Recombinant hybrid protease, DNA nucleotide sequence of said recombinant hybrid protease, process for obtaining said recombinant hybrid protease, and use thereof

Abstract

The present invention relates to a recombinant hybrid protease comprising fusion of the tobacco etch virus (TEV) protease with the catalytic site of the ULP1 protease, joined by a flexible linker. Additionally, the present invention relates to the DNA nucleotide sequence of said recombinant hybrid protease. Furthermore, the present invention relates to a process for obtaining said recombinant hybrid protease, comprising the steps of: (a) constructing the DNA nucleotide sequence of the recombinant hybrid protease of the present invention; (b) optimizing the codons for expression in yeast; (c) cloning the sequence obtained in step "b" into a bacterial vector; (d) expressing the hybrid protease in chemically competent E. coli bacteria; (e) purifying the hybrid protease after expression by affinity chromatography; and (f) dialyzing the hybrid protease in a dialysis buffer (50 mM Tris-HCl, pH 8.0; 0.2% Igepal; 300 mM NaCl; 1 mM DTT) to obtain it in soluble form. Finally, the present invention also relates to the use of said hybrid protease for removing fusion proteins from different proteins of interest, whether for research or clinical use.

Description

Recombinant Hybrid Protease, DNA Nucleotide Sequence of Said Recombinant Hybrid Protease, Process for Obtaining Said Recombinant Hybrid Protease and its Use. Field of Application:

[0001] The present invention falls within the field of genetic engineering, more specifically, in the area of ​​hybrid peptides, since it refers to a recombinant hybrid protease comprising the fusion of the tobacco etch virus (TEV) protease with the catalytic site of the ULP1 protease linked by a flexible linker. The present invention also refers to its DNA nucleotide sequence, the process for obtaining said hybrid protease, and its use. Background of the Invention and State of the Art:

[0002] The implementation of recombinant protein technology has revolutionized all aspects of biological sciences, as it has expanded the number of proteins that can be studied biochemically and structurally. Currently, it is possible to synthesize or isolate genes and clone them into an expression system suitable for large-scale production.

[0003] However, the production of soluble proteins is a common obstacle for many researchers. Therefore, there have been advances in optimizing the expression of recombinant proteins, including the development of fusion proteins. Some fusion proteins stand out for improving solubility, while others aid in the purification of recombinant proteins.

[0004] However, the presence of these affinity markers can affect important functions of the protein of interest. Therefore, efficient and reliable removal of the fusion protein after expression is necessary. Among the fusion proteins most commonly used for this purpose are maltose-binding protein (MBP), glutathione S-transferase (GST), and the fusion tail. of histidine (His-tag), Thyrodoxin (Trx), Transcription elongation protein NusA and the ubiquitin-like modifying protein (SUMO).

[0005] Currently, the most widely used protease for removing fusion proteins is the tobacco etch virus (TEV) protease, which recognizes a site composed of seven amino acids (ENLYFQG / S) and cleaves between the G and S residues. Thus, after cleavage of this site, the protein of interest will be released. Another protease for removing fusion proteins is SUMO 1 protease (Ulp1), which is specific for the ubiquitin-like modifier protein (SUMO), and is also widely used to aid in the solubilization of recombinant proteins. Therefore, the SUMO fusion protein can be removed from the protein of interest without the need to add a TEV site.

[0006] However, currently the proteases TEV (Thermofisher- 12575015) and ULP1 (Thermofisher- 12588018) are sold at high prices, since this involves the production costs of each protease (expression, purification and packaging).

[0007] In order to solve the existing technical problem, the present invention proposes obtaining a hybrid protease with a dual function, enabling both the cleavage of the TEV site and the SUMO fusion protein, thus reducing production costs, since it will involve only one expression, purification and packaging of the hybrid protease.

[0008] Some prior art documents describe hybrid recombinant proteins.

[0009] U.S. Patent Application No. 2022 / 056099 A1, published February 24, 2022, in the name of SHANGRAO CONCORD PHARMACEUTICAL CO., LTD, entitled: “TEV protease variant, fusion protein thereof, preparation method therefor and use thereof” describes a fusion protein obtained using a variant of the TEV protease that further comprises a purification marker, such as the SUMO marker, wherein said marker is inserted into the N-terminal portion of the TEV protease. However, the document departs from the present invention, as it is silent regarding the development of a hybrid protease comprising exactly the TEV and Ulp1 proteases, linked by a flexible linker.

[0010] International patent application No. PCT / US2021 / 056544, published under No. WO 2022 / 093741 A1 on May 5, 2022, in the name of RESEARCH DEVELOPMENT FOUNDATION, entitled: “Mutant proteases and uses thereof” provides mutant TEV proteases that exhibit enhanced activity, wherein the mutant TEV exhibits increased efficiency and / or a K catincreased capacity for cleavage of an amino acid sequence. Although the mutant protease TEV exhibits 97.84% identity with the TEV protease used in the hybrid protease of the present invention, the aforementioned international patent application is silent regarding the development of a hybrid protease comprising exactly the TEV and Ulp1 proteases, linked by a flexible linker, thus differing from the present invention.

[0011] Chinese patent application No. CN 102234640 A, published on November 9, 2011, in the name of HARBIN HAKELONG BIOLOGICAL PHARMACEUTICAL INSTITUTE, entitled: “Recombinant small ubiquitin-related modifier (SUMO) protease and preparation method as well as application thereof” describes a recombinant protease related to the ubiquitin-type modifier protein (SUMO) and a method of preparation, as well as its application. The recombinant protease is composed of a GST (glutathione-S-transferase) marker, a Ulp1 sequence (SUMO protease 1), and a poly-His (histidine) marker. Although the Ulp1 amino acid sequence of The aforementioned recombinant protease shown in SEQ ID NO:2 exhibits 100% identity with the Ulp1 protease used in the hybrid protease of the present invention. However, the aforementioned Chinese application is silent regarding the development of a hybrid protease that comprises exactly the TEV and Ulp1 proteases, linked by a flexible linker, thus differing from the present invention.

