# Patent

## Title
UBP1 protease mutant and its coding sequence, their applications and heterogonous protein expression system

## Abstract

Invention relates to novel and improved UBP1 protease mutants with a substitution at position (754), a deletion of amino-acids at position (1-54) and at least a portion of the amino-acids found at position (55-98) and their coding sequence as well as their applications and heterogonous protein expression system comprising thereof.

## Background
This application is a §371 national stage of PCT International Application No. PCT/PL2005/000002, filed Jan. 10, 2005, the contents of which are hereby incorporated by reference into this application.
Ubiquitin is a protein commonly found in eukaryotic organisms. It has been shown that (R. Baker, Current Opinion in Biotechnology 1996, 7:541-546) it is a convenient carrier of heterologous proteins obtained through expression in Escherichia coli. Ubiquitin is made of 76 amino-acid residues with a total molecular mass of 8.8 kDa. This protein is an element of the universal protein modification system. Ubiquitination accompanies almost every metabolic process, from cell division and differentiation up to cellular death. Ubiquitin is involved in the regulation of gene expression, DNA repair, and influences chromatin activity. It also takes part in tumour transformation. It plays a fundamental role in the proteolysis of regulatory proteins with short half-lives, as well as of proteins with longer half-lives which for various reasons must be eliminated from the cell.
Protein ubiquitination does not occur in bacteria. It has been proven that proteins bound to ubiquitin are expressed in E. coli, and are more stable. Crystallography studies of ubiquitin using magnetic nuclear resonance have shown that it is a compact, globular molecule both in aqueous solution and in solid state (S. Vijay-Kumar, C. Bugg, W. Cook, J. Mol. Biol. 15 1987, 194:531-544). The hydrophobic core of ubiquitin is composed of five parallel lengths of polypeptide chain, bound with regularly spaced hydrogen bonds which form a so-called β-pleated sheet. The edges of its surface are held by lengths of polypeptide chain containing 3.5 twists of an α-helix. Such a structure gives the ubiquitin molecule uncommonly high resistance to temperature, a wide range of pH and environmental polarity changes (M. M. Harding, D. H. Williams, D. N., Woolfson Biochemistry 1991, 30:3 120-3128).
Protease UBP1 is an enzyme isolated from yeast, which cleaves ubiquitin from proteins fused to its C-end. The enzyme was described in 1991 (J. Tobias, A. Varshaysky, J. Biol. Chem. 1991, 266; 12021-12028) and is the subject of the patent application WO91/17245 (European Patent EP 531 404). Its activity and culture conditions were described in E. coli. In accordance to the contents of the description, it is a cysteine protease, which binds ubiquitin with an ester bond during the course of the reaction. UBP1 is 809 amino-acids long. The enzyme's activity is dependent on its ability to cleave the ubiquitin peptide from a polypeptide fused to its C-end, regardless of the amino-acid sequence of the N-end of the polypeptide being digested off.
Application No. WO93/09235 describes other yeast proteins belonging to the same family of proteases, namely UBP2 and UBP3. These proteins exhibit similar activity (see also U.S. Pat. No. 5,494,818, U.S. Pat. No. 5,212,058, U.S. Pat. No. 5,683,904).
There are expression systems known, in which fusion proteins are obtained composed of ubiquitin or its derivative and a polypeptide of interest, and then, using a ubiquitin removing enzyme (e.g. UBP1) the protein of interest is recovered (for examples see: U.S. Pat. No. 5,132,213, U.S. Pat. No. 6,018,102). This method has many advantages such as improved quality and efficiency of obtaining the protein, as well as simplification of purification, which is of great importance in the industrial production of recombinant proteins (for example see: WO03/010204). Using an enzyme which removes ubiquitin and appropriate fusion proteins one may also obtain N-terminally modified polypeptides (for example: U.S. Pat. No. 5,847,097).
The international submission published as WO 2004/097011 describes UBP1 protease deletion mutants, containing a deletion of at least a portion of the initial 54 amino-acids from the amino-acid sequence of the UBP1 protease, and also describes some point mutations, which improve the expression level of such a protease in microbiological expression systems.
The application of an enzyme which removes ubiquitin in technological processes requires large amounts of this protein, which should also exhibit the maximal proteolytic activity level. A majority of known methods do not facilitate the efficient expression of this enzyme, which has greatly limited its applicability, especially in industrial processes. The purity and activity of the enzyme are important, if it is to be used in a subsequent stage of the production of a particular protein. Despite the solutions presented in WO 2004/097011, there is still a need to obtain a protein for cleaving UBP1, which could be produced in an efficient manner, for example through the expression in known microbiological systems, which protein would also exhibit improved specific proteolytic activity characteristics.
The goal of the present invention is provide an efficient method for the production of a protein for removing ubiquitin which could be successfully used in the industrial production of recombinant proteins in bacterial cells, in the form of fusion proteins with ubiquitin, as well as a more efficient method of production of proteins with UBP1 activity. Thus, the goal of the present invention is also to produce a nucleotide sequence facilitating the efficient expression of an enzyme with UBP1 activity. The goal of the present invention is also to produce a new, improved polypeptide producing a protein with UBP1 activity. The next goal of the present invention is to produce a new enzyme which cleaves ubiquitin, which would exhibit specific proteolytic activity of an improved character.
The topic of the present invention is a mutant of the UBP1 protease characterised in that it contains an amino-acid sequence containing the following modifications:

