WO2005040800A1

WO2005040800A1 - Method for producing and using a new protein array, said protein array and the applications thereof

Info

Publication number: WO2005040800A1
Application number: PCT/ES2004/070088
Authority: WO
Inventors: Fernando Martin Gordillo; José Luis CASTRILLO DIEZ
Original assignee: Consejo Superior De Investigaciones Científicas
Priority date: 2003-10-23
Filing date: 2004-10-21
Publication date: 2005-05-06
Also published as: WO2005040800B1

Abstract

The invention relates to a method for producing and using protein arrays and other types of arrays which are resistant to denaturalization of its components due to dehydration and/or temperature. The method is based in the sequential use of concrete solvents, trihalose and trimethylamine N-oxide (TMAO), respectively. The invention also relates to the array thus produced and its applications in the fields of biology and biomedicine. The method can be used to protect the characteristics of the protein arrays and other types of arrays under ambient conditions without requiring the use of short-term cold storage or cold preservation, which reduces storage and delivery costs and makes it possible to obtain better quality products.

Description

TITLE

PROCEDURE FOR MANUFACTURING AND USING A NEW ARRAY OF

PROTEINS, SUCH ARRAY OF PROTEINS AND ITS APPLICATIONS.

SECTOR OF THE TECHNIQUE: The invention has its most evident application in the area of biology and biomedical, being indicated as a procedure and array for the determination of protein-protein interactions and their expression patterns in the discovery of molecular mechanisms, validation of targets Therapeutics, drug validation, clinical validation and clinical diagnosis.

STATE OF THE TECHNIQUE The identification of the functions and structures of proteins encoded by the human genome is one of the great challenges facing the current biomedicine. Until relatively recently, protein analyzes were performed using techniques such as western blotting that individually studied each of them. To carry out these studies on a large scale, new methods and technologies are required, such as those offered by matrices (hereinafter "arrays"). For this, a large number of different proteins are deposited in small quantities in various supports. This technology allows different experiments to be carried out in parallel, with which we can compare expression patterns or discover protein-protein interactions. In principle, this technology was successfully developed using DNA sequences, although it is currently adapting to different molecules, including proteins. Through these devices it is possible to study, among others, protein-protein interactions and expression patterns. Its applications go through the determination of molecular mechanisms, validation of therapeutic targets, drug validation, clinical validation and clinical diagnosis. Some patents describing protein microarrays are: US-6,197,599, US-6,475,808, US-2002/0049152 (Nock, S. et al.) And US-2003/0003599. They describe the chemical immobilization of proteins and antibodies on supports without specifying additional mechanisms. One of the difficulties that protein arrays have to overcome in order to become a really useful tool is the denaturation of the proteins that compose it (Talapatra et al. Pharmacogenomics. (2001) 3 (4), 527-536;). This differential fact against DNA arrays derives from the high structural complexity of proteins, as well as their chemical diversity. Protein arrays are frequently subjected