ES2306541B1 - FUNCTIONAL TRANSPLANT OF THE MACHINERY OF SUMOILATION TO ESCHERICHIA COLI. - Google Patents

Abstract

Functional transplantation of the sumoylation machinery to Escherichia coli .

The present invention provides a method of in vivo sumoylation of proteins susceptible to sumoylation by transforming host cells with the components of the eukaryotic sumoylation machinery E1, E2, of SUMO and with said protein, chimeric or not, susceptible to sumoylation. This method allows to obtain sumoylated proteins that are involved in multiple mechanisms of biological action so they are very useful in processes of the biotechnology-pharmaceutical industry, and also allows its application for the expression, obtaining and purification of proteins of commercial interest, within a chimeric protein susceptible to sumoylation, being a new method of gene expression.

Description

Functional transplantation of the sumoylation machinery to Escherichia coli .

Technical sector

The present invention is encompassed within the field of genetic engineering. More specifically, this invention provides materials and methods for expression, Separation and selective purification of proteins.

Prior art

The modification Post-translational protein is an important means of regulating the activity, stability or location of proteins Recently, several proteins have been identified with some similarity in its sequence to ubiquitin, which post-translationally modify target proteins by conjugation. SUMO protein conjugation (sumoylation) plays an important role in the regulation of the function protein

Ubiquitin and ubiquitin-like proteins are  widely known in the literature. SUMO (small ubiquitin related modifier), also known as Sentrin, SMT3, PIC1, GMP1 and UBL1, is one of these ubiquitin-like proteins, presenting 18% identity in its sequence with ubiquitin. In mammals, 3 members of the SUMO family have been described: SUMO-1, SUMO-2 and SUMO-3.

In order for any of the members of the SUMO family to be competent for conjugation with a target protein, they must be previously proteolyzed in their C-terminal, losing their last four residues, so that it will end with a glycine. After the SUMO C-terminal has been trimmed, the enzyme El, SUMO activating enzyme, comes into play, which is composed of two proteins, Aos1 and Uba2 (in humans) (Johnson, Schwienhorst et al. Embo J 1997 16, 5509-5519), which has the ability to join SUMO forming a thioester bond, intermediate SUMO-Uba2. Once this has happened, SUMO is then transferred to the enzyme E2, Ubc9 (Johnson and Blobel J Biol Chem 1997 272, 26799-26802; Desterro, Rodriguez et al . J Biol Chem 1999 274, 10618-10624), which is conjugated to SUMO, through a thioester link. It has been shown that, once attached to SUMO, Ubc9 can transfer SUMO directly to its corresponding in vitro substrate (Okuma, Honda et al . Biochem Biophys Res Commun 1999 254, 693-698; Sampson, Wang et al . J Biol Chem 2001 276, 21664-21669). So, only the presence of enzymes E1 and E2 is required, and a substrate protein for in vitro conjugation. Although they can be omitted in vitro , a number of E3 enzymes have been identified for in vivo sumoylation (Melchior, Schergaut et al . Trends Biochem Sci 2003 28, 612-618). Once a protein is sumoylated, there are many SUMO specific proteases and isopeptidases in the cell that can release SUMO from the targets to which it was conjugated, which plays an important role in cell regulation (Melchior, Schergaut et al . Trends Biochem Sci 2003 28, 612-618).

SUMO and ubiquitin are synthesized as precursors that need to be processed before protein conjugation. The mechanism for SUMO conjugation is also analogous to that of the ubiquitin system, such as the use of the enzyme cascade E1, E2 and E3. However, the biological consequence of the SUMO modification is absolutely different from that of the ubiquitin system. While the ubiquitination of most proteins is a path to degradation, the modification by SUMO of the proteins is involved in the nuclear direction, the formation of subnuclear structures, the regulation of transcriptional activities or DNA binding capabilities of the transcription factors and the stability control of the
protein.

Sumoylation antagonizes ubiquitin conjugation (ubiquitination), thus preventing the degradation of proteins and complexes (Desterro, JM, MS Rodriguez, et al . Mol Cell 1998, 2 (2): 233-9, Buschmann, T., SY Fuchs, et al . Cell 2000, 101 (7): 753-62), serves as a signal for nuclear localization (Pichler, A. and F. Melchior Traffic 2002, 3 (6): 381-7, Seeler, JS and A. Let Nat Rev Mol Cell Biol 2003, 4 (9): 690-9), activate or deactivate enzymes) Ulrich, HD Cell Cycle 2004, 3 (1): 15-8), prevents or stimulates DNA binding or activity of transcription factors (Bies, J., J. Markus, et al . J Biol Chem 2002, 277 (11): 8999-9009, Gill, G. Curr Opin Genet Dev 2003, 13 (2): 108- 13, Verger, A., J. Perdomo, et al . EMBO Rep 2003, 4 (2): 137-42), plays a role in higher order chromatin structures and nuclear organization (Jackson, PK Genes Dev 2001, 15 (23): 3053-8, Eskiw, CH and DP Bazett-Jones Biochem Cell Biol 2002, 80 (3): 301-10, Pinsky, BA and S. Biggins Dev Cell 2002, 3 (1):
4-6).

Certain critical proteins for the regulation of the cell have been shown to undergo SUMO modification in some stadium. Examples of these are p53, PCNA, Rho, Jun, CREB, c / EBP, cMyb, I? B, MDM2, Mek1, HDAC, etc. (Seeler, J. S. and A. Let Nat Rev Mol Cell Biol 2003, 4 (9): 690-9), Table 1 shows the main SUMO substrates grouped by function.