    
    
        a substitution at position 754 of the amino-acid sequence of UBP1,
        deletions of amino-acids found at positions 1 to 54 of the UBP1 sequence and at least a portion of the amino-acids found at positions 55 to 98 of the UBP1 sequence.
    
    


It was unexpectedly found that glutamine found at position 754 has a significant effect on the UBP1 protein expression, particularly in microbiological expression systems. The introduction of a substitution at position 754 of the amino-acid sequence of UBP1 resulted in the improvement in the expression of the mutant obtained, without an undesirable reduction in its activity level. This effect is presented in the examples below. The mentioned substitution at position 754 may be the result of replacing glutamine at this position with any other amino-acid. In a particular case, this will be a natural amino-acid, and the introduction of the change will consist of introducing an appropriate codon change in the sequence used to express the mutant produced. In a particularly preferential embodiment, the characteristics of the introduced amino-acid are significantly different than those of glutamine. Thus, basic, neutral or hydrophobic amino-acids will be preferred. In a particular embodiment of the present invention, the amino-acid replacing glutamine at position 754 of the amino-acid sequence of UBP1 may be an amino-acid containing a neutral amino-acid residue, for example selected from among Ala, Val, Ile, Leu, or Gly. A mutant was described in an example embodiment, in which the glutamine at position 754 of the UBP1 sequence was replaced with leucine.
Also unexpectedly, it was found that preferential properties of UBP1 deletion mutants can be obtained through the simultaneous contraction of the sequence at the N-end of UBP1. Extensive mutations, in an extreme case encompassing all of the 98 amino-acids found at the N-end of UBP1, yield a protein which is produced faster and more efficiently, while still maintaining the desirable ubiquitin-cleaving enzymatic activity.
Therefore, according to the preferential embodiment of the present invention, the deletion encompasses all amino-acid found at positions 1 to 98 of the UBP1 sequence.
The mutants obtained can also contain additional mutations, published in WO2004/097011, which improve the properties of the mutants obtained. Preferentially, a mutant according to the present invention contains an amino-acid sequence containing at least one of the following modifications:

    
    
        replacement of the proline at position 415 of the UBP1 sequence with leucine,
        replacement of the phenylalanine at position 739 of the UBP1 sequence with leucine,
        fusion of the ubiquitin polypeptide to the N-terminal amino-acid with a peptide bond,
        fusion of a marker sequence to the C-terminal amino-acid of the amino-acid sequence,
        attachment with a peptide bond of a fusion polypeptide to the N-terminal aminoacid, where the fusion polypeptide is composed of a peptide containing the marker aminoacid sequence and the ubiquitin fused to its C-end, and preferentially the fusion polypeptide is 6HisUbi whose sequence is represented as SEQ ID NO: 2.
    