to conditions that induce the denaturation of their components, mainly derived from dehydration and temperature. Hydration is very important to preserve the three-dimensional structure of proteins and their function, in fact they lose their activity in the absence of water (Dean, A. et al. Archives of Biochemistry and Biophysics (1999) 365, 289-298). Therefore, maintaining the structure of proteins in these conditions is crucial to preserve the characteristics of the array. Currently, these situations are avoided by short-term consumption or cold storage, which causes additional costs that make the product more expensive. Protein stability is the result of a balance between macromolecular interactions of protein functional groups and their interactions with the surrounding solvent. The addition of cosolvents to the medium can modify this balance favoring the native state of the proteins. Some of these cosolvents are naturally present in various organisms that are subject to extreme conditions (dehydration, cold, high salt concentrations, etc.). Among them are sugars, polyalcohols, amino acids and certain salts. Two of them are trehalose and trimethylamine N-oxide. Trehalose disaccharide has been described as a good stabilizer of the structure and function of several macromolecules by virtue of its ability to form hydrogen bonds with membranes or its ability to modify the solvation layer of proteins, serving as a water substitute ( Sola-Penna, M. and and Meyer-Fernandez, J. Arch. Biochem. Biophys. (1998) 360, 10-14). This sugar is especially effective in protecting enzymes against dehydration and thermal inactivation and is frequently used in lyophilization processes. Trehalose has been described in patents as a stabilizer of dry protein reagents (WO-89/00693, Tills, D. et al., 1989), and dry forms of reagents with enzymatic activity (EP-192.320, Drake, RAL and Whiteley , SC, 1986). It has also been described as a protective agent for denaturation during drying of biological macromolecules (US-4,891,319, Roser, BJ, 1987). It has been observed on some occasions that, although trehalose exerts a protective effect during dehydration, sometimes its three-dimensional conformation of the native protein is not fully recovered after rehydration (Miller, DP et al. Pharm Res (1998) 15 ( 8), 1215-21). Trimethylamine N-oxide (TMAO) is an osmolyte that also occurs in several species in nature and that functions as a chemical bucket inducing protein folding from its denatured state, or stabilizing the native state (Baskarov, I. and Bolen, DW JBiol Chem (1998) 279 (9), 4831-4834). This is due to the increase in the free energy of the denatured state, disfavoring the transition from the native state (Hill, CM JStructBiol. (2002) 139 (l), 13-26). In summary, the current start of the development of protein and derivative arrays is playing an increasingly important role both in the discovery of biological molecular pathways and in the area of clinical diagnosis and therapy through the parallel analysis of a high number of proteins. . However, these instruments encounter a series of difficulties to overcome inherent in the complexity and chemical nature of proteins. One of these problems consists in their lability against dehydration and / or temperature conditions. This particularity causes the loss of the intrinsic characteristics of the protein and the uselessness of the array as an analysis tool and forces the product to be used in a very short period of time or to keep it cold.