TABLE 1

one

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One of the characteristics of sumoylation is that it is normally directed by a consensus sequence in the target proteins. This sequence is \ PsiKXE, where \ Psi represents a hydrophobic amino acid (for example leucine, isoleucine, valine or alanine), K represents lysine, X represents any amino acid and E represents glutamic acid (Rodriguez, MS, C. Dargemont, et al . J Biol Chem 2001, 276 (16): 12654-9, Sampson, DA, M. Wang, et al . J Biol Chem 2001, 276 (24): 21664-9).

As mentioned earlier, there are three types of SUMO in human cells. One difference between them is that SUMO-2 and SUMO-3 but not SUMO-1 have in their sequences, near the N-terminal, the above-mentioned sumoylation sequence. It has been shown that as a consequence of the presence of this sequence, they can form poly-SUMO chains both in vitro and in vivo (Tatham, MH, E. Jaffray, et al . J Biol Chem 2001, 276 (38): 35368-74).

At present, some in vitro sumoylation works of target proteins using previously purified enzymatic sumoylation cascade components are known. However, the selective obtaining of pure sumoylated proteins from eukaryotic cells is not easy due to the dynamic nature of this modification and the large number of sumoylated proteins in vivo .

Therefore, it would be of interest to obtain a method that allows quantitative sumoylated proteins to be obtained in vivo in prokaryotic cells, as well as a way of obtaining large amounts of specifically sumolated target proteins with a high degree of purity, from the generation of proteins. of fusion not limited to N or C-terminal addiction and / or polymerization of proteins by means of covalent bonds.

Explanation of the invention.

The invention faces the problem of provide new procedures for expression, production and purification of heterologous proteins of commercial interest.

The solution provided by this invention is based on the fact that the inventors have demonstrated that this new described method is capable of moving the eukaryotic sumoylation machinery to Escherichia coli to study the functionality and / or obtain and / or purify sumoylated proteins directly from E. coli . This method of coexpression of the enzymes involved in the sumoylation of proteins (El and E2) together with SUMO and the sumoylated proteins in E. coli uses an inducible system, without the need to previously purify any of the components of the sumoylation system, and It can be used in a manner compatible with the most common expression vectors, resulting in the sumoylation of target proteins in vivo .

In accordance with a first aspect of the present invention, an in vivo sumoylation method of sumoylation susceptible proteins is provided characterized in that it comprises:

to)

the transformation of the host cell by introducing The components of the eukaryotic sumoylation machinery E1, E2, a DNA sequence encoding eukaryotic SUMO or any of its derivatives, and a DNA sequence that encodes the protein susceptible to sumoylation under the action of a suitable promoter for its expression in one or more expression vectors and,

b)

its subsequent induction.

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As used in the present invention the term "sumoylation susceptible proteins" refers to proteins that are naturally belonged belonging, between others, by way of illustration and without limiting the scope of the invention, to the following group: lef-1, p53, PCNA, Rho, Jun, CREB, c / EBP, cMyb, I? B, MDM2, Mek1 and HDAC (for further expansion of potential heterologous proteins susceptible to sumoylation can be taken as a reference illustrative Table 1 of the present invention). On the other hand, the term "sumoylation susceptible proteins" includes also to all those proteins susceptible to sumoylation chimeric or commercial interest mergers that comprise the Protein domain susceptible to sumoylation. As used in  the present invention the term "proteins susceptible to chimeric or fusion sumoylation of commercial interest "se refers to any chimeric protein that contains a protein or peptide of commercial interest, of different origin (eukaryotic, prokaryotic, viral, etc.), belonging among others, and in title illustrative and without limiting the scope of the present invention, to Next group: enzymes, monoclonal antibodies, factors of growth, cytokines, hormones, proteins and application peptides in the food sector, for example pepsin, transglutaminase, etc; therapeutic agents, for example IGF-I, immunogenic antigens for vaccine production, etc.

As used in the present invention the term "components of the sumoylation machinery eukaryotic "refers, both in the case of components The (Aos1 and Uba2 proteins) and E2 (ubc9 protein) as in the case of SUMO (SUMO-I proteins, protein SUMO-2 and SUMO-3 protein), to any of the eukaryotic protein forms thereof, preferably of vertebrate animals, more preferably mammalian animals, and much more preferably of a human being, and to its analogous forms. The term "analogous forms" is intended include any protein that can be isolated or constructed from artificial form, for example, by introducing conservative or non-conservative nucleotide substitutions, including the insertion of one or more nucleotides, the addition of one or more nucleotides at either end of the molecule or the deletion of one or more nucleotides at either end or in the inside the DNA sequence encoding said protein. In the meaning used in this description, the expression "analogous" means that the amino acid sequences of the proteins in question have a degree of identity, at the level of nucleotides of at least 60%, preferably of at least one 85%, or more preferably, at least 95%.

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In a preferred embodiment of the present invention, the in vivo sumoylation method of sumoylation susceptible proteins comprises:

to)

the transformation of the host cell with an expression system gene consisting of:

a.1.-

an expression vector containing the sequence of DNA coding for the machinery components of eukaryotic sumoylation E1 (proteins Aos1 and Uba2) and E2 (protein ubc9),

a.2.-

an expression vector containing the sequence of DNA encoding eukaryotic SUMO or any of its derivatives, Y

a.3.-

an expression vector containing the sequence of DNA encoding the protein susceptible to sumoylation,

all of them under the action of a promoter / s is suitable for its expression, Y

b)

its subsequent induction.