[0012] Chinese patent application No. CN 116410961 A, published on July 11, 2023, in the name of CHONGQING AILIBI BIO TECH CO LTD, entitled: “Tobacco etching virus protease fusion protein as well as preparation method and application thereof” describes a fusion protein containing tobacco etch virus protease (TEV enzyme), which exhibits 97% identity with the TEV protease used in the hybrid protease of the present invention. However, like the cited prior art documents, the aforementioned Chinese document is silent regarding the development of a fusion protein that comprises exactly the TEV and Ulp1 proteases, linked by a flexible linker.

[0013] The article entitled “Effect of Linker Length and Flexibility on the Clostridium thermocellum Esterase Displayed on Bacillus subtilis Spores” by Chen H. et al., published on December 8, 2016, reveals that the linker used in the hybrid protease of the present invention is rich in glycine and serine residues that can provide flexibility, while glutamate and lysine residues can improve the solubility of the fusion protein. However, the aforementioned article is only illustrative of the state of the art, since it is silent regarding the development of a fusion protein comprising the TEV and ULP1 proteases, linked by the flexible linker.

[0014] Therefore, based on the aforementioned state of the art, obtaining new recombinant proteins is a common technical problem. Understanding the use of TEV protease or Ulp1 protease in order to reduce costs in fusion protein removal processes. In this sense, the present invention is an alternative solution to the existing technical problem of the prior art.

[0015] Due to the importance of these two proteases in removing fusion proteins, advantageously, the present invention aims to clone and express Ulp1 and TEV proteases in E. coli bacteria in a fused form containing a linker between them to allow greater folding flexibility. In this way, in a single expression, it will be possible to obtain a hybrid protease that can effectively offer a dual function, allowing both the removal of the TEV site and the SUMO fusion protein, reducing costs and time for obtaining the protease.

[0016] Thus, no prior art document reveals a hybrid protease obtained by fusing the TEV sequence with the catalytic site of ULP1, linked by a flexible linker to allow mobility between the proteases, increasing the possibilities of correct folding of said hybrid protease. Therefore, it was possible to obtain a hybrid protease (TEV / ULP1) that, using only a cleavage buffer, allowed both the cleavage of the TEV protease site (ENLYFQG / S) and the removal of SUMO (small ubiquitin-like modifier). Summary of the invention:

[0017] The present invention will provide significant advantages in melt protein removal processes.

[0018] In one aspect, the present invention relates to a recombinant hybrid protease comprising the fusion of the tobacco etch virus (TEV) protease with the catalytic site of the ULP1 protease linked by a flexible linker.

[0019] In a second aspect, the present invention relates to a recombinant hybrid protease DNA nucleotide sequence of the present invention. The invention comprises fusing the DNA sequence of the TEV protease with the catalytic site of the ULP1 protease DNA sequence, linked by a flexible linker.

[0020] In a third aspect, the present invention relates to a process for obtaining the recombinant hybrid protease of the present invention, which comprises the steps of: (a) constructing the nucleotide DNA sequence of the recombinant hybrid protease of the present invention; (b) optimizing the codons for expression in yeast; (c) cloning the sequence obtained in step “b” into a bacterial vector; (d) expressing the hybrid protease in chemically competent E. coli bacteria; (e) purifying the hybrid protease after expression by affinity chromatography; and (f) dialyzing the hybrid protease obtained in soluble form using dialysis buffer (50mM Tris-HCl, pH 8.0; 0.2% Igepal; 300mM NaCl, 1mM DTT).

[0021] In a fourth aspect, the present invention relates to the use of the hybrid protease of the present invention to remove fusion proteins from different proteins of interest, whether for research or clinical use. Brief description of the figures:

[0022] The present invention, along with its additional advantages, can be better understood by referring to the attached images and the following description.

[0023] Figure 1 illustrates the pET24b(+) vector used for cloning the hybrid protease sequence (Ulp1_TEV) into the restriction sites of the NheI and XhoI enzymes.

[0024] Figure 2A shows an SDS-PAGE gel image displaying a band corresponding to the expression of Ulp1_TEV obtained from E. coli Bl21Star (DE3). This expression was performed at 30°C with 0.1mM IPTG induction for 4h, where (M) refers to the molecular weight marker, (1) refers to the bacterial extract before IPTG induction; (2) refers to the bacterial extract after IPTG induction; and (3) refers to Ulp1_TEV. purified and dialyzed.

[0025] Figure 2B represents the purification graph performed on an AKTA Start chromatograph (Cyntiva) using a Ni-Sepharose HiisTrap column (Cyntiva) in an imidazole gradient (0-500mM). The peak represents the retention time of the recombinant Ulp1_TEV protein.

[0026] Figure 3 comprises an SDS-PAGE gel showing the digestion assay of phospholipase D (LgRec2) fused with SUMO (SUMO_LgRec2), in which the hybrid protease Ulp1_TEV was used to remove the SUMO fusion protein. The digestion reaction was carried out at 30°C for 4 hours; and (M) refers to the molecular mass marker; (1) refers to Ulp1_TEV; (2) refers to SUMO_LgREc2; and (3) refers to the digestion product of LgREc2-SUMO with the protease Ulp1_TEV, where the release of LgRec2 can be observed.

[0027] Figure 4 corresponds to an SDS-PAGE gel showing the digestion test of the recombinant Knutiz-type inhibitor fused with GST (GST_Kunitz). Due to the presence of the TEV site between the Knutiz and GST proteins, the hybrid protease Ulp1_TEV was used to remove the GST fusion protein. The digestion reaction was carried out at 30°C for 4 hours; and (M) refers to the molecular mass marker; (1) refers to Ulp1_TEV; (2) refers to Kunitz_SUMO; and (3) refers to the digestion product of LgREc2-SUMO with the Ulp1_TEV protease, where the release of LgRec2 can be observed.

