Purification of Rubisco from Leaves.

Rubisco fixes CO2 through the carboxylation of ribulose 1,5-bisphosphate (RuBP) during photosynthesis, enabling the synthesis of organic compounds. The natural diversity of Rubisco properties represents an opportunity to improve its performance and there is considerable research effort focusing on better understanding the properties and regulation of the enzyme. This chapter describes a method for large-scale purification of Rubisco from leaves. After the extraction of Rubisco from plant leaves, the enzyme is separated from other proteins by fractional precipitation with polyethylene glycol followed by ion-exchange chromatography. This method enables the isolation of Rubisco in large quantities for a wide range of biochemical applications.

In vitro activity of reconstituted rubisco enzyme from Gloeobacter violaceus.

RuBisCO (Ribulose 1,5 bisphosphate carboxylase/oxygenase) by virtue of its dual specificity towards oxygen and carbon dioxide is an important rate-limiting step in photosynthesis and is believed to be the key factor for limited productivity of higher plants and algae. The photoautotrophic growth rate of cyanobacteria is a culmination of several factors including, rates of photosynthetic reactions, stress combating mechanisms and basic biomass generation metabolism in combination with optimal nutrient availability, irradiance, gaseous environment, etc. In case of cyanobacteria, the effect of RuBisCO in affecting the multiplication rate has been observed to show varied response. The current paper presents the RuBisCO activity of an early diverging cyanobacterium, Gloeobacter violaceus PCC 7421 and also compares the growth rates and RuBisCO activity of various cyanobacteria. A spectrophotometric estimation in a coupled enzyme assay system of the heterologous expressed G. violaceus PCC 7421 RuBisCO in E. coli, upon purification, revealed a carboxylation activity of LSu to be 5 nMol of phosphoglycerate min-1 mg-1 of protein, which is in coherence with the organism's slow growth rate. Further, the in vitro complementation of RbcL with RbcS in presence of RbcX of G. violaceus facilitated partial reconstitution of the protein and was hence found to cause a four-fold enhancement in its specific activity. The unique characteristics of the primitive cyanobacteria, such as, absence of thylakoids, lack of several photosystem constituting genes, slow carboxylation rate, pose limitation for its rapid multiplication. The RuBisCO carboxylation rate is observed as not the sole but an important parameter for obtaining optimal cell multiplication rates in photo-autotrophically multiplying cyanobacteria.

Improved recombinant expression and purification of functional plant Rubisco.

Improving the performance of the key photosynthetic enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) by protein engineering is a critical strategy for increasing crop yields. The extensive chaperone requirement of plant Rubisco for folding and assembly has long been an impediment to this goal. Production of plant Rubisco in Escherichia coli requires the coexpression of the chloroplast chaperonin and four assembly factors. Here, we demonstrate that simultaneous expression of Rubisco and chaperones from a T7 promotor produces high levels of functional enzyme. Expressing the small subunit of Rubisco with a C-terminal hexahistidine-tag further improved assembly, resulting in a ~ 12-fold higher yield than the previously published procedure. The expression system described here provides a platform for the efficient production and engineering of plant Rubisco.

Extraction of RuBisCO to Determine Catalytic Constants.

RuBisCO enables net carbon fixation through the carboxylation of RuBP during photosynthesis. Its complex biochemistry and catalytic diversity found among different plants make characterization of RuBisCO properties useful for investigations aimed at improving photosynthetic performance. This chapter reports methods for rapid extraction of soluble proteins to examine RuBisCO catalytic properties, and for large-scale purification of RuBisCO from leaves to measure the specificity of the enzyme toward its gaseous substrates.

Isolation of Arabidopsis Leaf Peroxisomes and the Peroxisomal Membrane.

