FR2876106A1 - PROCESS FOR EXTRACTING PROTEINS FOR THEIR ELECTROPHORESIS SEPARATION - Google Patents

Abstract

The object of the invention is a process for extracting proteins from a cell extract with a view to their separation by electrophoresis, characterized in that cyclodextrins are added to a buffer solution and said cell extract in this buffer solution supplemented with cyclodextrins. The invention also covers the extraction buffer used.

Description

2876106 1

  The present invention relates to a method of extracting proteins, based on the use of cyclodextrins, to obtain a preparation capable of being discriminated by electrophoresis.

  The invention also covers the extraction buffer used.

  Electrophoresis is one of the most used techniques in molecular biology. It makes it possible to separate charged molecules of very different types and sizes, such as proteins, peptides, amino acids, nucleic acids or even nucleotides.

  Electrophoresis is based on the principle of differential motion.

  The molecules to be separated are placed in an electric field and move towards a pole of sign opposite to their charge and at a speed proportional to this charge. They are generally deposited on a suitable support porous and impregnated with buffer. For this purpose, for example, paper, cellulose acetate, polyacrylamide gel, agarose gel, starch gel or silica gel may be used.

  Most current electrophoresis techniques use a gel or equivalent. The gel behaves like a molecular sieve, the molecules migrating as fast as they are bigger.

  Several factors therefore influence the migration rate and thus the separation of the molecules during electrophoresis. These factors are the charge and the size of the molecules as well as their affinity for the matrix and the pH of the medium.

  Proteins are molecules commonly separated by electrophoresis.

  The separation of proteins by electrophoresis is essentially in gel, and more particularly in polyacrylamide gel. The proteins to be separated can be native or denatured by agents such as sodium dodecyl sulphate (SDS). There are several gel electrophoresis techniques.

  We call for the rest of the description:

  native gel, a gel for electrophoresis in which undenatured proteins, SDS gel, an electrophoresis gel are migrated in which denatured proteins are migrated by SDS, IEF gel, an electrophoresis gel in which the proteins according to the technique of isoelectric focusing.

  Native gel electrophoresis allows the proteins to be separated according to their molecular weight and charge, while allowing maintenance of protein-protein interactions and biological activity.

  There is also SbS polyacrylamide gel electrophoresis, the principle of which is to use SDS, which imposes a fixed charge on all proteins. Thus, the proteins are separated only according to their molecular weight.

  IEF gel electrophoresis is still known which consists of separating the proteins as a function of their isoelectric point, that is to say the pH for which the sum of their positive and negative peripheral charges cancel each other out. Finally it is possible to combine two electrophoreses with respect to each other and achieve what is called a two-dimensional electrophoresis. This technique consists in carrying out a second electrolytic transport perpendicular to the first on the separated components obtained during the first electrophoresis.

  Currently these different separation methods are commonly used for proteomic studies applicable in many fields.

  However, these techniques have a limit: they do not make it possible to study all the proteins. Indeed, some classes of proteins or proteins present in very small quantities can not be separated by gel electrophoresis.

  Among the proteins that are difficult to detect by electrophoresis, mention may in particular be made of hydrophobic proteins. This phenomenon is explained by the presence of interactions between the hydrophobic residues of the same protein, or of several proteins, resulting in modifications of the configuration of the protein which folds, aggregates and can no longer migrate into the gel . This is particularly the case for membrane proteins. But these membrane proteins are crucial because they are involved in many interactions and constitute a "gateway" for the cell.

  There are also other proteins which are difficult to detect by electrophoresis and which play a decisive role in the understanding of cellular functioning, in particular of very high molecular weight proteins, such as certain factors involved in the biosynthesis of proteins.

  There remains therefore a need for a tool that would improve the power of detection of proteins by electrophoresis.

  In order to meet them, the present invention proposes to increase the quality of extraction of the proteins and to improve their solubilization in the electrophoresis gel, by introducing, before the electrophoretic separation step, cyclodextrins into the buffer. extraction.