[0028] Figure 5A shows an SDS-PAGE gel image displaying a band corresponding to the expression of TEV_Ulp1 obtained from E. coli Bl21Star (DE3). This expression was performed at 30°C with 0.1mM IPTG induction for 4h, where (M) refers to the molecular weight marker, (1) refers to the bacterial extract before IPTG induction; (2) refers to the bacterial extract after IPTG induction; and (3) refers to purified and dialyzed TEV_ULP1.

[0029] Figure 5B represents the purification graph performed on an AKTA Start chromatograph (Cyntiva) using a Ni-Sepharose HisTrap column (Cyntiva) in an imidazole gradient (0-500mM). The peak represents the retention time of the recombinant Ulp1_TEV protein.

[0030] Figure 6 corresponds to an SDS-PAGE gel showing the digestion test of the recombinant Knutiz-type inhibitor fused with GST (GST_Kunitz). Due to the presence of the TEV site between the Knutiz and GST proteins, the hybrid protease TEV_ULP1 was used to remove the GST fusion protein. The digestion reaction was performed at 30°C for 4 hours; and (M) refers to the molecular mass marker; (1) refers to the Knutiz protein fused with GST (presence of two bands due to protein truncation); (2) refers to TEV_Ulp1; and (3) refers to the digestion product of Kunitz-GST with the protease TEV_Ulp1, where the release of GST (blue arrow) and the two bands referring to the truncated Kunitz (red arrows) can be observed; (4) refers to Ulp1_TEV; (5) refers to the digestion product of Kunitz-GST with Ulp1_TEV protease for comparison with TEV_Ulp1, where we can also observe the release of GST (blue arrow) and the two bands referring to truncated Kunitz (red arrows).Detailed description of the invention:

[0031] Although the present invention may be susceptible to different embodiments, a preferred embodiment is shown in the following detailed discussion, with the understanding that the present embodiment should be considered an exemplification of the principles of the invention and is not intended to limit the present invention to what has been described in this report.

[0032] The present invention relates to a recombinant hybrid protease comprising the fusion of the tobacco etch virus (TEV) protease as described by SEQ ID NO: 1 with the catalytic site of the ULP1 protease as described by SEQ ID NO: 2, linked by a flexible linker.

[0033] SEQ ID NO: 1 corresponds to the amino acids of the recombinant TEV protease capable of recognizing and cleaving the ENLYFQG / S sequence. The TEV protease DNA sequence corresponds to positions 6256 to 7000 base pairs (bp) of the sequence found in the database and registered under number M15239.1. This DNA sequence (6256 to 7000 bp) was optimized for expression in yeast.

[0034] SEQ ID NO: 2 corresponds to amino acids 403 to 621 of the amino acid sequence of SUMO protease 1 (ULP1), which is specific for removing ubiquitin-like modifier protein (SUMO), also widely used to aid in the solubilization of recombinant proteins. The DNA sequence of ULP1 protease corresponds to positions 1207 to 1863 base pairs (bp) of the sequence found in the database registered under number NM_001183834.1. This sequence (1207 to 1863 bp) was optimized for expression in yeast.

[0035] The flexible linker comprises the amino acid sequence as established by SEQ ID NO: 3. This linker has the function of allowing flexibility between proteases after expression. In this way, the natural folding of one protease, in theory, would not interfere with that of the other.

[0036] It is worth noting that during the folding process, if there is rigidity between one protein and another, this can increase the chances of inactivity. Therefore, a sequence of 14 amino acids (flexible linker with SEQ ID NO: 3) was added between them, which has flexible properties, thus allowing greater mobility for both folding and facilitating their enzymatic activities.

[0037] In one embodiment, the ULP1 sequence (SEQ ID NO: 2) is inserted into the N-terminal portion of the TEV protease (SEQ ID NO: 1).

[0038] In one embodiment, the TEV protease sequence (SEQ ID NO: 1) is inserted into the N-terminal position of the ULP1 sequence (SEQ ID NO: 2).

[0039] Additionally, the SEQ ID NOs: 1 and 2 sequences are located between the amino acid pair alanine (A) and serine (S) and the amino acid pair leucine (L) and glutamate (E). These amino acids were added to the hybrid protease of the present invention due to the restriction enzymes used for cloning in the vector of interest.

[0040] Preferably, the vector of interest is pET24b(+).

[0041] Additionally, the aforementioned recombinant hybrid protease comprises 6 histidines at its tertiary end, which are derived from the vector from which the sequence is cloned. These histidines are intended to facilitate the purification of the hybrid protease by affinity chromatography using Ni-Sepharose resin.

[0042] In one embodiment of the present invention, said recombinant hybrid protease consists of the amino acid sequence as set forth by SEQ ID NO: 4.

[0043] In a preferred embodiment of the present invention, said hybrid protease comprises 479 amino acids, has a molecular mass of 54823.20 Da, and its theoretical isoelectric point (pI) is 7.79. These characteristics refer to both Ulp1_TEV and the TEV_Ulp1 construct.

[0044] Additionally, the present invention describes the recombinant hybrid protease DNA nucleotide sequence described above, which comprises the fusion of the TEV protease DNA sequence as defined by SEQ ID NO: 5 with the catalytic site of the ULP1 protease DNA sequence as defined by SEQ ID NO: 6, joined by a flexible linker.

[0045] The flexible linker preferably comprises the nucleotide sequence as established by SEQ ID NO: 7.

[0046] In a preferred embodiment of the present invention, the DNA sequence of the hybrid protease construct is cloned into the vector. bacterial pET24(+) as shown in Figure 1 between the NheI (GCTAGC nucleotides) and EcoRI (CTCGAG nucleotides) sites.

[0047] In a preferred embodiment of the present invention, the DNA sequence of the hybrid protease construct consists of the nucleotide sequence as set forth by SEQ ID NO: 8.

