A method for extracting protein from pectin rich plant material Field of the Invention The present invention relates to methods for extracting protein from plant material which is characterized by a high content of interfering compounds, specifically abundant polysaccharides like pectin. The inventive method applies a pectolytic treatment in preparation of the plant material sample prior to protein extraction. This optimized method results in high quality protein extracts compatible with further sensitive proteomic analyses specifically methods like two dimensional gel electrophoresis or mass spectrometry. The present invention further relates to the protein extracts obtained by the inventive method and the use of the inventive method in the preparation of protein samples. Also a protein extraction kit is disclosed for conducting the inventive method, as well as the use of the protein extraction kit in protein analysis and proteomics.

Background of the Invention

Proteomics is becoming a necessity in plant biology for deciphering the function and the role of genes in the ongoing plant genome sequencing projects. The applications of proteomics can be enormous in boosting up agricultural production. Different proteins of plant tissues with their own properties like molecular mass, solubility, pi, structure, etc. confer different roles that may provide us with tremendous achievements in food, pharmaceutical, cosmoceutical and textile industries. Sample preparation is a critical step in a two dimensional gel electrophoresis proteome approach and is absolutely essential for good results. Curculigo latifolia fruit is notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds such as pectin. A considerable portion of the fruit is occupied by seeds which contain large amounts of polyphenols which can also interfere with efficient protein extraction. This invention focused on finding the optimal protocol for protein extraction from the fruit of Curculigo latifolia which was quite challenging, due to the high concentration of pectin. Pectolytic enzyme pre-treatment successfully resolved this problem. The present invention will also play an important role in the analysis of the sequence data that is being produced by worldwide genome projects (Alexey, 2006). Recently plants have been considered as a source to provide higher proportion of proteins. Proteome analysis is becoming a powerful tool in the functional characterization of plant genomes (Jorrin, Maldonado et al. 2007). Identifying new proteins may allow us to construct new recombinant proteins which is an ongoing field of study in plant science nowadays. This, in turn could further lead to a significant revolution in above mentioned industries. If the identified proteins of plant tissues undergo protein engineering, the structural and catalytical properties of proteins could be improved or adopted according to specific needs.

Furthermore the results obtained from this project can be used as a foundation for further protein identification in other plants. In this study, extracting proteins from Curculigo latifolia fruit, is applied by using 7 different protocols, (eg: TCA-acetone based protocol, 3 different phenol based protocols, a combination of TCA-acetone and phenol based protocols, chloroform based protocol and hot SDS buffer protocol). Next, the determination of the most efficient protocol was judged by the optimal 2-DE pattern results. 2-DE is considered to be one of the most efficient and powerful methods for profiling of large sets of complex protein mixtures and comparative study between pairs of samples (Song, Braun et al. 2006). In principle, the three-dimensional structure of proteins makes them physically and chemically far more complicated than nucleic acids. In addition, the number of proteins is greater than the number of genes due to different RNA splicing and post-translational modifications (Kwon, Choi et al. 2006). At present, there is no amplification method for proteins and consequently the study of low copy number species is still a challenge. The most critical step in any protein profiling is sample extraction and preparation especially. This is particularly true for plant tissues containing high levels of interfering compounds, which are more problematic in the extraction than in other organisms. Preparation of high-quality protein samples from plant tissues for descriptive analysis of protein patterns represents a great challenge.

In addition to having relatively low protein concentrations, plant tissues are often rich in proteases and materials that severely interfere with subsequent protein separation and analysis, including cell wall, vacuoles and storage polysaccharides, lipids, pigments, phenolic compounds and a broad array of secondary metabolites. These contaminants are the reasons for horizontal and vertical streaking, smearing, and a reduction in the number of distinctly resolved proteins (Maldonado, et al. 2008). Plant proteomic studies are often involved in total protein populations. Unfortunately there is no single method of sample extraction that can be universally applied to all kinds of plant tissues. Over the years protein extraction protocols for proteomic analysis have proliferated, with specialization to plant organ (leaf, root, cell suspension), however TCA- acetone precipitation and phenol extraction has proven by Rossignol, et al in 2006 to be most generically useful. A chloroform based protocol was reported by Xie, et al. that gave reproducible result from protein extraction of perennial Bupleurum root in 2007. Ramu, S. et al. extracted protein from Tomato and suggested that the phenol protocol is highly effective with more recalcitrant tissues in 2004 and a combination of TCA-acetone and phenol methods provideed an enhanced 2-DE based proteomic analyses of most plant tissues.

Wang, et al. showed that TCA-acetone/phenol is an applicable protocol for extracting high- quality proteins from recalcitrant fruit and leaf tissues in 2008. Vincent, D. et al. showed that phenol extraction gave satisfactory results in recalcitrant plant tissues, such as mature grape berry clusters in 2006. Song J. et al. showed that a satisfactory result for banana proteins extracted by phenol extraction protocol in 2006. In contrast thereto Carpentier et al. found that classical TCA/acetone precipitation on banana and apple fruits was just as useful as phenol extraction in 2005.

