Recombinant Helianthus annuus NAD (P)H-quinone oxidoreductase subunit 4L, chloroplastic (ndhE)

What is Recombinant Helianthus annuus NAD(P)H-quinone oxidoreductase subunit 4L?

Recombinant Helianthus annuus NAD(P)H-quinone oxidoreductase subunit 4L, chloroplastic (ndhE) is a full-length protein (1-101 amino acids) derived from the common sunflower. It is expressed in E. coli with an N-terminal His tag and supplied as a lyophilized powder. This protein is a subunit of the NAD(P)H-quinone oxidoreductase complex that participates in electron transfer reactions within chloroplasts, playing an essential role in photosynthetic processes .

What are the key specifications of this product?

The product is supplied as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE. The full amino acid sequence consists of 101 amino acids (MMLEHVLVLSAYLFSVGLYGLITSRNMVRALMCLELILNAVNLNFVTFSDFFDSRQLKGA IFSIFVIAIAAAEAAIGLAIVSSIYRNRKSTRINQSNLLNK). It is fused to an N-terminal His tag for purification and detection purposes. The protein is expressed in E. coli and undergoes quality control testing to ensure consistency and reliability .

What are the synonyms or alternative names for this protein?

The protein is also known by several synonyms including: ndhE; NAD(P)H-quinone oxidoreductase subunit 4L, chloroplastic; NAD(P)H dehydrogenase subunit 4L; and NADH-plastoquinone oxidoreductase subunit 4L. In database records, it may be identified by its UniProt ID: Q1KXQ5 .

What is the functional role of NAD(P)H-quinone oxidoreductase in plants?

NAD(P)H-quinone oxidoreductases catalyze the transfer of electrons from NAD(P)H to quinones, which is essential for various cellular processes in plants. In chloroplasts, the ndhE subunit contributes to the NAD(P)H dehydrogenase complex involved in cyclic electron flow around photosystem I, photoprotection, and chlororespiration. This enzyme helps plants adapt to environmental stresses and participates in detoxification pathways. The electron transfer capability is critical for reducing harmful quinone compounds that may form during photosynthesis and other metabolic processes .

What is the relationship between chloroplast transcript processing and ndhE function?

The ndhE gene is encoded in the chloroplast genome and undergoes regulated transcription and RNA processing. Chloroplast transcription requires numerous quality control steps to generate the complex mixture of accumulating RNAs. The expression and processing of ndhE transcripts are part of this sophisticated interplay, with specific transcription start sites (TSS) and RNA termini. Research has identified numerous transcript termini in chloroplasts, including those for genes like ndhE, revealing clustering patterns of both 5′ and 3′ ends that suggest coordination in RNA processing machinery .

What is the recommended storage protocol for this product?

Upon receipt, the protein should be stored at -20°C/-80°C, with aliquoting necessary for multiple uses. For working solutions, store aliquots at 4°C for up to one week. The product is supplied in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0. Repeated freezing and thawing cycles should be avoided to maintain protein integrity and activity. For long-term storage, adding glycerol to a final concentration of 50% is recommended before storing at -20°C/-80°C .

How should the lyophilized protein be reconstituted?

It is recommended to briefly centrifuge the vial prior to opening to bring the contents to the bottom. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. To enhance stability for long-term storage, add glycerol to a final concentration of 5-50% (the default recommendation is 50% glycerol). After reconstitution, the solution should be aliquoted to avoid repeated freeze-thaw cycles .

What precautions should be taken when handling this protein?

Standard laboratory safety procedures should be followed when handling this protein. While handling, use appropriate personal protective equipment including gloves, lab coat, and eye protection. The product is for research use only and not for human consumption or diagnostic applications. When reconstituting and aliquoting the protein, maintain sterile conditions to prevent contamination. Working in a laminar flow hood is recommended when preparing solutions for sensitive applications .

What experimental applications is this protein suitable for?

This recombinant protein is suitable for a variety of biochemical and molecular biology applications, including but not limited to:

• Enzyme activity assays to study NAD(P)H-quinone oxidoreductase function

• Protein-protein interaction studies involving chloroplast electron transport chains

• Antibody production against ndhE for immunoassays

• Structural studies of NAD(P)H-quinone oxidoreductase complexes

• Functional characterization of electron transfer mechanisms

• Comparative analyses with homologous proteins from other plant species

Can this protein be used for studying quinone reduction mechanisms?