To date, less than 150 proteins have been located to plant peroxisomes, indicating that unbiased large-scale approaches such as experimental proteome research are required to uncover the remaining yet unknown metabolic functions of this organelle as well as its regulatory mechanisms and membrane proteins. For experimental proteome research, Arabidopsis thaliana is the model plant of choice and an isolation methodology that obtains peroxisomes of sufficient yield and high purity is vital for research on this organelle. However, organelle enrichment is more difficult from Arabidopsis when compared to other plant species and especially challenging for peroxisomes. Leaf peroxisomes from Arabidopsis are very fragile in aqueous solution and show pronounced physical interactions with chloroplasts and mitochondria in vivo that persist in vitro and decrease peroxisome purity. Here, we provide a detailed protocol for the isolation of Arabidopsis leaf peroxisomes using two different types of density gradients (Percoll and sucrose) sequentially that yields approximately 120 µg of peroxisome proteins from 60 g of fresh leaf material. A method is also provided to assess the relative purity of the isolated peroxisomes by immunoblotting to allow selection of the purest peroxisome isolates. To enable the analysis of peroxisomal membrane proteins, an enrichment strategy using sodium carbonate treatment of isolated peroxisome membranes has been adapted to suit isolated leaf peroxisomes and is described here.

Rubimetide, humanin, and MMK1 exert anxiolytic-like activities via the formyl peptide receptor 2 in mice followed by the successive activation of DP1, A2A, and GABAA receptors.

Rubimetide (Met-Arg-Trp), which had been isolated as an antihypertensive peptide from an enzymatic digest of spinach ribulose-bisphosphate carboxylase/oxygenase (Rubisco), showed anxiolytic-like activity prostaglandin (PG) D2-dependent manner in the elevated plus-maze test after administration at a dose of 0.1mg/kg (ip.) or 1mg/kg (p.o.) in male mice of ddY strain. In this study, we found that rubimetide has weak affinities for the FPR1 and FPR2, subtypes of formyl peptide receptor (FPR). The anxiolytic-like activity of rubimetide (0.1mg/kg, ip.) was blocked by WRW4, an antagonist of FPR2, but not by Boc-FLFLF, an antagonist of FPR1, suggesting that the anxiolytic-like activity was mediated by the FPR2. Humanin, an endogenous agonist peptide of the FPR2, exerted an anxiolytic-like activity after intracerebroventricular (icv) administration, which was also blocked by WRW4. MMK1, a synthetic agonist peptide of the FPR2, also exerted anxiolytic-like activity. Thus, FPR2 proved to mediate anxiolytic-like effect as the first example of central effect exerted by FPR agonists. As well as the anxiolytic-like activity of rubimetide, that of MMK1 was blocked by BW A868C, an antagonist of the DP1-receptor. Furthermore, anxiolytic-like activity of rubimetide was blocked by SCH58251 and bicuculline, antagonists for adenosine A2A and GABAA receptors, respectively. From these results, it is concluded that the anxiolytic-like activities of rubimetide and typical agonist peptides of the FPR2 were mediated successively by the PGD2-DP1 receptor, adenosine-A2A receptor, and GABA-GABAA receptor systems downstream of the FPR2.

Differential proteome and secretome analysis during rice-pathogen interaction.

Substantial evidences implicate that sample preparation and protein extraction in proteomic studies of plant-pathogen interactions are critical to understand cross talk between host and pathogen. Therefore, interest is growing in applying proteomics techniques to investigate simultaneously secreted proteins from rice and pathogen. We have found, however, that most proteins of interest are low abundant so that proper prefractionation or extraction of secreted proteins from extracellular space (ECS) in the rice leaf is required to excavate relevant protein. This chapter describes the preparation of sample and extraction procedure to enrich the proteins interested before separation by 2-DE or LC-MS/MS. This method significantly increases the sensitivity of proteomic comparisons.

Application of an improved proteomics method for abundant protein cleanup: molecular and genomic mechanisms study in plant defense.

High abundance proteins like ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. To address this challenge, we developed and evaluated Polyethyleneimine Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shot-gun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. Insect resistance was induced by over-expressing either jacalin-like or cupin-like genes in rice. The results further highlighted that PARC can serve as an effective strategy for proteomics analysis and gene discovery.