Proteomics has proven to be a very valuable tool for assessing the substantial equivalence and the safety of food and feed derived from transgenic plants. Thus in view of the state of art cited above it is a major interest of the present invention to provide a novel protein extraction procedure that allows the preparation of high quality protein, characterized by a low content of interfering substances, specifically compounds such as pectin. Furthermore, the present invention intends to provide means for preparing a high standard protein sample for protein analysis processes which are dependent on high quality protein extracts, in particular two dimensional gel electrophoresis in proteome techniques. The above problem is solved in a first aspect by a method for extracting protein from plants, the method comprising the steps of

(1) providing a sample of plant material,

(2) pre-treating the sample with a pectolytic enzyme,

(3) performing a protein extraction with the pre-treated sample of step (2),

(4) recovering the protein from the extract obtained by step (3).

Preferred is that the plant material originates from root, stem or leaves of the plant. Most preferred is that the plant material is fruit. In the context of the present invention one preferred embodiment requires that the plant material from which an extraction of protein is planned, is provided in the form of a powder. In another embodiment the plant material is provided from the fruit of Curculigo latifolia.

In one embodiment, it is preferred that step (1) of the above method comprises that the plant material is cleaned, then dried and quick frozen in liquid nitrogen. The frozen material is then grinded into a fine powder. The powder product then constitutes the sample of plant material.

The pectolytic treatment surprisingly did not affect the protein composition of the final extract. This is important, specifically if the present inventive method is to be used in the context of proteome analysis. Therefore, it is preferred that the pectolytic treatment in context of the present invention does not interfere with the extracted proteins.

Yet another embodiment of the inventive method relates to the pectinase enzyme as the pectolytic enzyme. In this context the term "pectinases" refers to any kind of enzymes, such as pectolyase, pectozyme and polygalacturonase, commonly referred to in brewing as pectic enzymes, that are capable of degrading pectins. Especially suitable pectinases are mixtures of polygalacturonases and pectin methylesterases. On the other hand, all major wood species contain some pectins and some non-wood species are very rich in pectins, which are chemically known as polygalacturonic acids or galacturonans. Pectinases break down pectin, a polysaccharide substrate that is found in the cell walls of plants. One of the most studied and widely used commercial pectinases is polygalacturonase. They can be extracted from fungi such as Aspergillus niger. Also preferred is that the pectolytic enzyme is a pectolytic enxyme mix comprising at least one of the enzymes selected from the group consisting of pectintranseliminase, polygalacturonase, pectinesterase hemicellulases and cellulases.

In a preferred embodiment of the method according to the invention the pectolytic treatment is performed at a temperature of between 30°C to 70°C, preferably at 50°C. With regard to incubation times it is further a preferred method of the invention to perform the pectolytic treatment for at least 1 to 24 hours, preferably for 6 to 18 hours, and most preferred for about 6 hours, which usually is done by overnight incubation.

Preferred in the context of the present invention is further to use 500mg of plant material (e.g. fruit powder) as the initial starting material. Furthermore, one embodiment of the present invention relates to the pectolytic treatment wherein 1 to 50μ1 of pectinase is used, preferably wherein 5μ1 of pectinase is used. The inventors surprisingly found that the best conditions for the pectolytic treatment is 5 μΐ, of pectolytic enzyme at 50°C for 6h, at ΙΟΟμΙ of incubation buffer. The incubation buffer preferably comprises 100 mM Tris-Cl pH 8.8, 100 mM EDTA, 100 mM KC1, 2% B-marcaptoethanol, 2mM PMSF. In context of the present invention the reaction takes place in ΙΟΟΟμΙ of incubation buffer for l-500mg of plant material.

In a next embodiment, the present invention relates to a method wherein the extraction in step (2) is performed in an extraction buffer comprising 0.1 M KC1, 0.5 M Tris-HCl at pH 7.5, 0.1 M EDTA at pH 7.5, 2% β-mercaptoethanoland and 2mM PMSF as protease inhibitor.

Yet another extraction method is preferred, wherein the extraction in step (2) is performed by suspending the pre-treated plant material in 4ml of the extraction buffer, shaking the mixture on ice for 1 hour, centrifuging and recovering the supernatant. With respect to the actual protein extraction a method according to the invention is preferred, wherein a chloroform based protocol is used to recover the protein. Chloroform and phenol based protein extraction protocols are known in the art, also for plant material based protein extractions (see background art). However, as it is a preferred embodiment of the invention that the initial plant material is from a recalcitrant plant tissue, in particular wherein the plant material has a high content of interfering compounds, specifically a high concentration of polysaccharides like pectin, an inventive and modified protocol is preferred in the context of the present invention.