Yes, this protein is ideal for studying quinone reduction mechanisms. NAD(P)H-quinone oxidoreductases catalyze the transfer of electrons from NAD(P)H to various quinone substrates. Experimental evidence indicates that related enzymes have preferences for specific quinone substrates, with some showing stronger reduction activity towards large substrates like 9,10-phenanthrenequinone. This recombinant ndhE can be used to investigate substrate specificity, electron transfer kinetics, and structural determinants of quinone binding in sunflower NAD(P)H-quinone oxidoreductase .

How can this protein be detected in experimental systems?

The protein can be readily detected using the N-terminal His tag through various methods:

• Western blotting using anti-His antibodies

• Immunoprecipitation with anti-His affinity resins

• Nickel or cobalt affinity chromatography for purification

• Enzyme-linked immunosorbent assay (ELISA) using anti-His or anti-ndhE antibodies
  Additionally, enzymatic activity assays measuring NAD(P)H oxidation or quinone reduction can be used to functionally detect the protein in experimental systems .

What buffer conditions are optimal for assaying this protein's activity?

Based on studies with similar NAD(P)H-quinone oxidoreductases, optimal buffer conditions typically include:

• pH range of 7.0-8.0 (commonly Tris-HCl or phosphate buffers)

• Presence of divalent cations (typically 1-5 mM MgCl₂ or MnCl₂)

• Reducing environment (often including low concentrations of DTT or β-mercaptoethanol)

• NAD(P)H concentrations between 50-500 μM

• Appropriate quinone substrates (such as benzoquinone derivatives or physiologically relevant plastoquinones)

• Temperature range of 25-37°C, though plant enzymes may have broader temperature optima

What are common issues when working with recombinant NAD(P)H-quinone oxidoreductases?

Common challenges when working with these enzymes include:

• Loss of activity due to oxidation of critical cysteine residues - consider working under nitrogen or argon atmosphere

• Substrate solubility issues - quinones often have limited water solubility and may require organic solvents like DMSO or ethanol (keep final concentrations below 1%)

• NADPH stability concerns - prepare fresh solutions before experiments

• Protein aggregation - optimize buffer conditions and consider adding stabilizing agents

• Interference from endogenous reductases in complex samples - use appropriate controls and purification steps

Is this protein compatible with common assay detection methods?

Yes, this recombinant ndhE protein is compatible with various detection methods:

• Spectrophotometric assays - monitoring NAD(P)H oxidation at 340 nm

• Fluorometric assays - measuring changes in NAD(P)H fluorescence

• Colorimetric assays - using electron acceptors like dichlorophenolindophenol (DCPIP)

• Electrochemical detection - measuring electron transfer in electrode-based systems

• Radiolabeled substrate approaches - for highly sensitive measurements

When selecting detection methods, consider potential interference from buffer components, especially reducing agents that might interfere with colorimetric assays .

How does this protein contribute to understanding chloroplast function?

This protein provides valuable insights into chloroplast bioenergetics and electron transport pathways. The ndhE subunit is part of the NAD(P)H dehydrogenase complex in chloroplasts that contributes to cyclic electron flow around photosystem I. Studying this recombinant protein helps researchers understand how plants optimize photosynthetic efficiency, especially under stress conditions. Additionally, the protein's role in chloroplast RNA processing and quality control mechanisms illuminates the sophisticated regulatory networks governing chloroplast gene expression and function .

Can this protein be used to study evolutionary aspects of photosynthetic organisms?

Yes, this recombinant Helianthus annuus ndhE protein is valuable for evolutionary studies. By comparing its structure, function, and sequence with homologous proteins from diverse photosynthetic organisms, researchers can trace the evolutionary history of NAD(P)H-quinone oxidoreductases and chloroplast electron transport chains. Such comparative analyses reveal conservation patterns in active sites, substrate specificities, and regulatory mechanisms across plant lineages. This protein can serve as a representative of higher plant NAD(P)H dehydrogenase complexes in evolutionary studies of photosynthetic apparatus diversification .

What insights about transcript processing can be gained using this protein?

While the protein itself is not directly involved in transcript processing, research into chloroplast transcriptomes has revealed important connections between transcript processing and protein function. Studies using Terminome-seq have catalogued numerous transcript termini in chloroplasts, showing clustering patterns of both 5′ and 3′ ends. Understanding how ndhE transcripts are processed provides insights into chloroplast gene expression regulation. The recombinant protein can be used alongside transcript analysis to correlate RNA processing events with protein expression levels and functional outcomes in photosynthetic organisms .