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In accordance with another preferred embodiment of the present invention, in the in vivo sumoylation method of proteins susceptible to sumoylation, purification of any of the system components is not necessary.

In accordance with another preferred embodiment of the present invention, the host cell is an E. coli cell.

According to another preferred embodiment of the present invention, the promoter can be a constitutive promoter or regulated.

According to another embodiment even more preferred, the promoter is selected from the group formed by T7, TRC, Ptac and Plac.

According to another preferred embodiment, the expression vector containing a DNA sequence that encodes for eukaryotic SUMO or any of its derivatives also contains a nucleic acid sequence encoding a sequence of determined amino acids fused to the sequence encoding the SUMO molecule.

According to another embodiment even more preferred, the amino acid sequence fused to the sequence that encodes the eukaryotic SUMO molecule, is selected from the following group formed by polyhistidine, Strep, E-tag, HA, FLAG, GST, binding protein calmodulin, maltose binding protein, a binding protein cellulose, thioredoxin, antibody type molecules, enzymes, fluorescent proteins, kinases, proteases, binding proteins DNA, RNA binding proteins, glycosyltransferases, proteins membrane, serum albumin and toxins.

In accordance with a second aspect of the present invention, a method of obtaining, separating and / or providing selective purification of proteins susceptible to sumoylation, comprising the transformation of the host cell by the Introduction of the components of the sumoylation machinery E1, E2 and a DNA sequence encoding SUMO or any of its derivatives under the action of a promoter for expression in one or more expression vectors and their subsequent induction.

According to a preferred embodiment of the present invention, the method of obtaining, separating and selective purification of sumoylation susceptible proteins comprises the transformation of a host cell with a vector of expression that contains the components of the machinery of sumoylation E1 and E2, along with another expression vector that contains a DNA sequence that encodes SUMO or any of its derivatives under the action of a promoter of its expression and its subsequent induction.

According to another preferred embodiment of the present invention, in the method of obtaining, separating and purifying Selective protein susceptible to sumoylation is not necessary the purification of any of the system components.

According to another preferred embodiment, an E. coli cell is used as a host cell in the method of obtaining, separating and purifying proteins susceptible to sumoylation .

According to another preferred embodiment in the method of obtaining, separation and selective purification of proteins susceptible to sumoylation, the promoter used in the method of separation and purification of proteins susceptible to sumoylation, it is a constitutive promoter or a regulated promoter.

In an even more preferred embodiment in the method of obtaining, separation and selective purification of proteins susceptible to sumoylation, the promoter is selected between the group formed by T7, TRC, Ptac and Plac.

According to another preferred embodiment in the method of obtaining, separation and selective purification of proteins susceptible to sumoylation, the expression vector that contains a DNA sequence that encodes SUMO or any of its derivatives also contain a nucleic acid sequence encoder of a given amino acid sequence, for the subsequent purification of said vector, fused to the sequence which encodes the SUMO molecule.

According to another embodiment even more preferred in the method of obtaining, separating and purifying Selective protein susceptible to sumoylation, the sequence of  amino acids fused to the sequence encoding the molecule of SUMO, is selected from the group formed by polyhistidine, Strep, E-tag, HA, FLAG, GST, protein binding calmodulin, maltose binding protein, a binding protein cellulose and thioredoxin.

A third aspect of the present invention provides a method of obtaining protein chains susceptible to sumoylation covalently linked through SUMO molecules, characterized in that it comprises the transformation of the host cell by introducing the components of the sumoylation machinery E1, E2 and a sequence of DNA encoding SUMO or any of its derivatives under the action of a promoter for its expression in one or more vectors of expression and its subsequent induction.

According to a preferred embodiment of the method of obtaining protein chains susceptible to sumoylation covalently linked through SUMO molecules, said method comprises the transformation of a host cell with an expression vector containing the components of the eukaryotic sumoylation machinery E1 and E2, along with another vector expression that contains a DNA sequence that codes for SUMO or any of its derivatives under the action of a promoter of its expression and its subsequent induction.

According to another preferred embodiment of the present invention, in the method of obtaining protein chains susceptible to sumoylation covalently linked through SUMO molecules, purification of any of the components of the sumoylation machinery.

According to a preferred embodiment, the method of obtaining sumoylation chains of covalently bound proteins through SUMO molecules employs an E. coli cell as the host cell.

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According to another preferred embodiment in the method of obtaining chains of proteins susceptible to sumoylation covalently linked through SUMO molecules, the promoter used in the protein separation and purification method susceptible to sumoylation, is it a constitutive promoter or a regulated promoter.

In an even more preferred embodiment in the method of obtaining protein chains susceptible to sumoylation covalently linked through SUMO molecules, the promoter is selected from the group consisting of T7, TRC, Ptac and Plac.

According to another preferred embodiment in the method of obtaining protein chains susceptible to sumoylation covalently linked through SUMO molecules, the expression vector containing a DNA sequence that encodes for SUMO or some of its derivatives it also contains a sequence of nucleic acid encoding an amino acid sequence determined, fused to the sequence that encodes the molecule of SUMO.

According to another embodiment even more preferred in the method of obtaining protein chains susceptible to sumoylation covalently linked through SUMO molecules, the amino acid sequence fused to the sequence encoding the SUMO molecule, is selected from the group formed by polyhistidine, Strep, E-tag, HA, FLAG, GST, calmodulin binding proitein, a binding protein maltose, cellulose binding protein, thioredoxin, type molecules antibody, enzymes, fluorescent proteins, kinases, proteases, DNA binding proteins, RNA binding proteins, glycosyltransferases, membrane proteins, serum albumin, toxins

A fourth aspect of the present invention provides a method to increase the expression levels of a  sumoylation protein of interest in a cell host, which comprises the transformation of the host cell by introducing the components of the machinery of sumoylation E1, E2 and a DNA sequence encoding SUMO or any of its derivatives under the action of a promoter for its expression in one or more expression vectors and their subsequent induction.