[0048] In addition, the present invention relates to a process for obtaining the recombinant hybrid protease described above, which comprises the steps of: a) Constructing the nucleotide DNA sequence of the recombinant hybrid protease by fusing the DNA sequence of the TEV protease as established by SEQ ID NO: 5 with the catalytic site of the DNA sequence of the ULP1 protease as established by SEQ ID NO: 6, joined by a flexible linker; b) Optimizing the codons for expression in yeast; c) Cloning the sequence obtained in step “b” into a bacterial vector; d) Expressing the hybrid protease in chemically competent E. coli bacteria; e) Purifying the hybrid protease after expression by affinity chromatography; f) Dialyzing the obtained hybrid protease in soluble form using dialysis buffer (50mM Tris-HCl, pH 8.0; 0.2% Igepal; 300mM NaCl, 1mM DTT).

[0049] Preferably, the bacterial vector for step “c” is the pET24(+) vector.

[0050] Preferably, the chemically competent E. coli bacterium from step “d” is the commercial bacterium known as One Shot™ BL21 Star™ (DE3) (Thermofisher).

[0051] In step “d”, the protein is preferentially expressed at 30°C. For 4 hours, with induction of 0.1 mM IPTG. After induction, the bacteria are disrupted by sonication and the hybrid protease is purified by affinity chromatography in the next step.

[0052] Preferably, after expression, the protease is purified in step “e” by affinity chromatography using Ni-sepharose columns using the ÄKTA start chromatography system (Cytiva).

[0053] Preferably, after the dialysis performed in step “f”, the present process comprises an additional step “g” which refers to the addition of 50% glycerol to the hybrid protease for freezing at -20ºC. For this purpose, freezing is carried out at concentrations ranging from 200 µg / mL to 600 µg / mL.

[0054] In this context, it is worth noting that, in general, both TEV protease and ULP1 are described in the literature as having insolubility problems. In this sense, initially, the hybrid protease of the present invention also presented these problems, but after tests with different dialysis and freezing buffers, this problem was overcome, as described in the process of the present invention.

[0055] Thus, through the process described here, it was possible to obtain a stable hybrid protease capable of both performing the activities of the Ulp1 protease (removal of the SUMO fusion protein) and cleaving TEV protease sites (ENLYFQG / S).

[0056] Additionally, the present invention relates to the use of the hybrid protease of the present invention to remove fusion proteins from different proteins of interest, whether for research or clinical use.

[0057] Therefore, in order to elucidate the present invention, experimental results and embodiments of the invention are presented below to demonstrate the inventive step of using the hybrid protease of the present invention. Embodiments of the invention

[0058] In one embodiment of the present invention, the hybrid protease was obtained by fusing the TEV sequence (SEQ ID NO: 1) with the catalytic site of ULP1 (SEQ ID NO: 2), linked by a flexible linker (SEQ ID NO: 3) to allow flexibility between the proteases. This construct had its codon optimized for expression in yeast, but was cloned into the bacterial vector pET24(+). The hybrid protease was expressed in chemically competent E. coli bacteria One Shot™ BL21 Star™ (DE3). After expression, the protease was purified by affinity chromatography using Ni-sepharose columns using the ÄKTA start purification system with a flow rate of 1 mL / minute, a pressure of 0.3 MPa, and an imidazole gradient of 0 to 500 mM.

[0059] The protease was obtained in soluble form, and after dialysis in phosphate-buffered saline (PBS), more than 60% of the protein precipitated. However, after trials with different buffers, dialysis buffer (50mM Tris-HCl, pH 8.0; 0.2% Igepal; 300mM NaCl, 1mM DTT) was used, which was able to reduce precipitation to less than 10%.

[0060] After achieving stability of the hybrid protease, initial tests were conducted to analyze its activity in cleaving the TEV site (ENLYFQG / S) placed between a fusion protein (Glutathione S-transferase (GST)) and a Kunitz-type inhibitor (GST-Kunitz). Subsequently, the protease was tested for its ability to remove SUMO fused with phospholipase D (SUMO-LgRec2), and complete cleavage of both the Kunitz-type inhibitor and phospholipase D from their fusion proteins (GST and SUMO) was verified. Implementation of the Ulp1_linker_TEV hybrid protease: The protease ULP1 (SEQ ID NO: 2) is inserted into the N-terminal portion of the protease TEV (SEQ ID NO: 1).

[0062] In one embodiment of the present invention, the protein sequence of the recombinant hybrid protease is represented below. (Ulp1_linker_TEV – SEQ ID NO:4). - SEQ ID NO: 4 ASEFGSLVPELNEKDDDQVQKALASRENTQLMNRDNIEITVRDFKTLAPR RWLNDTIIEFFMKYIEKSTPNTVAFNSFFYTNLSERGYQGVRRWMKRKKT QIDKLDKIFTPINLNQSHWALGIIDLKKKTIGYVDSLSNGPNAMSFAILTDL QKYVMEESKHTIGEDFDLIHLDCPQQPNGYDCGIYVCMNTLYGSADAPL DFDYKDAIRMRRFIAHLILTDALKEGKSSGSGSESKSTGESLFKGPRDYN PISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVF KVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTN FQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGI HSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHK VFMDKPEEPFQPVKEATQLMNSRLEHHHHHH

[0063] The amino acids added due to the restriction enzymes used for cloning in pET24b(+) are indicated in bold. In the N-terminal portion, we have the amino acids ASEFGS before the Ulp1 sequence, and in the C-terminal portion, the amino acids SRLE after the TEV sequence. In italics is the amino acid sequence of the catalytic domain of the Ulp1 protease (corresponding to amino acids 403 to 621). In bold and underlined is the protein sequence of the linker that connects the two proteases; in italics and underlined is the amino acid sequence of the TEV protease; and in italics and bold are the 6 histidines (from the vector in which the sequence was cloned). These histidines are intended to provide purification of the hybrid protease by affinity chromatography using Ni-Sepharose resin.