  The present invention relates to an extraction method which consists of placing a cell extract in extraction buffer containing cyclodextrins, so as to obtain a preparation capable of being discriminated by electrophoresis.

  By cyclodextrins is meant all natural or modified cyclodextrins. This is advantageously modified cyclodextrins.

  in the case of mono-modified cyclodextrins, the modified cyclodextrins are preferably selected on the primary hydroxyl and in particular 6A- (3-Aminopropylamino) -6A-desoxy-cyclomatoheptaose (propane diamine- (3-CD) and 6A- (3-Aminoethylamino) -6A-desoxycyclomatoheptaose (ethane diamine- (3-CD) described in the patent FR2714067.

  In the case of poly-modified cyclodextrins, 6I- (3a, 7a, 12-trihydroxy-5l3-cholan-24-amido) -6H-deoxy-2H-O-methyl hexakis may be mentioned as particularly effective for the present invention. (2II-VII, 6II-VII-di-O-methyl) cyclomaltoheptaose (DIMEB-cholic acid) described in patent FR2792942.

  It is believed that the improvement in the extraction capacity of the proteins extracted according to the present invention is due to the interaction of the cyclodextrins present in the extraction solution with the hydrophobic residues of the proteins. Thus cyclodextrins compete with the interactions that may exist between these residues, prevent the aggregation of proteins and thus allow their visualization in the gel.

  The proteins extracted according to the present invention can then be separated by any type of electrophoresis.

  Advantageously, to increase the effect obtained by the present invention, the electrophoresis can be carried out in a gel which itself contains cyclodextrins. Cyclodextrins can be introduced either directly into the gel at the time of manufacture or into the rehydration solution in the case of a ready-to-use gel.

  Other features and advantages will emerge from the examples which follow, examples given with reference to the appended figures in which: FIG. 1A represents the results of a standard two-dimensional IEF / SDS electrophoresis separation of total bacterial proteins, the proteins having been extracted in a standard way.

  FIG. 1B represents the results of a standard two-dimensional IEF / 5D5 electrophoresis separation of total bacterial proteins, the proteins having been extracted according to the invention in a buffer containing propane diamine-13-CD.

  FIG. 2A shows the results of a standard IEF / SDS two-dimensional electrophoresis separation of proteins from a membrane fraction of Escherichia coli, the proteins having been extracted in a standard manner.

  FIG. 2B represents the results of a standard two-dimensional IEF / SDS electrophoresis separation of proteins from a membrane fraction of E. coli. coli, the proteins that have been extracted according to the invention in a buffer containing propane diamine-13-CD.

  FIG. 2C represents the results of a two-dimensional electrophoresis separation IEF / SDS - the IEF gel used for the first dimension containing cyclodextrins - of proteins of a membrane fraction of E. coli. coli, the proteins having been extracted according to the invention in a buffer containing propane diamine- (3-CD.

  FIG. 3A represents the results of a standard IEF / 5D5 two-dimensional electrophoresis separation of proteins from a bacterial membrane fraction, the proteins having been extracted in a standard manner.

  FIG. 3B represents the results of a standard two-dimensional IEF / SD5 electrophoresis separation of proteins from a membrane fraction of bacteria, the proteins having been extracted according to the invention in a buffer containing DIMEB-cholic acid.

  EXAMPLE 1 Extraction of Total Proteins from Bacteria in the Presence of Diamine-p-CD-propane E. coli cells are used.

  The cells are lysed and treated with DNase I and RNase I. The resulting solution is then treated with an extraction buffer containing: - 8 M urea, - 50 mM dithiothreitol (DTT), - 10 to 120 mM propane diamine-p-CD, more particularly 20 to 70 mM, -2% CHAPS, and 0.2% ampholyte (pH 3-10).

  The solution obtained is finally clarified by centrifugation and stored at -80 C.

  This solution contains proteins that can be separated by electrophoresis.