According to a preferred embodiment of the method to increase the expression levels of a protein susceptible to sumoylation of interest in a host cell, said method comprises the transformation of a host cell with an expression vector containing the components of the Sumoylation machinery El and E2, along with another vector of expression that contains a DNA sequence that codes for SUMO or any of its derivatives under the action of a promoter of its expression and its subsequent induction.

According to another preferred embodiment of the present invention, in the method of increasing expression levels of a sumoylation protein of interest in a cell host, purification of any of the components of the sumoylation machinery.

According to a preferred embodiment, the method for increasing the expression levels of a sumoylation protein of interest in a host cell, uses an E. coli cell as the host cell.

According to another preferred embodiment in the method to increase the expression levels of a susceptible protein of sumoylation of interest in a host cell, the promoter used in the protein separation and purification method susceptible to sumoylation, is it a constitutive promoter or a regulated promoter.

In an even more preferred embodiment in the method to increase the expression levels of a protein susceptible to sumoylation of interest in a host cell, the promoter is selected from the group consisting of T7, TRC, Ptac and Plac.

According to another preferred embodiment in the method to increase the expression levels of a protein susceptible to sumoylation of interest in a host cell, the expression vector containing a DNA sequence that encodes for SUMO or some of its derivatives it also contains a sequence of nucleic acid encoding an amino acid sequence determined, fused to the sequence that encodes the molecule of SUMO.

According to another embodiment even more preferred in the method to increase expression levels of a sumoylation protein of interest in a cell host, the amino acid sequence fused to the sequence which encodes the SUMO molecule, is selected from the group formed by polyhistidine, Strep, E-tag, HA, FLAG, GST, calmodulin binding proitein, maltose binding protein, cellulose binding protein, thioredoxin, type molecules antibody, enzymes, fluorescent proteins, kinases, proteases, DNA binding proteins, RNA binding proteins, glycosyltransferases, membrane proteins, serum albumin, toxins

A fifth aspect of the present invention provides a kit comprising an expression vector that Contains the components of the sumoylation machinery E1 and E2, together with another expression vector that contains a sequence of DNA encoding SUMO or any of its derivatives under the action of a promoter of expression, and a multiple cloning site able to clone a nucleic acid that encodes the protein of interest in the framework of the nucleic acid sequence encoding the SUMO molecule.

According to a preferred embodiment, the kit also comprises host cells suitable for the expression of said vector.

According to an even more preferred embodiment, the host cells contained in the kit are E. coli cells .

A specific embodiment of the invention is a host cell consisting of E. coli strain BL21 (DE3) containing plasmids pBADE12 and pKRHSUM. A culture of this bacterium E. coli BL21 (DE3) has been deposited in the Spanish Type Culture Collection (CECT), University of Valencia, Research Building, Burjasot Campus, 46100 Burjasot, Valencia, Spain, on April 1, 2004, corresponding to it, the deposit number CECT 5694.

According to another preferred embodiment in the kit, the employed promoter is a constitutive promoter or a promoter regulated.

In an even more preferred embodiment in the kit, the promoter is selected from the group consisting of T7, TRC, Ptac and Plac.

According to another preferred embodiment in the kit, the expression vector that contains a DNA sequence that code for SUMO or any of its derivatives also contains a nucleic acid sequence encoding a sequence of determined amino acids, fused to the sequence encoding the SUMO molecule.

According to another embodiment even more preferred in the kit, the amino acid sequence fused to the sequence encoding the SUMO molecule, is selected from the group formed by polyhistidine, Strep, E-tag, HA, FLAG, GST, calmodulin binding proitein, a binding protein maltose, cellulose binding protein, thioredoxin, type molecules antibody, enzymes, fluorescent proteins, kinases, proteases, DNA binding proteins, RNA binding proteins, glycosyltransferases, membrane proteins, serum albumin, toxins

According to another preferred embodiment of the present invention, the kit also contains a composition containing at least one protease capable of releasing the molecule from SUM of fusion protein.

Other reagents can be included in the kits  provided by the present invention, such as lysis, washing and elution buffers.

The system for obtaining sumoylated proteins according to the present invention, it allows to increase the expression of poorly expressed proteins, increase the solubility of proteins, protect proteins specifically from degradation by intracellular proteases by SUMO fusion.

The sumoylation of certain proteins involved in cell regulation such as p53, PCNA, Rho, Jun, CREB, c / EBP, cMyb, I? B?, MDM2, Mek1 and HDAC, allows therefore control their function, so according to An aspect of the present invention provides a method of regulation of the function of proteins susceptible to sumoylation

Brief description of the drawings

Fig. 1. Expression of sumoylation machinery in E. coli . The cells were incubated at 37 ° C for 4 hours in the presence of 0.5 mM of IPTG. After this, 1 ml of the culture was centrifuged, resuspended and placed on a 15% acrylamide SDS-PAGE. Proteins were transferred and the membrane was tested with a 1/5000 dilution of anti his tag - peroxidase antibody. Vectors alone or containing the genes to express the indicated proteins, were placed in the corresponding bacterial culture. His-tag was present in the Aos1 protein as in SUMO.