[0064] Below is the DNA sequence of the hybrid protease construct named Ulp1_linker_TEV, which was cloned into the bacterial vector pET24(+) (Figure 1). - SEQ ID NO: 8 GCTAGCGAATTCGGATCCTTGGTTCCAGAGTTGAATGAAAAAGATGA TGACCAGGTGAAAAAGCCTTGGCGTCCCGTGAAATACCCAGTTGAT GAATCGTGACAATATCGAAATCACCGTTCGGTATTTCAAGACCTTGGCT CCCGGTCGTTGGCTGAATGATACCATTATCGAGTTTTTTCATGAAGTACA TTGAAAAGAGCACCCCGAACACCGTTGCTTTTTAACAC CAATCTGAGCGAACGTGGTTACCAAGGTGTTTCGTCGTTGGATGAAGCG TAAGAAACCCCAGATCGACAAGCTGGATAGATTTTTACCCCGATCAAAT CTGAACCAAAGCCACTGGGCTCTGGGTATTATCGACCTGAAAAAGAAG ACCATTGGTTACGTTGACAGCTTGAGCAATGGTCCGAATGCGATGCT TTGCGATCCTGACCGATTTGCAAAAGTACGTTATGGAGGAAAGCAAGCA TACCATTGGTGGAAGACTTCGACCTGATCCACCTGGATTGCCCGCAGCA ACCGAACGGTTACGATTGCGGTATCTACGTTTGCATGAATACCCTGTAT GGTAGCGCGGACGCTCCGCTGGATTTTGACTACAAGGATGCGATTCGT ATGCGTCGTTTTATCGCGCATCTGATCTTGACCGATGCTCTGAAAGAG GGTAAAAGCAGCGGTAGCGGTAGCGAAAGCAAAGCACCGGT GAAAGCTTGTTTCAAGGGTCCGCGTGATTACAACCCGATCAGCAGCA CCATTTGCCACTTGACCAATGAAAGCGACGGTCATACCACCAGCTTG TACGGTATCGGTTTCGGTCCGTTTTATTACCCAACAAGCACTTGTT CCGTCGTAAACAATGGTACCTTGCTGGTTCAAAGCTTGCACGGTGTT TTAAGGTTAAGAAACACCACCACCTTGCAACAGCATTTGATTGATGGT CGTGACATGATCATCATTCGTATGCCGAAGGATTTCCCGCCGTTTCCCGCAAAAGCTGAAGTTCCGTGAACCGCAACGTGAGGAACGTATTTGC CTGGTTACCACCAATTTCCAGACCAAAAGCATGAGCAGCATGGTTAG CGATACCAGCTGCACCTTTCCGAGCAGCGATGGTATCTTCTGGAAAC ACTGGATTCAGACCAAAGACGGTCAGTGCGGTAGCCCGTTGGTTAG CACCCGTGATGGTTTCATCGTTGGTATCCATAGCGCGAGCAATTTTA CCAACACCAACAATTATTTCACCAGCGTTCCGAAGAACTTCATGGAG CTGTTGACCAACCAAGAGGCTCAGCAATGGGTTAGCGGTTGGCGTT TGAATGCTGATAGCGTTTTGTGGGGTGGTCATAAGGTTTTTATGGAC AAACCAGAAGAGCCGTTCCAGCCAGTGAAAGAAGCGACCCAGTTGA TGAATTCTAGACTCGAG

[0065] Underlined in bold in the 5' portion are the restriction enzymes NheI (gctagc), EcoRI (gaattc), and BamHI (ggatcc), respectively. In the 3' portion are the enzymes XbaI (tctaga) and Xho I (gagctc), respectively. The Ulp1 domain sequence, corresponding to positions 1207 to 1863 base pairs (bp) of the GenBank sequence NM_001183834.1, is highlighted in italics. The sequence encoding the flexible ligand is underlined in bold, and the sequence of the tobacco etch virus protease TEV, corresponding to positions 6256 to 7000 base pairs (bp) of the GenBank sequence M15239.1, is also in italics and bold. As previously mentioned, this sequence was optimized for expression in yeast, but cloned into the bacterial vector pET24(+) and expressed in E. coli Bl21(DE3). Implementation of the hybrid protease TEV_linker_Ulp1_:

[0066] In another embodiment, the TEV protease sequence (SEQ ID NO: 1) is inserted into the N-terminal portion of the ULP1 protease sequence (SEQ ID NO: 2).

[0067] Abaixo represents a sequence of protection against the recombinant hibernating protease (TEV_linker_Ulp1 – SEQ ID NO:9). - SEQ ID NO: 9 ASEFGSGESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKH LFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQ KLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTK DGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQ WVSGWRLNADSVLWGGHKVFMDKPEEPFQPVKEATQLMNEGKSSGSG SESKSTLVPELNEKDDDQVQKALASRENTQLMNRDNIEITVRDFKTLAPR RWLNDTIIEFFMKYIEKSTPNTVAFNSFFYTNLSERGYQGVRRWMKRKKT QIDKLDKIFTPINLNQSHWALGIIDLKKTIGYVDSLSNGPNAMSFAILTDL QKYVMEESKHTIGEDFDLIHLDCPQQPNGYDCGIYVCMNTLYGSADAPL DFDYKDAIRMRRFIAHLILTDALKSRLEHHHHHH

[0068] The amino acids added due to the restriction enzymes used for cloning in pET24b(+) are indicated in bold. In the N-terminal portion, we have the amino acids ASEFGS before the Ulp1 sequence, and in the C-terminal portion, the amino acids SRLE after the TEV sequence. The amino acid sequence of the TEV protease is in italics and underlined, and the protein sequence of the linker that connects the two proteases is in bold and underlined. The amino acid sequence of the catalytic domain of the Ulp1 protease (corresponding to amino acids 403 to 621) is also in italics. Following this, in italics and bold, are the 6 histidines (from the vector in which the sequence was cloned). These histidines are intended to purify the hybrid protease using affinity chromatography with Ni-Sepharose resin.