Depletion of abundant plant RuBisCO protein using the protamine sulfate precipitation method.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant plant leaf protein, hampering deep analysis of the leaf proteome. Here, we describe a novel protamine sulfate precipitation (PSP) method for the depletion of RuBisCO. For this purpose, soybean leaf total proteins were extracted using Tris-Mg/NP-40 extraction buffer. Obtained clear supernatant was subjected to the PSP method, followed by 13% SDS-PAGE analysis of total, PS-supernatant and -precipitation derived protein samples. In a dose-dependent experiment, 0.1% w/v PS was found to be sufficient for precipitating RuBisCO large and small subunits (LSU and SSU). Western blot analysis confirmed no detection of RuBisCO LSU in the PS-supernatant proteins. Application of this method to Arabidopsis, rice, and maize leaf proteins revealed results similar to soybean. Furthermore, 2DE analyses of PS-treated soybean leaf displayed enriched protein profile for the protein sample derived from the PS-supernatant than total proteins. Some enriched 2D spots were subjected to MALDI-TOF-TOF analysis and were successfully assigned for their protein identity. Hence, the PSP method is: (i) simple, fast, economical, and reproducible for RuBisCO precipitation from the plant leaf sample; (ii) applicable to both dicot and monocot plants; and (iii) suitable for downstream proteomics analysis.

Gelation in protein extracts from cold acclimated and non-acclimated winter rye (Secale cereale L. cv Musketeer).

A protein gel is a three-dimensional network consisting of molecular interactions between biopolymers that entrap a significant volume of a continuous liquid phase (water). Molecular interactions in gels occur at junction zones within and between protein molecules through electrostatic forces, hydrogen bonding, hydrophobic associations (van der Waals attractions) and covalent bonding. Gels have the physicochemical properties of both solids and liquids, and are extremely important in the production and stability of a variety of foods, bioproducts and pharmaceuticals. In this study, gelation was induced in phenol extracted protein fractions from non-acclimated (NA) and cold-acclimated (CA) winter rye (Secale cereale L. cv Musketeer) leaf tissue after repeated freeze-thaw treatments. Gel formation only occurred at high pH (pH 12.0) and a minimum of 3-4 freeze-thaw cycles were required. The gel was thermally stable and only a specific combination of chemical treatments could disrupt the gel network. SDS-PAGE analysis identified ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) as the major protein component in the gel, although Rubisco itself did not appear to be a factor in gelation. Raman spectroscopy suggested changes in protein secondary structure during freeze-thaw cycles. Overall, the NA and CA gels were similar in composition and structure, with the exception that the CA gel appeared to be amyloidic in nature based on thioflavin T (ThT) fluorescence. Protein gelation, particularly in the apoplast, may confer protection against freeze-induced dehydration and potentially have a commercial application to improve frozen food quality.

Determination of ¹5N-incorporation into plant proteins and their absolute quantitation: a new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions.

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

Evidence of form II RubisCO (cbbM) in a perennially ice-covered Antarctic lake.

The permanently ice-covered lakes of the McMurdo Dry Valleys, Antarctica, harbor microbially dominated food webs. These organisms are adapted to a variety of unusual environmental extremes, including low temperature, low light, and permanently stratified water columns with strong chemo- and oxy-clines. Owing to the low light levels during summer caused by thick ice cover as well as 6 months of darkness during the polar winter, chemolithoautotrophic microorganisms could play a key role in the production of new carbon for the lake ecosystems. We used clone library sequencing and real-time quantitative PCR of the gene encoding form II Ribulose 1, 5-bisphosphate carboxylase/oxygenase to determine spatial and seasonal changes in the chemolithoautotrophic community in Lake Bonney, a 40-m-deep lake covered by c. 4 m of permanent ice. Our results revealed that chemolithoautotrophs harboring the cbbM gene are restricted to layers just above the chemo- and oxi-cline (≤ 15 m) in the west lobe of Lake Bonney (WLB). Our data reveal that the WLB is inhabited by a unique chemolithoautotrophic community that resides in the suboxic layers of the lake where there are ample sources of alternative electron sources such as ammonium, reduced iron and reduced biogenic sulfur species.

Efficiency of ligation-mediated PCR and TAIL-PCR methods for isolation of RbcS promoter sequences from green microalgae Ankistrodesmus convolutus.