    


The mentioned amino-acid marker sequence is a sequence which facilitates the isolation of polypeptides containing it, particularly using affinity chromatography. A person skilled in the art would be able to design a series of such sequences based on common knowledge, which could be used to design an isolation system for the protein produced using affinity chromatography. For example, the introduction of a sequence recognized by an antibody facilitates the isolation of the protein using this antibody. Other examples are amino-acid sequences with affinity for particular nucleotide sequences. Yet another example are techniques basing on the well known phenomenon of complex formation between certain amino-acid residues and transition metal atoms. The best known example of such a system are complexes of Zinc and His. All such systems composed of an amino-acid marker sequence and of a substance for which the sequence has a sufficiently strong affinity can be used to design an isolation system for proteins containing the marker sequence. Usually, this will be affinity chromatography in a medium containing the mentioned substance. Due to the above, in one of the preferential embodiments of the present invention, the marker amino-acid sequence contains a sequence of six histidines (His6).
Preferentially, a mutant according to the present invention is selected from among UBI::UBP1ΔC (SEQ ID NO:4), UBP1ΔC2 (SEQ ID NO:6).
Another subject of the present invention is a nucleotide sequence coding the UBP1 protease mutant defined above, characterised in that it contains the following mutations:

    
    
        replacement of the glutamine codon at position 754 of the amino-acid sequence of UBP1 for a different amino-acid codon,
        deletion of the initial 54 codons of the sequence coding UBP1 and at least a portion of the codons found at positions to 98 in the sequence coding UBP1. Preferentially, the deletion encompasses the initial 294 nucleotides of the sequence coding UBP1.
    
    


Equally preferentially, a nucleotide sequence according to the present invention additionally contains at least one of the following mutations:

    
    
        replacement of the proline codon at position 415 of the amino-acid sequence of UBP1 with a leucine codon,
        replacement of the phenylalanine codon at position 739 of the amino-acid sequence of UBP1 with a leucine codon,
        fusion of a ubiquitin-coding sequence, preferentially in the beginning region of the reading frame,
        fusion of a sequence coding a marker amino-acid sequence, preferentially in the end region of the reading frame,
        attachment of a sequence coding a fusion polypeptide to the beginning region of the reading frame, where the sequence coding the fusion polypeptide is composed of a sequence coding the marker amino-acid sequence and a sequence coding ubiquitin, where the sequence coding the fusion polypeptide preferentially is the sequence coding 6HisUbi represented as SEQ ID NO: 2, where the marker amino-acid sequence is a sequence facilitating the isolation of the polypeptide containing it, particularly using affinity chromatography. In one example embodiment of the present invention, the sequence coding the marker amino-acid sequence contains a sequence composed of six consecutive histidine codons.
    
    


To improve the expression level in the selected expression system, a nucleotide sequence according to the present invention additionally contains codon changes which account for the preferences of the planned expression system. Preferentially, the planned expression host is E. coli, and codon changes encompass a change selected from the following:

    
    
        replacement of at least one arginine codons at positions 96, 334, 425, 476, 482, 487, 613, 702, 705, 710, 796, 801 of the amino-acid sequence of UBP1 with either a CGT or CGC codon,
        replacement of at least one leucine codons at positions 354, 356, 258, 367, 369, 372, 373, 479, 480 of the amino-acid sequence of UBP1 with the CTG codon.
    