Therefore, in a specific embodiment of the present invention steps (3) and (4) of the inventive method are conducted by the following protocol. 500mg of fruit powder is pretreated with pectolytic enzyme (pectinase) and suspended in 4 mL of extraction buffer (0.1 M KCl, 0.5 M Tris-HCl at pH 7.5, 0.1 M EDTA at pH 7.5 2% β-mercaptoethanoland, 2mM PMSF as protease inhibitor). The mixture is then shaken on ice for 1 h and centrifuged at 13 OOOg, 4°C for 20 minutes. The supernatant is then transferred to a new tube and an equal volume of water saturated chloroform is added. The tube is then gently shaken on ice for 30 min, then stored on ice for 5 min before centrifugation at 13 OOOg, 4°C for 20 minutes. After centrifugation, the upper and bottom phases are transferred to new tubes. Then 4 mL of water is added to the interphase together with an equal volume of water saturated chloroform, and the mixture is shaken on ice for 30min. This mixture is allowed to stand on ice for 5 min and centrifuged at 13 OOOg, 4°C for 20 minutes. This treatment is repeated twice. The interphase is washed with ice- cold acetone three times, dried in vacuum (for 15 min) and re-dissolved in rehydration buffer. The lower phase id air-pumped until 500μ£ of solution remained. Five volumes of acetone are added and stored at - 20C overnight. The pellet is recovered by centrifugation at 13 OOOg, 4°C for 20 minutes and washed by ice-cold acetone, dried, re-dissolved.

The upper phase is treated with an equal volume of Tris-HCl pH 8-saturated phenol solution and the mixture is shaken on ice for 30 minutes and finally centrifuged at 13 OOOg, 4°C for 30 min. The upper buffer phase is removed. The phenol at the bottom is re-extracted twice with extraction buffer. Then proteins are precipitated from the final phenol phase by adding five volumes of 100% methanol containing 0.1M ammonium acetate and left overnight at -20C. The pellet is generated through centrifugation at 13 OOOg for 15 min Yet a further embodiment of the present invention is directed at a method wherein the extracted protein is low in interfering compounds such as cell wall, vacuoles and storage polysaccharides, lipids, pigments, phenolic compounds and a broad array of secondary metabolites. The above described problems are further solved in a second aspect of the present invention by a use of the afore described method, in the preparation of a sample for protein analysis, in particular for proteome analysis. In one embodiment of this second aspect the protein analysis involves protein separation, preferably a one or most preferably a two dimensional gel electrophoresis. These methods require in particular pure and high quality protein extracts in order to provide a good resolution of protein bands or spots. Furthermore included is the use of the protein extraction method for the preparation of samples usable in mass spectrometry.

Two dimensional gel electrophoresis begins with a first dimension electrophoresis but then separates the molecules by a second property in a direction of 90 degrees from the first. In the first dimension electrophoresis, proteins (or other molecules) are separated in one dimension, so that all the proteins/molecules will lie along a lane but that the molecules are spread out across a two dimensional gel. Because it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively separated in two dimensional electrophoresis than in one electrophoresis. For example, proteins are first separated on the basis of their Isoelectric pH in the first dimension and then separated according to their molecular weight in the second dimension. This could be protein complex mass in the native state or protein mass.

Yet another aspect of the present invention relates to the protein extract produced with a method according to any one of the aforementioned embodiments. It is further preferred that in this aspect the protein extract according to the invention is low in interfering compounds such as cell wall, vacuoles and storage polysaccharides, lipids, pigments, phenol compounds and a broad array of secondary metabolites; preferably wherein the protein extract is low in pectin.

The problems are also overcome by providing in a further aspect of the invention a protein extraction kit comprising, a pectolytic enzyme and at least two or more reaction buffers. Preferred in this respect is that the protein extraction kit comprises a group of pectinases as the pectolytic enzyme.

Furthermore one embodiment relates to the protein extraction kit, wherein the one or more reaction buffers are selected from the group comprising a pectinase incubation buffer and protein extraction buffer.

A preferred pectinase incubation buffer according to another embodiment comprises 100 mM Tris-Cl pH 8.8, 100 mM EDTA, 100 mM KC1, 2% B-marcaptoethanol and 2mM PMSF.

A preferred extraction buffer according to a next embodiment comprises 0.1 M KG, 0.5 M Tris- HC1 at pH 7.5, 0.1 M EDTA at pH 7.5, 2% β-mercaptoethanoland and 2mM PMSF.

Yet another embodiment of the invention relates to a protein extraction kit, further comprising an instruction manual showing a procedure for carrying out the method according to any one of the aforementioned embodiments of the present invention

In a final aspect the problems of the present invention are solved by a use of the protein extraction kit according to the aforementioned embodiments, in the preparation of a sample for protein analysis, in particular for proteome analysis. Preferred is that the protein analysis involves protein separation, preferably a one or most preferably a two dimensional gel electrophoresis.

