function relationship of psbN may reveal opportunities to enhance photosystem stability under stress conditions. Recombinant psbN can also serve as an antigen for generating specific antibodies, enabling detailed studies of photosystem dynamics in various orchid species and other plants.

The protein could potentially be utilized in biosensor development, particularly for detecting herbicides or environmental contaminants that target photosystem II. Additionally, comparative studies of psbN across Coelogyne species with differing environmental adaptations may reveal sequence variations that correlate with photosynthetic efficiency under various conditions, informing crop improvement strategies beyond orchids.

Future applications may include incorporation of optimized psbN variants into synthetic biology platforms designed to capture light energy for biotechnological processes, extending beyond natural photosynthesis.

What emerging technologies are transforming research on chloroplast-encoded proteins like psbN?

Several emerging technologies are revolutionizing research on chloroplast-encoded proteins like psbN. CRISPR/Cas9-based chloroplast genome editing, though technically challenging, is advancing rapidly and allows precise modification of chloroplast genes in their native context, enabling functional studies through targeted mutagenesis . This approach circumvents many challenges associated with recombinant expression.

Cryo-electron microscopy has transformed structural biology of membrane protein complexes, allowing visualization of photosystem components in near-native states at near-atomic resolution without crystallization. This technique could reveal psbN's position and interactions within the complete PSII complex.

Nanopore sequencing technologies enable direct sequencing of native DNA without amplification, allowing rapid sequencing of entire chloroplast genomes from limited material with long reads that simplify assembly. This facilitates comparative genomics across multiple Coelogyne species and ecotypes to correlate sequence variations with functional adaptations.

Advanced mass spectrometry techniques, particularly hydrogen-deuterium exchange mass spectrometry (HDX-MS), can map protein-protein interaction surfaces and conformational changes in membrane proteins like psbN under various conditions, providing dynamic structural information complementary to static structural models.

Single-molecule fluorescence approaches allow tracking of individual protein molecules during complex assembly, potentially revealing the temporal sequence of psbN incorporation into developing photosystems. These technologies collectively provide unprecedented insights into chloroplast protein function that were inaccessible with conventional approaches.