Fig. 2. Purification of sumoylated proteins . Strains of bacteria transformed with plasmids expressing his-SUMO and any of hisAos1-Uba2 (C) or hisAos-Ubc9-Uba2 (Sum) were incubated in the presence of IPTG, lysed in the presence of 6 M Urea and the soluble fraction was collected and purified on a column of Cobalt TALON. The bands marked with arrows were identified by MALDI-TOF.

Fig. 3. Expression of the sumoylation machinery together with a specific target in E. coli . The cells were incubated at 37 ° C for four hours in the presence of 0.5 mM IPTG. After this, 1 ml of the culture was collected and centrifuged, resuspended and 1/30 of the original amount was loaded onto 15% acrylamide SDS-PAGE. The proteins were transferred and the membrane tested with a 1/5000 dilution of anti-histidine tag-peroxidase antibody. Vectors alone or containing the genes that express the indicated proteins were placed in the corresponding bacterial cultures. The histidine sequence was present in the Aos1 protein as well as in LEF1.

Fig. 4. Expression of sumoilable peptides fused to GST together with the sumoylation machinery . The gel was stained with Coomasie blue (right panel) or after transfer, the proteins were tested with anti-SUMO antibodies (left panel). Vectors alone or containing the genes expressing the indicated proteins were present in the corresponding bacterial strains. In all cases the sumoylation machinery was present.

Fig. 5. Protein purification . Bacterial strains transformed with plasmids expressing the indicated proteins were incubated for 4 hours in the presence of 0.5 mM of IPTG, lysed under moderate conditions and soluble proteins were collected and purified on TALON Cobalt columns for LEF1 fused to the histidine sequence or envelope Glutathione columns for GST derivatives. The purified proteins were resolved by 12% acrylamide SDS-PAGE and the gels were stained with Coomasie blue (right panels) or transferred on immobilon membranes, the proteins being detected with anti-SUMO antibodies (central panels), anti-GST antibodies ( bottom, left) or anti-his-tag (top, left). The arrows mark the bands that reacted with two of the antibodies and were detected by Coomasie staining.

Figure 6. Mass Spectrometry Detection of the peptides obtained in the gel digestion of the bands of coomasie shown in figure 5. A) Bands of the line corresponding to the purification of LEF1 fused to the sequence of histidines. B) Bands of the line corresponding to the GST-PML fusion purification. C) Bands of the line corresponding to the purification of the fusion GST-I \ kappaB.

The present invention is illustrated with the following non-limiting examples.

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Examples of the invention

Example one

Plasmid Construction

DNA manipulations were performed of according to standard techniques, known in the state of the art.

The initial polycistronic construction expressing enzymes E1 and E2 was performed as follows:  plasmid pET28a (Novagen) containing Aos1 (human sequence) modified with histidine in its N-terminal was cut with EcoRI and HindIII and we cloned between these sites a amplified PCR fragment of plasmid pET23aUbc9 with the oligos, Ubc9Eco of SEQ ID NO 1 to introduce an EcoRI site in the 5 'terminal and Ubc9Hind of SEQ ID NO 2 to enter a site HinDIII in the 3 'terminal and cut with enzymes corresponding.

Another fragment of the PCR was generated using the plasmid pET11Uba2 as a model and the UbaNot oligos of SEQ ID NO 3 to introduce a Not site in the 5 'terminal and UbaXho of SEQ ID NO 4 to enter an XhoI in the 3 'terminal. This fragment was cut with NotI and XhoI and cloned between the NotI and XhoI sites of the plasmid obtained previously to generate pETE12 (SEQ ID NO 5) which contains Aos1, Ubc9 and Uba2 cloned together under a T7 promoter (human sequence of Aos1 and Uba 2, and mouse sequence from Ubc9).

Similarly, a plasmid expressing Aos1 and Uba2 was constructed, the two components of the enzyme E1 (Vec Aos1 / uba2) Subsequently, plasmid pET11aSUMO was cut with SphI and BamHI and cloned the 641 bp fragment containing SUMO (human sequence of SUMO-I) at the SphI and BamHI sites of plasmid pRXAv to obtain pRSUM (SEQ ID NO 6). This was constructed by cloning an XylR derivative into plasmid pJB866 (Blatny et al ., Plasmid 1997 38, 35-51) (RK2 replicon, with Ampicillin resistance) between the SphI and BamHI sites. A hexahistidine modification of plasmid pER28a was added to the N-terminal of SUMO.

With the intention of facilitating the co-expression of different targets together with the sumoylation machinery, the vectors that expressed the constructions mentioned above were changed. Plasmid pETE12 was cut with MluI and XhoI and cloned between the MluI and SalI sites of pBAD33 to obtain pBADE12 (SEQ ID NO 7) (p15A replicon, with chloramphenicol resistance). The SphI-BamHI fragment containing SUMO without tag was also cloned into plasmid pJB861 (Blatny, Brautaset et al . Plasmid 1997 38, 35-51) to obtain plasmid pKRSUM (SEQ ID NO 8) (RK2 replicon, kanamycin resistance) cut with SphI and BamHI.

GST mergers were built starting of plasmid PGEX2T (Amersham Biosciences) and cloning among its BAMHI and EcoRI sites the oligos of SEQ ID NO 9, direct and of SEQ Reverse ID NO 10 for I \ KappaB and SEQ ID NO 11 direct and SEQ ID NO 12 reverse, for PML. The new plasmids pGST-I \ KappaB (SEQ ID NO 13) and pGST-PML (SEQ ID NO 14) were verified by sequencing

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Example 2

Analysis of the presence of SUMO in lysates of E. coli by Western blotting

Once the BL21 strain (DE3) of E. coli was transformed with two of the plasmids obtained in the previous example, 1 ml of bacterial culture was centrifuged and the supernatant was subsequently removed and resuspended in 300 µl of protein loading buffer. Protein samples for SDS-PAGE were prepared under denaturing conditions (1% SDS, 100 ° C 10 min) and reducing (140 mMβ-mercaptoethanol) and 10 µl were separated on SDS (0.1%), containing polyacrylamide gels (12 15%) using standard methods (Ausubel, Brent et al Current Protocols in Molecular Biology (New York, John Wiley & Sons), 1994).