    


In a preferential embodiment of the present invention, the nucleotide sequence contains one of the nucleotide sequences represented as the sequence coding UBP1ΔC (SEQ ID NO:4), or the sequence coding UBP1ΔC2 (SEQ ID NO:6).
The next subject of the present invention is the application of the UBP1 protease mutant according to the present invention defined above to obtain ubiquitin-cleaving enzymatic activity.
In a particular embodiment of this aspect of the present invention, the ubiquitin-cleaving enzyme obtained is used to obtain a protein of interest from a hybrid protein composed of a peptide containing ubiquitin and the protein of interest.
Preferentially, the UBP1 protease mutant and the peptide containing ubiquitin contain an amino-acid marker sequence which facilitates their isolation from a reaction mixture, wherein the protein of interest is a medicinal protein, such as interleukin, interferon, growth hormone, insulin or erythropoietin.
The next subject of the present invention is a heterologous protein expression system in bacterial cells, characterised in that it contains:

    
    
        an expression cassette coding a hybrid protein composed of a peptide containing ubiquitin and the heterologous protein sequence, and
        a UBP1 protease mutant, according to the present invention, as defined above.
    
    


Preferentially, the UBP1 protease and peptide containing ubiquitin contain a marker amino-acid sequence, and the nucleotide sequence of the expression box contains codon changes taking into account the preferences of the planned expression system.
The next subject of the present invention is an application of the nucleotide sequence according to the present invention, as defined above, to obtain an enzyme cleaving ubiquitin.
The next subject of the present invention is an expression vector, characterised in that it contains a nucleotide sequence coding a UBP1 protease mutant according to the present invention, as defined above. In one of the example embodiments of this aspect of the present invention, the nucleotide sequence coding the UBP1 protease mutant is contained on plasmid pT7-7ArgStop.
The next subject of the present invention is a host cell transformed with an expression vector defined above.
The next subject of the present invention is a method of obtaining a protein cleaving ubiquitin, characterised, in that a host cell is cultured which has been transformed with an expression vector containing with an expression vector containing a nucleotide sequence coding the UBP1 protease mutant according to the present invention defined above, and subsequently the enzyme or the fraction containing it is isolated. In a particular embodiment of the method according to the present invention, the amino-acid sequence of the UBP1 protease mutant contains a sequence of six consecutive histidines (His6), and the proteases mutant produced is isolated using the well known affinity chromatography technique.
The next subject of the present invention is a method of obtaining a heterologous protein, characterised in that a host cell is cultured, transformed with an expression vector containing an expression cassette coding a hybrid protein composed of a peptide containing ubiquitin and a heterologous protein sequence under conditions favourable to the production of the hybrid protein. The hybrid protein or a fraction containing it is isolated, and then the hybrid protein thus obtained is digested with the UBP1 protease mutant according to the present invention, as defined above. Next, the UBP1 protease mutant is removed from the reaction mixture along with the peptide containing ubiquitin and the heterologous protein is isolated. In a particular embodiment of the method according to the present invention, the UBP1 protease mutant and the peptide containing ubiquitin contain a sequence of six consecutive histidines (His6) and are removed from the mixture using the well known affinity chromatography technique.
Unexpectedly, it turned out that the new UBP1 mutants proposed in the present invention retain the fundamental activity of cleaving ubiquitin, and are easier to obtain. The means and methods presented facilitate the easy and efficient expression of an enzyme with UBP1 activity, for example in the well understood system based on E. coli cells. Due to this the mutant according to the present invention is suitable for industrial use, for example in the synthesis of recombinant proteins encompassing the expression of hybrid proteins containing ubiquitin.
A heterologous protein contained in a hybrid protein according to the present invention mentioned above may be an arbitrary known protein, whose coding sequence is known. For example, this may be a medicinal protein, whose production is desirable due to its therapeutic proteins. Based on the instructions contained in the present description common knowledge, a professional will be able to design a sequence coding a hybrid protein containing a sequence coding the desired heterologous protein. Sequences of known heterologous proteins may for example be obtained from the GenBank database, available at the URL www.ncbi.nlm.nih.gov/Genbank/index.html, which contains the sequences of known genes. To increase the expression level of the heterologous protein in a bacterial system, one may use methods which include the use of strong promoters, transcription enhancer sequences or codons preferred by the bacterial cell.