  The obtained proteins are separated by standard two-dimensional IEF / SD5 electrophoresis.

  Figures 1A and 1B allow us to compare the separation of total proteins from E. coli. coli, extracted standard or in the presence of propane diamine-p-CD.

  It is found that the intensity and number of spots are larger in Figure 1B than in Figure 1A. Thus the presence of propane diamine-p-CD promotes the extraction of proteins and therefore increases the detection limit and the separation power of electrophoresis.

  EXAMPLE 2 Extraction of Membrane Proteins from Bacteria in the Presence of Propane Diamine-5-CD The proteins to be separated are extracted into a solution consisting of: 8 M urea, 50 mM DTT, 2% CHAPS, 10 to 100 mM propane diamine-13-CD, more particularly 20 to 70 mM, and 0.2% ampholyte (pH 3-10).

  This solution is incubated for two hours at 25 ° C.

  The insoluble material is then removed by centrifugation at 25 ° C. for one hour.

  The proteins thus extracted can then be separated by standard two-dimensional IEF / SDS electrophoresis.

  Comparing the results obtained in FIGS. 2A and 2B, it is found that the presence of cyclodextrins in the protein extraction solution makes it possible to obtain new spots and a better resolution. The limit of detection and the separation capacity of the membrane proteins by gel electrophoresis is therefore increased in the presence of propane diamine-13-CD.

  In FIG. 2C, it is observed that the addition of propane diamine-13-CD both in the protein extraction solution and in the electrophoresis gel makes it possible to significantly increase the limit of detection and the separation power. membrane proteins by electrophoresis.

  EXAMPLE 3 Extraction of Bacterial Membrane Proteins in the Presence of DIMEB-Cholic Acid The proteins to be separated are extracted into a solution consisting of: - 8 M urea, - 50 mM bTT, 2% CHAPS, - 1 to 80 mM of DIMEB-cholic acid, more particularly 5 to 30 mM, and - 0.2% of ampholyte (pH 3-10).

  This solution is incubated for two hours at 25 ° C.

  The insoluble material is then removed by centrifugation at 25 ° C. for one hour.

  The proteins thus extracted can then be separated by standard two-dimensional IEF / SDS electrophoresis.

  When comparing the results obtained in FIGS. 3A and 3B, the results of FIG. 3B show new spots and a better resolution. The presence of DIMEB-cholic acid in the extraction solution thus makes it possible to detect the membrane proteins by gel electrophoresis.

Claims (6)

  1. Process for extracting proteins from a cell extract for the purpose of separating them by electrophoresis, characterized in that cyclodextrins are added to a buffer solution and said cell extract is placed in this added buffer solution cyclodextrins.   2. Process for extracting proteins from a cell extract for the purpose of separating them by electrophoresis according to claim 1, characterized in that the cyclodextrins are chosen from natural or modified cyclodextrins.   3. Process for extracting proteins from a cell extract for the purpose of separating them by electrophoresis according to claim 1 or 2, characterized in that the cyclodextrins are chosen from cyclodextrins which are mono-modified at the level of the primary hydroxyl.   4. Process for extracting proteins from a cell extract for electrophoretic separation according to claim 1, 2 or 3, characterized in that the cyclodextrins are of the form 6A- (3Aminopropylamino) -6A-desoxy-cyclomatoheptaose or 6A- (3-Aminoethylamino) -6A-desoxy-cyclomatoheptaose.   5. Process for extracting proteins from a cell extract for the purpose of separating them by electrophoresis according to claim 1 or 2, characterized in that the cyclodextrins are chosen from polyimorphosed cyclodextrins of the 6I- (3a, 7a, 12a-trihydroxy (3-cholan-24-amido) -6H-deoxy-2H-O-methylhexakis (2H-VII, 6H-VII-di-O-methyl) cyclomaltoheptaose.   Extraction buffer obtained by carrying out the process according to any one of the preceding claims, characterized in that it contains 1 to 100 mM of cyclodextrins.