[0069] Below is depicted the DNA sequence of the construct of the said hybrid protease called TEV_linker_Ulp1 which was cloned into the bacterial vector pET24(+). - SEQ ID NO: 10 GCTAGCGGTGAAAGCTTGTCCAAGGGTCCGCGTGATTACAACCCGA TCAGCAGCACCATTTGCCACTTGACCAATGAAAGCGACGGTCATACC ACCAGCTTGTACGGTATCGGTTTCGGTCCGTTTTATCACCAACAA GCACTTGTTCCGTCGTGACAATGGTTGCTTAGTCTAGTCTA ACGGTGTTTTTAAGGTTAAGAACACCACCACCTTGCAACAGCATTTG ATTGATGGTCGTGACATGATCATCATTCGTATGCCGAAGGATTTCCC GCCGTTTCCGCAAAAGCTGAAGTTCCGTGAACCGCAACGTGAGGAA CGTATTTGCCTGGTTACCACCAATTCCAGACCAAGAGCATGAGCAT CATGGTTAGCGATACCAGCTGCACCTTTCCGAGCAGCGATGGTATCT TCTGGAAACACTGGATTCAGACCAAAGACGGTCAGTGCGGTAGCCC GTTGGTTAGCACCCGTGATGGTTTCATCGTTGGTATCCATAGCGA TTCATGGAGCTGTTGACCAACCAAGAGGCTCAGCAATGGGTTAGCG GTTGGCGTTTGAATGCTGATAGCGTTTTGTGGGGTGGTCATAAGGTT TTTATGGACAAACCAGAAGAGCCGTTCCAGCCAGTGAAAGAAGCGA CCCAGTTGATGAATGAGGGTAAAAGCAGCGGTAGCGGTAGCGA AAGCAAAAGCACCGAATTCGGATCCTTGGTTCCAGAGTTGAATGAA AAAGATGATGACCAGGTGCAAAAAGCCTTGGCGTCCCGTGAAAATACC CAGTTGATGAATCGTGACAATATCGAAATCACCGTTCGTGATTTCAAGA CCTTGGCTCCGCGTCGTTGGCTGAATGATACCATTATCGAGTTTTTCAT GAAGTACATTGAAAAGAGCACCCCGAACACCGTTGCTTTTAACAGCTTC TTTTACACCAATCTGAGCGAACGTGGTTACCAAGGTGTTCGTCGTTGGA TGAAGCGTAAGAAAACCCAGATCGACAAGCTGGATAAGATTTTTACCCC GATCAATCTGAACCAAAGCCACTGGGCTCTGGGTATTATCGACCTGAAA AAGAAGACCATTGGTTACGTTGACAGCTTGAGCAATGGTCCGAATGCG ATGAGCTTTGCGATCCTGACCGATTTGCAAAAGTACGTTATGGAGGAAA GCAAGCATACCATTGGTGAAGACTTCGACCTGATCCACCTGGATTGCC CGCAGCAACCGAACGGTTACGATTGCGGTATCTACGTTTGCATGAATAC CCTGTATGGTAGCGCGGACGCTCCGCTGGATTTTGACTACAAGGATGC GATTCGTATGCGTCGTTTTATCGCGCATCTGATCTTGACCGATGCTCTG AAATCTAGACTCGAG

[0070] In bold at the 5' end, we have the restriction enzymes NheI (gctagc), EcoRI (gaattc), and BamHI (ggatcc), respectively. At the 3' end, the enzymes XbaI (tctaga) and XhoI (gagctc), respectively. In italics and bold, the sequence of the TEV protease from the tobacco etch virus – corresponding to positions 6256 to 7000 base pairs (bp) of the GenBank sequence: M15239.1. In bold and underlined, we have the sequence that encodes the flexible ligand. Following that, in italics, is the highlighted sequence of the Ulp1 domain, which corresponds to positions 1207 to 1863 base pairs (bp) of the GenBank sequence: NM_001183834.1. As previously mentioned, this sequence was optimized for expression in yeast, but cloned into the bacterial vector pET24(+) and expressed in E. coli Bl21(DE3).

[0071] After cloning both the Ulp1_linker_TEV and TEV_linker_Ulp1 sequences into the pET24b(+) vector, this was transformed into E. coli Bl21Star (Thermofisher) bacteria and the protein was expressed at 30°C for 4h with 0.1 mM IPTG induction. After induction, the bacteria were disrupted by sonication and the hybrid protease was purified by affinity chromatography. In the gel shown in Figure 2, the expression of Ulp1_linker_TEV can be observed, while in Figure 5, we can observe the expression of TEV_linker_Ulp1, where we also observe the presence of a smaller band (arrow).

[0072] After purification, both proteases Ulp1_linker_TEV and TEV_linker_Ulp1 showed instability after being dialyzed with PBS (phosphate-saline) buffer commonly used for this purpose. However, after testing with other dialysis buffers, an ideal buffer was found (50mM Tris-HCl, pH 8.0; 0.2% Igepal; 300mM NaCl, 1mM DTT) that stabilized their solubility.

[0073] It is important to highlight that the only difference between the proteases Ulp1_linker_TEV and TEV_linker_Ulp1 is that, after purification by affinity chromatography, the protease TEV_linker_Ulp1 (~60 kDa) also showed a smaller band (~25 kDa), possibly some partial degradation of TEV_linker_Ulp1. It is noteworthy that this does not occur with the hybrid protease Ulp1_linker_Tev. Experimental results

[0074] The activity of the hybrid protease was initially tested in order to evaluate the ability of the ULP1 fraction, present in the hybrid protease, to remove the SUMO fusion protein from the protein of interest.

[0075] For this purpose, a phospholipase D cloned from the venom gland of the Loxosceles gaucho spider was used as a model protein of interest; this protein needed to be fused to the SUMO protein to be expressed. This fused phospholipase was named SUMO_LgRec2, which It was also expressed in E. coli Bl21Star(DE3) bacteria.

[0076] As can be seen in Figure 3, ULP1_linker_TEV was able to cleave SUMO fused to phospholipase D (column 2) and promote its release (column 3). We also observed that the protease TEV_liker_Ulp1 also showed the ability to cleave the TEV site when incubated with the Kuniz protein fused to GST containing a TEV site (Figure 6).