Isolation of promoter sequences from known gene sequences is a tedious task in genome-related research. An efficient method of obtaining the promoter sequences is necessary in order to successfully use targeted promoters for genetic manipulations. Here, efficiency and usefulness of two PCR-based methods, namely: ligation-mediated PCR and thermal asymmetric interlaced (TAIL) PCR, for isolation of promoter sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) gene from green microalgae Ankistrodesmus convolutus (A. convolutus) were evaluated. The results showed that the amplification efficiency of TAIL-PCR was higher than that of the ligation-mediated PCR method, i.e. the amplified promoter fragments of 1.2 and 0.8 kb in length or promoter sequences of 813 and 606 bp (after eliminating the unreadable sequences). The use of TAIL-PCR described here presents a low cost and efficient strategy for the isolation of promoter sequences of known genes, especially in GC-rich regions, and species with little or no available genome information such as A. convolutus.

Automated flow-based anion-exchange method for high-throughput isolation and real-time monitoring of RuBisCO in plant extracts.

In this work, a miniaturized, completely enclosed multisyringe-flow system is proposed for high-throughput purification of RuBisCO from Triticum aestivum extracts. The automated method capitalizes on the uptake of the target protein at 4°C onto Q-Sepharose Fast Flow strong anion-exchanger packed in a cylindrical microcolumn (105 × 4 mm) followed by a stepwise ionic-strength gradient elution (0-0.8 mol/L NaCl) to eliminate concomitant extract components and retrieve highly purified RuBisCO. The manifold is furnished downstream with a flow-through diode-array UV/vis spectrophotometer for real-time monitoring of the column effluent at the protein-specific wavelength of 280 nm to detect the elution of RuBisCO. Quantitation of RuBisCO and total soluble proteins in the eluate fractions were undertaken using polyacrylamide gel electrophoresis (PAGE) and the spectrophotometric Bradford assay, respectively. A comprehensive investigation of the effect of distinct concentration gradients on the isolation of RuBisCO and experimental conditions (namely, type of resin, column dimensions and mobile-phase flow rate) upon column capacity and analyte breakthrough was effected. The assembled set-up was aimed to critically ascertain the efficiency of preliminary batchwise pre-treatments of crude plant extracts (viz., polyethylenglycol (PEG) precipitation, ammonium sulphate precipitation and sucrose gradient centrifugation) in terms of RuBisCO purification and absolute recovery prior to automated anion-exchange column separation. Under the optimum physical and chemical conditions, the flow-through column system is able to admit crude plant extracts and gives rise to RuBisCO purification yields better than 75%, which might be increased up to 96 ± 9% with a prior PEG fractionation followed by sucrose gradient step.

Isolation of ribulose-1,5-bisphosphate carboxylase/oxygenase from leaves.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a multifunctional enzyme that catalyzes the fixation of CO2 and O2 in photosynthesis and photorespiration, respectively. As the rate-limiting step in photosynthesis, improving the catalytic properties of Rubisco has long been viewed as a viable strategy for increasing plant productivity. Advances in biotechnology have made this goal more attainable by making it possible to modify Rubisco in planta. To properly evaluate the properties of Rubisco, it is necessary to isolate the enzyme in pure form. This chapter describes procedures for rapid and efficient purification of Rubisco from leaves of several species.

The construction, fouling and enzymatic cleaning of a textile dye surface.

The enzymatic cleaning of a rubisco protein stain bound onto Surface Plasmon Resonance (SPR) biosensor chips having a dye-bound upper layer is investigated. This novel method allowed, for the first time, a detailed kinetic study of rubisco cleanability (defined as fraction of adsorbed protein removed from a surface) from dyed surfaces (mimicking fabrics) at different enzyme concentrations. Analysis of kinetic data using an established mathematical model able to decouple enzyme transfer and reaction processes [Onaizi, He, Middelberg, Chem. Eng. Sci. 64 (2008) 3868] revealed a striking effect of dyeing on enzymatic cleaning performance. Specifically, the absolute rate constants for enzyme transfer to and from a dye-bound rubisco stain were significantly higher than reported previously for un-dyed surfaces. These increased transfer rates resulted in higher surface cleanability. Higher enzyme mobility (i.e., higher enzyme adsorption and desorption rates) at the liquid-dye interface was observed, consistent with previous suggestions that enzyme surface mobility is likely correlated with overall enzyme cleaning performance. Our results show that reaction engineering models of enzymatic action at surfaces may provide insight able to guide the design of better stain-resistant surfaces, and may also guide efforts to improve cleaning formulations.