For immunoblot, the gels were transferred to polyvinyldene difluoride membranes (PVDF) (Immobilon-P; Millipore) using a device semi-dry electrophoresis transfer (Bio-Rad). For the immunodetection of proteins, an anti-tag monoclonal antibody of peroxidase-conjugated histidine (Clontech) was diluted 1 to 5000 in blocking buffer (PBS, Tween-20 0.1%, 3% skimmed milk) Anti-SUMO antibodies (anti-GMP1) was obtained from Zymed and used at a 1/2000 dilution. IgG anti-mouse conjugates peroxidase (Boehringer Manheim) was used as an antibody secondary to a 1/10000 dilution. For the detection of GST, used anti-GST peroxidase conjugates (Amersham Biosciences) at a 1/5000 dilution. For the development of membranes, the chemiluminescence substrate was used to BM immunoblot from Roche. After 1 minute incubation, the membranes were exposed to x-ray films.

In Figure 1 you can see that in the lines of control, 1 to 4, that the corresponding expression occurs  proteins (SUMO and Aos1) and their respective mobilities are They correspond to their molecular weight. When the system was expressed integer (lane 5), many proteins were found along the band that are linked with the histidine sequence. A model very similar bands was observed when the experiment with an untagged version of SUMO and we probed the immunoblot with anti-SUMO antibodies (no shown).

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Example 3

Protein purification

For protein purification, they were induced 50 ml of cultures (OD 0.7) for 4 hours with 0.5 mM IPTG, subsequently the cells were harvested by centrifugation. To carry out purification under conditions denaturing, the cells were lysed in Buffer B (10 mM of Phosphate buffer pH 8, 8 M Urea, 300 mM NaCl), the sample was centrifuged at 14000G for 10 minutes, the residue was discarded and the supernatant was placed on a column of TALON resin, the column was washed with 20 volumes of Buffer C (same as Buffer B but pH 6.3), and the proteins were eluted with 3 elution buffer volumes (same as buffer C, supplemented with 150 mM imidazole). To purify the proteins under native conditions, the cells were lysed by sonication in 1 ml of Buffer A (PBS 2X) plus 1% Triton X-100, 1 mM β-mercaptoethanol, 1 mM PMSF and 5 mM imidazole in case the proteins were marked with his. The lysate was centrifuged and the waste discarded. 300 µl of the supernatant for 2 hours at 4 ° C with a bed of 50 µl of the corresponding resins, TALON (Clontech) for proteins with histidine tag and Glutation-Sepharose 4B (Amersham Biosciences) for GST derivatives, in one volume 1 ml final obtained by adding 600 µl of Buffer B. The resin was washed 3 times with 1 ml of Buffer B and proteins were eluted by the addition of 150 µl of Buffer B plus 150 mM of imidazole for TALON resin and Buffer B plus 10 mM Glutathione reduced in the case of GST mergers. Eluted proteins were mixed 1: 1 with elution buffer and stored at -20 ° C.

The results are shown in Figure 5, finding that all purifications were performed with sufficient yields to be detected by staining with blue from Coomasie. The bands marked with arrows show those positions detected using antibodies anti-histidine or anti-GST respectively and also detected with the antibody anti-sum. Therefore, this indicates that sumoylated proteins can be purified under conditions native to obtain high amounts of protein, being the way monosumoilada the most abundant.

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Example 4

LEF1 transcription factor sumoylation

In order to use the most common plasmids used to express proteins in E. coli (colE1 replicon, with ampicillin resistance), we move the constructs to express E1 and E2 to a vector with the p15A replicon and chloramphenicol resistance and the gene SUMO without tag was cloned into a vector with the RK2 replicon and kanamycin resistance. The transcription factor LEF1 fused with the histidine sequence was used as the sumoylation target. As shown in Figure 3, when SUMO and LEF1 were expressed in the absence of the rest of the machinery, the band corresponding to LEF1 was detected, but when all the machinery plus SUMO plus LEF1 was expressed, the appearance of additional number of bands. One of them corresponds to the molecular weight of LEF1 conjugated to two SUMO molecules and the rest of the bands at the top of the line can be additions of SUMO to the SUMO molecule already conjugated. So the components of the sumoylation machinery of our system are recognizing the target protein and performing the sumoylation on it.

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Example 5

Fusion protein sumoylation

It has been shown in vitro that some peptides containing the consensus sequence necessary for sumoylation can be fused to the GST C-terminal for sumoylation. For this purpose, two fusions of GST to I? B peptides (SEQ ID NO 15) and PML (SEQ ID NO 16) were constructed. As shown in Figure 4, in cases where the sumoylation of the fusions between the peptide and GST was expressed a different pattern of bands appeared compared to the lines without GST or GST without the fusion of the sumoilable peptide, when analyzed with an anti-SUMO antibody. The new bands corresponded to GST conjugated to one or two SUMO molecules. In the Coomasie blue staining of the SDS-PAGE gel, the bands corresponding to GST and the monosumoilated forms can be seen. This indicates that the sumoylation is quantifiable and can be used to purify large amounts of sumoilable proteins.