[0077] After verifying that the ULP1 portion of the hybrid protease of the present invention was active, the activity of the TEV portion was verified.

[0078] To this end, a Kunitz-type inhibitor cloned from the salivary gland of the insect Simulium pertinax was used as a model and fused to the fusion protein Glutathione S-transferase (GST) to obtain this protein in soluble form. A recognition site (ENLYFQG / S) for the protease TEV was inserted between the Kunitz inhibitor and GST. In this way, after cleavage, the recombinant Kunitz inhibitor would be released from the fusion with GST.

[0079] As can be seen in Figure 4, the TEV fraction present in the hybrid protease (ULP1_linker_TEV) was able to cleave the ENLYFQG / S site that was added between GST and the Kunitz inhibitor, thus releasing the Kunitz inhibitor from GST. In Figure 5 (line 3) we can observe that the protease TEV_linker__Ulp1 was also able to perform this cleavage in a similar way to the protease Ulp1_linker_TEV (line 5).

[0080] Therefore, through the process of the present invention, it was possible to obtain a hybrid protease comprising TEV and ULP1 proteases with the effective ability to remove fusion proteins that possess the TEV protease recognition site (ENLYFQG / S) before the protein of interest and / or the SUMO fusion protein bound to the protein of interest.

[0081] It is worth noting that when one protein fuses with another, Precipitation (insolubility) of the new hybrid protein can occur, especially when the proteins involved contain cysteine ​​residues. These cysteines tend to form bonds; therefore, a cysteine ​​from one protein can bond with cysteines from the other molecule, leading to the inactivity of both. Furthermore, even if a soluble hybrid protein is obtained, one or both may cease to exhibit activity due to hydrophobic interactions between their amino acids and / or the aforementioned cysteine ​​bonds.

[0082] In this context, it is worth emphasizing that the hybrid protease (Ulp1_linker_TEV and TEV-linker_Ulp1) of the present invention has 7 cysteines (Ulp1 with 3 and TEV with 4), which would increase the possibilities of forming incorrect bonds during the folding process.

[0083] However, the present invention solved the problem of instability after dialysis by using a suitable buffer and obtained a stable hybrid protein possessing effective enzymatic activities.

[0084] In summary, the invention relates to the following aspects, as defined in the following numbered items: 1. Recombinant hybrid protease comprising the fusion of the tobacco etch virus (TEV) protease as defined by SEQ ID NO: 1 with the catalytic site of the ULP1 protease as defined by SEQ ID NO: 2 joined by a flexible linker. 2. Recombinant hybrid protease, according to item 1, wherein the flexible linker comprises the amino acid sequence as defined by SEQ ID NO: 3. 3. Recombinant hybrid protease, according to item 1 or 2, wherein the ULP1 sequence (SEQ ID NO: 2) is inserted into the N-terminal portion of the TEV protease (SEQ ID NO: 1). 4. Recombinant hybrid protease, according to items 1 or 2, wherein the TEV protease sequence (SEQ ID NO: 1) is inserted into the position N-terminal of the ULP1 sequence (SEQ ID NO: 2). 5. Recombinant hybrid protease, according to any of items 1 to 4, wherein the sequences SEQ ID NOs: 1 and 2 are located between the amino acid pair alanine (A) and serine (S) and the amino acid pair leucine (L) and glutamate (E). 6. Recombinant hybrid protease, according to any of items 1 to 5, wherein it is cloned into a bacterial vector, preferably pET24b(+). 7. Recombinant hybrid protease, according to item 6, wherein it additionally comprises 6 histidines at its terminal end (C-terminal) derived from the vector in which the sequence is cloned. 8. Recombinant hybrid protease, according to any of items 1 to 7, comprising 485 amino acids, has a molecular mass of 55486.89 Da, and its theoretical isoelectric point (pI) is 7.79. These characteristics refer to both the Ulp1_TEV and TEV_Ulp1 constructs. 9.10. Recombinant hybrid protease DNA sequence, according to any one of items 1 to 8, consisting of the amino acid sequence as defined by SEQ ID NO: 4 or 9. 11. DNA nucleotide sequence of the recombinant hybrid protease as defined in any one of items 1 to 9, comprising the fusion of the TEV protease DNA sequence as defined by SEQ ID NO: 5 with the catalytic site of the ULP1 protease DNA sequence as defined by SEQ ID NO: 6 joined by a flexible linker. 12. DNA nucleotide sequence, according to item 10, wherein the flexible linker preferably comprises the nucleotide sequence as defined by SEQ ID NO: 7. 13. DNA nucleotide sequence, according to item 10 or 11, wherein the ULP1 sequence (SEQ ID NO: 6) is inserted into the N- portion. 13. DNA nucleotide sequence, according to item 10 or 11, in which the TEV protease sequence (SE ID NO: 5) is inserted at the N-terminal position of the ULP1 sequence (SEQ ID NO: 6). 14. DNA nucleotide sequence, according to either item 10 to 13, which is cloned into a bacterial vector, preferably pET24(+), between the NheI and EcoRI sites. 15. DNA nucleotide sequence, according to either item 10 to 14, which consists of the nucleotide sequence as established by SEQ ID NO: 8 or 10. 16.The process for obtaining the recombinant hybrid protease as defined in any of items 1 to 9 comprises the following steps: a) Constructing the nucleotide DNA sequence of the recombinant hybrid protease as defined in any of items 10 to 15; b) Optimizing the codons for expression in yeast; c) Cloning the sequence obtained in step “b” into a bacterial vector; d) Expressing the hybrid protease in chemically competent E. coli bacteria; e) Purifying the hybrid protease after expression by affinity chromatography; f) Dialyzing the obtained hybrid protease in soluble form using dialysis buffer (50mM Tris-HCl, pH 8.0; 0.2% Igepal; 300mM NaCl, 1mM DTT). 17. Process, according to item 16 or 17, in which in step “d”, the protein is expressed at 30°C for 4 hours, with induction of 0.1 mM IPTG, where after induction, the bacteria are disrupted by sonication. 18. Process, according to either of items 16 to 18. 18. Preferably, after expression, the protease is purified in step “e” by affinity chromatography using Ni-sepharose columns using a purification system. 19. Process, according to any of items 16 to 19, in which after dialysis performed in step “f”, the said process comprises an additional step “g” which refers to the addition of 50% glycerol to the hybrid protease for freezing at -20ºC, wherein the freezing is performed at concentrations ranging from 200 µg / mL to 600 µg / mL. 20. Use of the hybrid protease as defined in any of items 1 to 9 where it is for removing fusion proteins from different proteins of interest.