A rapid method for depletion of Rubisco from soybean (Glycine max) leaf for proteomic analysis of lower abundance proteins.

2-DE analysis of complex plant proteomes has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the low abundance proteins within leaf tissue is difficult when it is comprised of 30-50% of the CO(2) fixation enzyme Rubisco. Resolution can be improved through depletion of Rubisco using fractionation techniques based upon different physiological or biochemical principles. We have developed a fast and simple fractionation technique using 10 mM Ca(2+) and 10 mM phytate to precipitate Rubisco from soybean leaf soluble protein extract. This method is not only rapid, but also inexpensive, and capable of removing 85% of the extremely abundant Rubisco enzyme from soybean leaf soluble protein extract. This method allowed for roughly 230 previously inconspicuous protein spots in soybean leaf to be more easily detectable (3-fold increase in vol%) using fluorescent detection and allowed 28 phosphorylated proteins previously undetected, to be isolated and identified by MALDI-TOF-MS.

Rapid screening of surfactant and biosurfactant surface cleaning performance.

Surface Plasmon Resonance (SPR) and rubisco protein stain were used as tools to screen the effectiveness of detergent formulations in cleaning a protein stain from solid surfaces. Surfactant and biosurfactant-based formulations, with and without added protease, were screened for cleaning performance. Enzyme-free detergent formulations at 1500 ppm total surfactant were insufficient to cause complete surface cleaning, despite the high concentration of surfactant. The cleaning performance of a "home-made" formulation containing 2 ppm subtilisin A (SA) and 2 ppm sodium dodecyl benzyl sulphonate (SDOBS) was as efficient as the best amongst the three enzyme-free 1500 ppm formulations. The cleaning performance of 2 ppm SA in the absence of SDOBS was less effective than the combined formulation, even though 2 ppm SDOBS alone did not cause any protein removal. The observed synergistic performance was attributed to the cooperative mechanisms (chemical and physical attack) by which these two agents act on a rubisco stain. Replacing SDOBS in the enzyme-surfactant formulation with the same amount of surfactin biosurfactant (2 ppm) gave the best rubisco removal of all formulations examined in this study, irrespective of the surface chemistry underlying the protein film. It was found that 75% and 80% of immobilised rubisco stain could be removed from hydrophobic and hydrophilic surfaces, respectively, by the biosurfactant-SA formulation (compared with 60% and 65%, respectively, using the SDOBS-SA formulation). Our results suggest that it may be possible to generate fully renewable biochemical-based cleaning formulations that have superior cleaning performance to existing technologies. In developing optimised formulations, there is a pressing need for chip-based tools similar to that developed in this research.

Affinity chromatography reveals RuBisCO as an ecdysteroid-binding protein.

The aim of this work was to isolate plant ecdysteroid-binding proteins using affinity chromatography. Ecdysteroids as insect hormones have been investigated thoroughly but their function and the mechanism of action in plants and other organisms is still unknown although ecdysteroids occur in some plants in a relatively large amount. Therefore, 20-hydroxyecdysone was immobilized on a polymeric carrier as a ligand for affinity chromatography in order to isolate plant ecdysteroid-binding proteins from the cytosolic extract of New Zealand spinach (Tetragonia tetragonoides). Non-specifically bound proteins were eluted with a rising gradient of concentration of sodium chloride, and 3% (v/v) acetic acid was used for the elution of the specifically bound proteins. Using this method, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was isolated. The influence of ecdysteroids on RuBisCO was further studied. Our results show that ecdysteroids are able to increase the yield of RuBisCO-mediated reaction in which CO(2) is fixed into organic matter by more than 10%.