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Example 6

Mass spectrometry

To confirm the conjugation of SUMO to LEF1 and to GST derivatives, a Mass Spectrometry was performed on the bands marked on coomasie gels. The results shown in figure 6, confirm the presence of peptides triptychs corresponding to SUMO protein in the bands with an electrophoretic mobility corresponding to the sum of the molecular masses of SUMO plus LEF1 or SUMO plus GST. Together with the SUMO peptides were detected a series of corresponding peptides  unequivocally to LEF1 and GST derivatives respectively.

Protein gel digestion

Protein bands were cut manually and then automatically processed using a Investigator? ProGest protein digestion station (Genomic Solutions, Cambridgeshire, UK) (Houthaeve et al ., FEBS Lett 1995 376, 91-94). The digestion protocol used was that described in (Shevchenko, A et al ., Anal Chem 1996 68, 850-858).

Obtaining the peptide pattern by MALDI and searching the databases

An aliquot of 0.3 ml of solution was deposited matrix (5 g / l 2,5-dihydroxybenzoic acid in 33% acetonitrile-water and 0.1% trifluoro acid acetic) in a probe for MALDI AnchorChip? 400 mm and allowed to dry at room temperature. Then 0.3 ml was added of the extraction solution mentioned above and again left dry at room temperature.

The samples were measured on a spectrometer of Bruker Reflex ™ IV MALDI-TOF masses (Bruker-Franzen Analytic GmbH, Bremen, Germany) equipped with SCOUT ™ source in ion reflector mode positive using delayed extraction. Acceleration voltage of ions was 20 kV. The device was first calibrated externally using protonated mass signals that covered the 1000-3200 m / z range and subsequently each spectrum it was calibrated internally using selected signals obtained of trypsin self-protection to achieve accuracy typical for mass measurements of ± 30 ppm. Mass measures of tryptic peptides were transferred through the MS program BioTools ™ to perform a search in the database NCBInr using the Mascot ™ software (Matrix Science, London, UK). No restrictions were imposed on the species of protein origin and allowed molecular masses were between 1 and 200 kDal. A lack of a trypsin cut was allowed and 50 ppm accuracy was used in peptide searches triptychs

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25

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27

28

29

30

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31

32

33

3. 4

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Four. Five

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54

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<400> 16

55

Claims (40)

1. An in vivo sumoylation method of sumoylation susceptible proteins characterized in that it comprises the following steps:

to)

the transformation of the host cell by introducing The components of the eukaryotic sumoylation machinery E1, E2, a DNA sequence encoding eukaryotic SUMO or any of its derivatives, and a DNA sequence that encodes the protein susceptible to sumoylation under the action of a suitable promoter for its expression in one or more expression vectors and,

b)

its subsequent induction.

2. The method according to claim 1, characterized in that it comprises:

to)

the transformation of the host cell with an expression system gene consisting of:

a.1.-

an expression vector containing the sequence of DNA coding for the machinery components of eukaryotic sumoylation E1 (Aos1 and Uba2 proteins) and E2 (ubc9 protein),

a.2.-

an expression vector containing the sequence of DNA encoding eukaryotic SUMO or any of its derivatives, Y

a.3.-

an expression vector containing the sequence of DNA encoding the protein susceptible to sumoylation,

all of them under the action of a promoter / s is suitable for its expression, Y

b)

its subsequent induction.

3. The method according to any of the preceding claims 1 to 2, characterized in that purification of any of the system components is not necessary. 4. The method according to any one of the preceding claims 1 to 3, characterized in that the host cell is an E. coli cell. 5. The method according to any of the preceding claims 1 to 4, characterized in that the promoter can be a constitutive or regulated promoter. 6. The method according to claim 5, characterized in that the promoter is selected from the group consisting of T7, TRC, Ptac and Plac. The method according to any one of the preceding claims 1 to 6, characterized in that the expression vector containing a DNA sequence encoding SUMO or any of its derivatives also contains a nucleic acid sequence encoding a fused amino acid sequence. to the sequence encoding the SUMO molecule. 8. The method according to claim 7, characterized in that the amino acid sequence fused to the sequence encoding the SUMO molecule is selected from the group consisting of polyhistidine, Strep, E-tag, HA, FLAG, GST, calmodulin binding protein, maltose binding protein, cellulose binding protein, thioredoxin, antibody type molecules, enzymes, fluorescent proteins, kinases, proteases, DNA binding proteins, RNA binding proteins, glycosyltransferases, membrane proteins , serum albumin, toxins. 9. A method of obtaining, separating and purifying proteins susceptible to sumoylation, characterized in that it comprises the following steps:

to)

the transformation of the host cell by introducing the components of the sumoylation machinery E1, E2 and a DNA sequence encoding SUMO or any of its derivatives under the action of a promoter for expression in one or more expression vectors and;

b)

its subsequent induction.

10. The method according to claim 9, characterized in that it comprises the transformation of a host cell with an expression vector containing the components of the sumoylation machinery E1 and E2, together with another expression vector containing a sequence of DNA encoding SUMO or any of its derivatives under the action of a promoter of its expression and subsequent induction.