[0085] Thus, the embodiments presented in the present invention do not limit the totality of possibilities, and it will be understood that various omissions, substitutions and alterations may be made by a person skilled in the art, without departing from the scope of the present invention.

[0086] It is expressly provided that all combinations of elements that perform the same function substantially in the same way to achieve the same results are within the scope of the invention. Substitutions of elements from one described embodiment for another are also fully intended and contemplated.

[0087] Skilled individuals will appreciate the knowledge presented here and will be able to reproduce the invention in the embodiments shown and in other variants covered by the claims.

Claims

CLAIMS 1. Recombinant hybrid protease, characterized in that it comprises the fusion of the tobacco etch virus (TEV) protease as defined by SEQ ID NO: 1 with the catalytic site of the ULP1 protease as defined by SEQ ID NO: 2, joined by a flexible linker.

2. Recombinant hybrid protease, according to claim 1, characterized in that the flexible linker comprises the amino acid sequence as defined by SEQ ID NO:

3.

3. Recombinant hybrid protease, according to claim 1 or 2, characterized in that the ULP1 sequence (SEQ ID NO: 2) is inserted into the N-terminal portion of the TEV protease (SEQ ID NO: 1).

4. Recombinant hybrid protease, according to claim 1 or 2, characterized in that the TEV protease sequence (SE ID NO: 1) is inserted at the N-terminal position of the ULP1 sequence (SEQ ID NO: 2). 5.Recombinant hybrid protease, according to any one of claims 1 to 4, characterized in that the SEQ ID NOs: 1 and 2 sequences are located between the amino acid pair alanine (A) and serine (S) and the amino acid pair leucine (L) and glutamate (E).

6. Recombinant hybrid protease, according to any one of claims 1 to 5, characterized in that it is cloned into a bacterial vector, preferably pET24b(+).

7. Recombinant hybrid protease, according to claim 6, characterized in that it additionally comprises 6 histidines at its terminal end derived from the vector in which the sequence is cloned.

8. Recombinant hybrid protease, according to any one of claims 1 to 7, characterized in that it comprises 479 amino acids, has a molecular mass of 54823.20 Da, and its point... Theoretical isoelectric number (pI) being 7.

79.

9. Recombinant hybrid protease, according to any one of claims 1 to 8, characterized in that it consists of the amino acid sequence as set forth by SEQ ID NO: 4 or 9.

10. DNA nucleotide sequence of the recombinant hybrid protease as defined in any one of claims 1 to 9, characterized in that it comprises the fusion of the DNA sequence of the TEV protease as set forth by SEQ ID NO: 5 with the catalytic site of the DNA sequence of the ULP1 protease as set forth by SEQ ID NO: 6, joined by a flexible linker.

11. DNA nucleotide sequence, according to claim 10, characterized in that the flexible linker comprises the nucleotide sequence as set forth by SEQ ID NO:

7. 12.DNA nucleotide sequence according to claim 10 or 11, characterized in that the ULP1 sequence (SEQ ID NO: 6) is inserted into the N-terminal portion of the TEV protease sequence (SEQ ID NO: 5).

13. DNA nucleotide sequence according to claim 10 or 11, characterized in that the TEV protease sequence (SE ID NO: 5) is inserted into the N-terminal position of the ULP1 sequence (SEQ ID NO: 6).

14. DNA nucleotide sequence according to any one of claims 10 to 13, characterized in that it is cloned into a bacterial vector, preferably pET24(+), between the NheI and EcoRI sites.

15. DNA nucleotide sequence, according to any one of claims 10 to 14, characterized in that it consists of the nucleotide sequence as set forth by SEQ ID NO: 8 or 10.

16. Process for obtaining recombinant hybrid protease. as defined in any one of claims 1 to 9, characterized in that it comprises the steps of: a) Constructing the nucleotide DNA sequence of the recombinant hybrid protease as defined in any one of claims 10 to 15; b) Optimizing the codons for expression in yeast; c) Cloning the sequence obtained in step “b” into a bacterial vector; d) Expressing the hybrid protease in chemically competent E. coli bacteria; e) Purifying the hybrid protease after expression by affinity chromatography; f) Dialyzing the obtained hybrid protease in soluble form using dialysis buffer (50mM Tris-HCl, pH 8.0; 0.2% Igepal; 300mM NaCl, 1mM DTT).

17. Process, according to claim 16, characterized in that the bacterial vector of step “c” is the pET24(+) vector. 18.Process, according to claim 16 or 17, characterized in that in step “d”, the protein is expressed at 30°C for 4 hours, with induction of 0.1 mM IPTG, wherein after induction, the bacteria are disrupted by sonication.

19. Process, according to any of claims 16 to 18, characterized in that preferably, after expression, the protease is purified in step “e” by affinity chromatography using Ni-sepharose columns using a purification system.

20. Process, according to any of claims 16 to 19, characterized in that, after dialysis performed in step “f”, said process comprises an additional step “g” which refers to the addition of 50% glycerol to the hybrid protease for freezing at -20°C, wherein the... Freezing is carried out at concentrations ranging from 200 µg / mL to 600 µg / mL.

21. Use of the hybrid protease as defined in any of claims 1 to 9, characterized in that it is for removing fusion proteins from different proteins of interest.

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