To better illustrate the nature of the present invention, the description includes the following figures:
FIG. 1 (SEQ ID No: 3, SEQ ID No: 4) represents the UB1::UBP1ΔC gene and its various variants and derivatives. The deleted DNA fragment is in bold, whereas primer sequences, the C mutation and histidine residues are in underlined italics.
FIG. 2A (SEQ ID No. 5) represents the nucleotide sequence of UBP1ΔC2 protease (SEQ ID No. 5). FIG. 2B (SEQ ID No: 26) represents the amino-acid sequence of UBP1ΔC2 protease (SEQ ID No: 6) prior to deletion of the 98 N-terminal amino-acids found in the wild-type UBP1. Bold italics delineate mutation C at position 754 and fused histidine residues, the active centre is in italics. The sequence in bold is removed from the N-end.
FIG. 3 represents an image of an SDS-PAGE electrophoresis gel. The lanes contain, in turn: 1—lysate of E. coli BLD3 bacteria transformed with pT7-7ArgStopUBI::UBP1ΔC2, not induced with IPTG (from cultures maintained in 100 ml LB), 2—lysate of E. coli BLD3 bacteria transformed with pT7-7ArgStopUBI::UBP1ΔC2, induced with IPTG (from cultures maintained in 100 ml LB), 3—lysate of E. coli BLD3 bacteria transformed with pT7-7ArgStopUBI::UBP1ΔC2, not induced with IPTG (from cultures maintained in a fermentor containing 3,5 1 LB), 4—lysate of E. coli BLD3 bacteria transformed with pT7-7ArgStopUBI::UBP1ΔC2, induced with IPTG (from cultures maintained in a fermentor containing 3,5 1 LB), 5—sonicated supernatant (20 μl of samples from cultures maintained in a fermentor), 6—lysate of E. coli BLD3 bacteria transformed with pT7-7ArgStopUBI::UBP1ΔC, not induced with IPTG, 7—lysate of E. coli BLD3 bacteria transformed with pT7-7ArgStopUBI::UBP1ΔC, induced with IPTG, 8—lysate of E. coli BLD3 bacteria transformed with pT7-7ArgStop+ubiquitin, not induced with IPTG, 9—lysate of E. coli BLD3 bacteria transformed with pT7-7ArgStop+ubiquitin, induced with IPTG, 10—molecular mass marker (kDa).
FIG. 4 represents an image of an SDS-PAGE electrophoresis gel. The lanes contain in turn: 1—molecular mass marker (kDa), 2—the substrate, UB1::KALA digested with ubi::UBP1ΔC2 (SEQ ID No. 10), 3—the substrate, UB1::KALA.
FIG. 5 represents an image of an SDS-PAGE electrophoresis gel. The lanes contain in turn: 1—the substrate UB1::ElisaC digested with ubi::UBP1ΔC (SEQ ID No. 4), 2—the substrate UB1::ElisaC digested with ubi::UBP1ΔC2 (SEQ ID No. 10), 3—the undigested substrate UBI::ElisaC, 4—molecular mass marker (kDa), 5—the substrate UB1::KALA digested with ubi::UBP1ΔC2 (SEQ ID No. 10), 6—the substrate UB1::KALA digested with ubi::UBP1ΔC, 7—the undigested substrate UB1::KALA.
FIG. 6 represents the 6HisUbi nucleotide sequence (SEQ ID No. 1) and amino acid sequence (SEQ ID No. 2).
FIG. 7 represents a map of the expression vector pT7-7RHUC.
FIG. 8 represents the sequence of 6HisUBI::ElisaC (SEQ ID No. 7, SEQ ID No. 8).
FIG. 9 represents example results of the application the system according to the present invention to express the ElisaC protein (see example 3). An image of an SDS-PAGE gel is presented in which the lanes contain, in turn: 1—molecular mass marker (kDa), 2—6HisUbi::ElisaC (SEQ ID No. 8) purified in a chromatography column containing Ni-NTA medium, 3—6HisUbi::ElisaC (SEQ ID No. 8) digested with protease UBP1ΔC2 (SEQ ID No. 6), 4 and 5—ElisaC protein purified in a chromatography column filled with Ni-NTA medium, with 6HisUbi removed.