While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the invention within the principles and scope of the broadest interpretations and equivalent configurations thereof. Description of the Drawings

Figure 1 : shows a flow diagram of the extraction method procedures of the present invention.

Figure 2: shows a comparison of SDS-PAGE results between 6 different protocols. In the figure the lanes are as follows: 1- Hot SDS protocol; 2- Interphase chloroform; 3- Upper phase chloroform; 4- phenol based protocol (1); 5- phenol based protocol (2); 6- phenol based protocol (3); 7-TCA-acetone/phenol; 8- pectinase; 9- standard marker.

Figure 3: shows the 1-DE and 2-DE results of fruit proteins extracted by the chloroform based protocol. Enclosed as well is a 1-DE result of the pectinase enzyme as a comparison to the 1 -DE result of the protein extracted by the chloroform based protocol

Figure 4-7: shows the two dimensional electrophoresis results using protein extracts provided by other protocols than the herein used chloroform based protocol (shown in figure 3). The pectinase 2-DE gel shows that the pecto lytic treatment does not interfere with the fruit proteins. Figure 4: 2-DE profile of protein extrated by TCA- acetone/phenol; Figure 5: 2-DE profile of protein extrated by two different phenol based protocol; Figure 6: 2-DE profile of protein extrated by HOT-SDS buffer- TC A/acetone; Figure 7: 2-DE profile of pectinase.

Detailed Description of the Invention

Example I: Selection of optimal extraction protocol The general protocol for the present invention is presented in a flow chart in figure 1. The results of seven common extraction protocols were evaluated first in a one dimensional gel (figure 2)2- DE was performed to evaluate the most efficient method/protocol in terms of protein quality and number of spots., (see figures 3 and 4). The modified chloroform based protocol was identified to be the most effective method for extracting proteins from fruit and may provide enhanced proteomic information for all tissues with abundant pectin.

Example II: Pectinase optimization and protein extraction

500 mg of fruit powder were incubated with 1000 μΐ. of incubation buffer containing 100 mM Tris-Cl pH 8.8, 100 mM EDTA, 100 mM KC1, 2% B-marcaptoethanol, 2mM PMSF and different volume of pectolytic enzyme mix (2.5,5,10,20,40) μ Fruits with different volume of pectolytic enzyme mix were incubated at 37°C over night and also 50°C for lh, 3h and 6h to determine the efficient amount of enzyme, incubation time and temperature. 5 μΙ_, of pectolytic enzyme in lOOul of incubation buffer at 50°C for 6h was found to be the most efficient optimization.

Example III: Protein extraction 500mg of fruit powder which was pretreated with pectolytic enzyme mix (pectinase) was suspended in 4 mL of extraction buffer (0.1 M KC1, 0.5 M Tris-HCl at pH 7.5, 0.1 M EDTA at pH 7.5 2% β-mercaptoethanoland, 2mM PMSF as protease inhibitor). The mixture was then shaken on ice for 1 h and centrifuged at 13 OOOg, 4°C for 20 minutes. The supernatant was then transferred to a new tube and an equal volume of water saturated chloroform was added. The tube was gently shaken on ice for 30 min, then stored on ice for 5 min before centrifugation at 13 OOOg, 4°C for 20 minutes. After centrifugation, the upper and bottom phases were transferred to new tubes. Then 4 mL of water was added to the interphase together with an equal volume of water saturated chloroform, and the mixture was shaken on ice for 30min. This mixture was allowed to stand on ice for 5 min and centrifuged at 13 OOOg, 4°C for 20 minutes. This treatment was repeated twice. The interphase was termed A. The lower phases and upper phases were collected and pooled together. They were termed B and C respectively.

The interphase (A) was washed with ice-cold acetone three times, dried in vacuum (for 15 min) and re-dissolved in rehydration buffer.

The lower phase (B) was air-pumped until 500μΤ of solution remained. Five volumes of acetone were added and stored at - 20C overnight. The pellet was recovered by centrifugation at 13 OOOg, 4°C for 20 minutes and washed by ice-cold acetone, dried, re-dissolved.

The upper phase (C) was treated with an equal volume of Tris-HCl pH 8-saturated phenol solution and the mixture was shaken on ice for 30 minutes and finally centrifuged at 13 OOOg, 4°C for 30 min. The upper buffer phase was removed. The phenol at the bottom was re-extracted twice with extraction buffer. Then proteins were precipitated from the final phenol phase by adding five volumes of 100% methanol containing 0.1M ammonium acetate and left overnight at -20°C. The pellet was generated through centrifugation at 13 OOOg for 15 min.