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11. The method according to any of the preceding claims 9 to 10, characterized in that purification of any of the system components is not necessary. 12. The method according to any of the preceding claims 9 to 11, characterized in that the host cell is an E. coli cell . 13. The method according to any of the preceding claims 9 to 12, characterized in that the promoter can be a constitutive or regulated promoter. 14. The method according to claim 13, characterized in that the promoter is selected from the group consisting of T7, TRC, Ptac and Plac. 15. The method according to any of the preceding claims 9 to 14, characterized in that the expression vector containing a DNA sequence encoding SUMO or any of its derivatives also contains a nucleic acid sequence encoding a given amino acid sequence. , for the subsequent purification of said vector, fused to the sequence encoding the SUMO molecule. 16. The method according to claim 15, characterized in that the purification amino acid sequence is selected from the group consisting of polyhistidine, Strep, E-tag, HA, FLAG, GST, calmodulin binding protein, binding protein to maltose, cellulose binding protein, thioredoxin. 17. A method of obtaining sumoylation chains of covalently bonded proteins through SUMO molecules, characterized in that it comprises the following steps:

to)

the transformation of the host cell by introducing the components of the sumoylation machinery E1, E2 and a DNA sequence encoding SUMO or any of its derivatives under the action of a promoter for expression in one or more expression vectors and;

b)

its subsequent induction.

18. The method according to claim 17, characterized in that it comprises the transformation of a host cell with an expression vector containing the components of the sumoylation machinery E1 and E2, together with another expression vector containing a sequence of DNA encoding SUMO or any of its derivatives under the action of a promoter of its expression and subsequent induction. 19. The method according to any of the preceding claims 17 to 18, characterized in that purification of any of the system components is not necessary. 20. The method according to any of the preceding claims 17 to 19, characterized in that the host cell is an E. coli cell . 21. The method according to any of the preceding claims 17 to 20, characterized in that the promoter can be a constitutive or regulated promoter. 22. The method according to claim 21, characterized in that the promoter is selected from the group consisting of T7, TRC, Ptac and Plac. 23. The method according to any one of the preceding claims 17 to 21, characterized in that the expression vector containing a DNA sequence encoding SUMO or any of its derivatives also contains a nucleic acid sequence encoding a given amino acid sequence. , fused to the sequence encoding the SUMO molecule. 24. The method according to claim 23, characterized in that the amino acid sequence is selected from the group consisting of polyhistidine, Strep, E-tag, HA, FLAG, GST, calmodulin binding protein, maltose binding protein , cellulose binding protein, thioredoxin, antibody type molecules, enzymes, fluorescent proteins, kinases, proteases, DNA binding proteins, RNA binding proteins, glycosyltransferases, membrane proteins, serum albumin, toxins. 25. A method to increase the levels of expression of a sumoylation protein of interest in a host cell comprising:

to)

Insert into said host cell a DNA sequence that encodes SUMO or any of its derivatives under the action of a promoter of their expression, together with E1 and E2 proteins;

b)

promoter induction.

26. The method according to claim 25, characterized in that it comprises the transformation of a host cell with an expression vector containing the components of the sumoylation machinery E1 and E2, together with another expression vector containing a sequence of DNA encoding SUMO or any of its derivatives under the action of a promoter of its expression and subsequent induction. 27. The method according to any of the preceding claims 25 to 26, characterized in that purification of any of the system components is not necessary. 28. The method according to any one of the preceding claims 25 to 27, characterized in that said host cell is E. coli. 29. The method according to any of the preceding claims 25 to 28, characterized in that the promoter can be a constitutive or regulated promoter. 30. The method according to claim 29, characterized in that the promoter is selected from the group consisting of T7, TRC, Ptac and Plac. 31. The method according to any one of the preceding claims 25 to 30, characterized in that the expression vector containing a DNA sequence encoding SUMO or any of its derivatives also contains a nucleic acid sequence encoding a given amino acid sequence. , fused to the sequence encoding the SUMO molecule. 32. The method according to claim 31, characterized in that the amino acid sequence is selected from the group consisting of polyhistidine, Strep, E-tag, HA, FLAG, GST, calmodulin binding protein, maltose binding protein , cellulose binding protein, thioredoxin, antibody type molecules, enzymes, fluorescent proteins, kinases, proteases, DNA binding proteins, RNA binding proteins, glycosyltransferases, membrane proteins, serum albumin, toxins. 33. A kit comprising an expression vector which contains the components of the sumoylation machinery El and E2, together with another expression vector that contains a sequence of DNA encoding SUMO or any of its derivatives under the action of a promoter of expression, and a multiple site of cloning capable of cloning a nucleic acid encoding the protein of interest in the framework of the nucleic acid sequence encoding the SUMO molecule. 34. The kit according to claim 33, characterized in that the promoter can be a constitutive or regulated promoter. 35. The kit according to claim 34, characterized in that the promoter is selected from the group consisting of T7, TRC, Ptac and Plac. 36. The kit according to any of the preceding claims 33 to 35, characterized in that it also contains a composition containing a protease or proteases capable of releasing the SUMO molecule from the fusion protein. 37. The kit according to any one of the preceding claims 33 to 36, characterized in that it further comprises host cells suitable for the expression of said vector. 38. The kit according to claim 37, characterized in that said host cells are from E. coli. 39. The kit according to any one of the preceding claims 33 to 38, characterized in that the expression vector may also contain a nucleic acid sequence encoding a particular amino acid sequence, fused to the sequence encoding the SUMO molecule. 40. The kit according to claim 39, characterized in that the amino acid sequence is selected from the group consisting of polyhistidine, Strep, E-tag, HA, FLAG, GST, calmodulin binding protein, maltose binding protein , cellulose binding protein, thioredoxin, antibody type molecules, enzymes, fluorescent proteins, kinases, proteases, DNA binding proteins, RNA binding proteins, glycosyltransferases, membrane proteins, serum albumin, toxins.