DESCRIPTION OF THE INVENTION Brief Description An object of the present invention is a method of manufacturing and using protein arrays and other types of arrays resistant to denaturation of its components by dehydration and / or temperature, based on the sequential use of specific solvents, trehalose and trimethylamine N-oxide (TMAO), respectively. Likewise, the array thus manufactured and its applications in the field of biology and biomedicine form part of the present invention. The procedure can be used to protect the characteristics of protein and other arrays. types of arrays in environmental conditions without the need for short-term use or cold storage. This point lowers your storage and shipping costs and allows you to obtain higher quality products.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the fact that the inventors have observed that the use of solvents, trehalose and TMAO, sequentially in the manufacture and use of protein arrays allows the conservation and subsequent renaturation of the proteins deposited in said array reaching a resistance level of 95% at limiting conditions such as dehydration and / or temperature. Thus, an object of the present invention is a method of manufacturing and using, hereinafter method of the invention, protein arrays and other types of arrays resistant to denaturation of its components by dehydration and / or temperature, based on the sequential use of specific solvents and consisting of two parts: i) manufacturing process of said array that includes, at least, the dispensing of proteins in a buffer containing trehalose sugar at a concentration between 80 mM and 300 mM , preferably 80 mM (minimum sufficient concentration), and its subsequent dehydration; and ii) a method of using the array of i) which includes an incubation thereof with trimethylamine N-oxide (TMAO) at a concentration between 1 M and 2.5 M, preferably 1 M (minimum sufficient concentration), at the time prior to its use. The term "array" as used in the present invention refers to an ordered array of biological molecules of natural or artificial origin arranged on a support. The term "proteins" as used in the present invention refers to any molecule that contains a polypeptide chain of natural or artificial origin, such as, for example, enzymes, hormones, structural proteins, antibodies or fragments thereof, receptors, peptides, peptide nucleic acids, etc. As used in the present invention, the term "other types of arrays" refers to any type of array in whose composition proteins appear, for example, and without limiting the scope of the invention, cellular arrays, tissue arrays, arrays mixed cDNA / proteins and arrays of small molecules. As used in the present invention, the term "method of manufacturing an array of proteins" refers to any of the different conventional methods of manufacturing arrays and which are referenced in the following documents by way of examples, without limiting the scope of the invention: MacBeath, G. and Schreiber, SL (2000) "Printing proteins as icroarrays for high-throughput function determination" Science 289, 1760-1763); and, Haab, BB et al. "Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions" (2001) Genome Biol. 2, 4.1-4.13. In general, existing technologies today are simple adaptations of those developed for cDNA arrays applications. No similar sequential treatment is collected in scientific articles known to date. Trehalose has been used individually as a stabilizer in lyophilization and vacuum drying processes of restriction enzymes and various proteins. Both the mechanism and the stabilization procedure do not include in any case the use of this treatment proposed in the object of the invention. In the article by Moerman et al. (Anal. Chem. (2001) 73 (10), 2183-2189), a protein electrospray technique for the production of arrays is described. Trehalose is used as stabilizer in this process. The use of trehalose is used as a protein stabilizer to exclusively resist the electrospray process, not for the ultimate purpose of stabilizing the array under conditions of temperature dehydration. Likewise, it does not employ at any time a sequential treatment with trehalose and trimethylamine N-oxide as described in this invention, basic for the correct obtaining of an array in perfect conditions for its use. There are also patents related to trehalose protection, among which WO-89/00693 (Tills, D. et al, 1989), EP-192,320 (Drake, RAL and Whiteley, SC, 1986) and US-4,891,319 (Roser, BJ, 1987). In the first one, trehalose is used in the drying process of proteins applied on plastic. There is no reference to an additional renaturation with TMAO or that it is an array. In addition, the substrate is limited to plastic, when the supports on which the technology derived from our invention can be used range from nitrocellulose to glass. EP-192,320 describes the storage of an enzymatic agent under stable conditions for an immunoassay. Trehalose is used optionally and, again, no further use of TMAO is mentioned. US Pat. No. 4,891,319 also makes no reference to the additional use of this compound that induces renaturation. Another object of the present invention is an array manufactured by the method of the present invention point i) and in which the proteins are dispensed in a buffer containing trehalose sugar at a concentration between 80 mM and 300 M, preferably 80 mM (minimum concentration sufficient). Finally, another object of the present invention is the use of the method and the array of the present invention in the manufacture and application of protein arrays. By way of example, and without limiting the scope of the present invention, by this method and these arrays it is possible to study, among others, protein-protein interactions (see Ge, Hui. "UPA, a universal protein array system for quantitative detection of protein-protein, protein-DNA, protein-RNA and protein-ligand interactions ". 2000. Nucleic Acids Research. 28, No. 2) and expression patterns (see Fung et al." Protein biochips for differential profiling ". 2001. Analytical Biotechnology, 12, 65-69). Its applications go through the determination of molecular mechanisms, validation of therapeutic targets, drug validation, clinical validation and clinical diagnosis. Some patents describing protein microarrays are: US-6,197,599, US-6,475,808, US-2002/0049152 (Nock, S. et al.) And US-2003/0003599. The preservation of the characteristics of the array until the moment of its use is of singular importance for the industry, since not being necessary special conditions of conservation helps to reduce the costs of storage and shipping. Likewise, the method and the array of the present invention represent an advantage in this type of products when they are especially directed to the field of pharmaceutical validation and clinical diagnosis, where the quality of the product is of vital importance. BRIEF DESCRIPTION OF THE CONTENT OF THE FIGURES Figure 1. Experiment in which an array of proteins (antibodies) was subjected to dehydration conditions (1 hour at 37 ° C and preservative with desiccant for two days in an airtight bottle).

Figure 2. Thermostability study provided by this treatment, recovering the activity of anti-Bag, anti-Bax-1 and anti-FAS antibodies (1, 2, and 3; respectively) even after supporting 3 hours at 37 ° C and 80 ° C. In the abscissa axis, the results obtained for arrays not submitted to the treatment object of the invention (1) and submitted thereto (2) are represented.

EXAMPLE OF EMBODIMENT OF THE INVENTION The process of manufacturing and using stabilized protein arrays in a specific example is described below. Ten rabbit polyclonal antibodies (anti-Bcl-2; anti-Bax-1; anti-Bag; anti-Caspase 3; anti-FAS; anti-FAS Ligand; anti-p53; and, anti-TRAIL) were selected which They were used as raw material in the construction of the pilot array. Equal volumes of antibodies were diluted with 160 mM PBS-trehalose (2x). Each sample was deposited in a microplate and microdispensed on a support. The arrays are dehydrated 1 hour at 37 ° C and stored for two days at room temperature until used in an airtight container together with a desiccant (silica gel). Similarly, similar results have been obtained in experiments for samples preserved under the same conditions for periods of at least two months. On the other hand, a protein extract was prepared from 10 ⁷ cells with modified RIPA buffer (50 mM Sodium Borate; 1% NP-40; 0.5% Sodium Deoxycholate; 150 M NaCl; lmM EDTA; lmM PMSF; Aprotinin, Leupeptin and Pepstatinalmg / ml c / u; Na ₃ VO 1 mM) of the U373MG cell line (derived from glioblastoma). 1 ml of it was labeled with 25 ml of biotincaproate-3 NHS (10 mg / ml) for twenty minutes and the reaction was stopped by adding 50 ml NHC1 and incubating fifteen minutes. Subsequently, the labeled proteins were purified by column filtration of Sephadex G-25, whose eluent was used in successive experiments. Prior to using the array, it was incubated overnight at room temperature with TBS containing 1 M trimethylamine N-oxide. After treatment, it was washed the membrane briefly with TBS-T and was blocked for 1 hour with 5% skim milk in the same buffer. The array was incubated with 10 mg / ml of the biotinylated protein extract in blocking solution for 2 hours, washed with TBS-T, and incubated for half an hour with a streptavidin-peroxidase solution. The signals with ECL were revealed. Likewise, control arrays, arrays not subjected to dehydration, and other arrays subjected to dehydration were performed but without the treatment of trehalose or TMAO or without any of them (Figure 1). The manufacture of them and their use were carried out under the same general conditions. Experiments have been carried out with different concentrations of these solvents, which suggest a range of concentrations ranging from 80 to 300 mM in the case of trehalose, and from 1 to 2.5 M in the case of TMAO. The results described in the figures correspond to 80 mM and 1M, respectively. Likewise, the test was carried out on different supports (nitrocellulose, nylon, PNDF and glass), offering the best results the use of nitrocellulose and glass. The use of this sequential procedure allows the array proteins to be obtained in their native conformational state just at the time of use. It is necessary to perform both sequential steps to obtain and use an array in optimal conditions for its use. Thus, the fall of the signal obtained in the untreated sample, the increase in sensitivity derived from greater stability obtained with this treatment (+ Trehalose + TMAO) can be observed (Figure 1). The addition of trehalose in the manufacturing process manages to preserve the proteins, providing the product with thermostability and resistance to dehydration. In this way, the array thus manufactured can be safely stored at room temperature and for long periods of time. However, after this first treatment only about 50-70% of the total deposited proteins are recovered (Figure 1). The incubation with trimethylamine óxido-oxide manages to induce the folding and renaturation of the denatured proteins, obtaining functional arrays in 90-95% (Figure 1). Figure 2 shows the increase in thermostability offered by the process object of the invention. Two arrays were produced for each experiment, one of which was manufactured by dispensing the trehalose samples under the conditions described above. Without mediating dehydration, they were subjected to different conditions of temperature (37 ° C and 80 ° C) for three hours. The graph representing the results obtained at 37 ° C shows how the signals obtained for untreated arrays (1) as treated (2) are similar, although higher values are already observed in the treated samples. At 80 ° C the untreated arrays (1) have almost completely lost their activity, while the arrays produced and treated by the process object of the invention retain significant activity values. The realization of the process of the invention can be carried out both from cell cultures and from tissues and fluids, simply varying the extraction method. In the case of tissues (DeSeau N, et al. J Cell Biochem. 1987 Oct; 35 (2): 13-28), prior homogenization thereof is necessary, continuing with a protocol similar to that described. For fluid analysis, samples should be dialyzed and used directly on the array (Bergquist J, et al. Mass Spectrom Rev. 2002 Jan-Feb; 21 (l): 2-15).

Claims

1.- Procedure of manufacture and use of protein arrays and other types of arrays resistant to the denaturation of its components by dehydration and / or temperature characterized by the sequential use of specific solvents and consisting of two parts: i) procedure for preparing said array which includes, at least, the dispensing of proteins in a buffer containing trehalose sugar at a concentration between 80 mM and mM, preferably 80 mM, and subsequent dehydration; and ii) a method of using the array of i) that includes an incubation thereof with trimethylamine N-oxide (TMAO) at a concentration between 1M and 2.5M, preferably 1M, in the moments prior to its use.

2. Ar y of proteins characterized in that it is manufactured by the method according to claim 1 i).

3. Use of the method according to claim 1 and the array according to claim 2 in the manufacture and application of protein arrays.