The following examples are only meant to present assorted embodiments of the present invention and should not be viewed as the whole of its scope.
EXAMPLE 1
New UBP1 Protease Mutants and their Coding Sequences
It has been determined that the active centre of the enzyme UBP1 probably begins from the amino-acid 102. The removal of amino-acids was planned. The following primers were designed to this end:









SKRUT 1



5′ AAAACCGCGGTGGTTTCATTGCTGGTTTA 3′



(SEQ ID No. 17)



         Sac II


 



SKRUT 2



5′ GGAAGAATTCTTGCGCGTCCTC 3′



(SEQ ID No. 18)



        Eco RI






The location of the primers is shown in bold in FIG. 1.
Primers: SKRUT1 (SEQ ID No. 17) and SKRUT2 (SEQ ID No. 18) were used to amplify a 468 bp fragment using PCR, on a template of pT7-7ArgStopUBI::UBP1ΔC6His. Next, the 468 bp fragment was cloned into the pBluscript (−) vector, between Eco RI and Sac II restriction sites. The UBP1ΔC protease fragment was sequenced and cloned into the pT7-7ArgStopUBI::UBP1ΔC6His plasmid digested with Eco RI and Sac II restrictases. Using this method, a sequence was formed coding the UBP1ΔC2 protease (SEQ ID No. 5) truncated by 294 bp, which corresponds to the UBP1ΔC2 protein (SEQ ID No. 6) truncated by 98 amino-acid. The sequence represents the gene of protease UB1::UBP1ΔC2 (see SEQ ID No. 9 and 10), which contains mutation C, meaning the replacement of a glutamine codon (CAG) with a leucine codon (CTG) at position 754 (see WO2004/097011). The hybrid protein UB1::UBP1ΔC2 (SEQ ID No. 10) is 796 amino-acids long, with a mass of 88.35 kDa. Changes introduced into the amino-acid sequence are also represented in FIG. 2.
The UBP1ΔC protease gene (e.g. SEQ ID No. 15) was additionally modified through the replacement of a portion of arginine codons, unfavourable to E. coli (AGA or AGG), for codons favourable to E. coli (CGT or CGC). The UBP1ΔC2 protease gene (SEQ ID No. 5) in its final form (vs. UBP1ΔC protease (SEQ ID No. 16)) has had the following arginine codons at positions 334, 425 and 613 replaced. The leucine codon, TTA, was also replaced with CTG at positions 354, 356, 358, 367, 369, 372, 373, 479 and 480. The changes introduced are delineated in FIG. 1 in bold.
EXAMPLE 2
Obtaining New UBP1 Mutants
Expression of the UBI::UBP1ΔC2 protease (SEQ ID No. 10) with six histidine residues attached at its C-end was achieved in competent E. coli BLD3 and E. coli T7ples cells. The results are shown in FIG. 3, in which its expression results are shown along with the expression results of the gene UB1::UBP1ΔC (patent WO2004097011 and SEQ ID No: 3). The culture was maintained until OD=1 (ca. 9h) at 25° C. in LB medium with amphicilin. Following centrifugation, the bacteria were sonicated. The supernatant was cleaned using ion affinity chromatography. The activity of the UBI::UBP1ΔC2 protease (SEQ ID No. 10) was assayed using a standard method using the hybrid protein substrate UBT::KALA (where KALA is an 18-amino-acid peptide with the amino-acid sequence AspProGlyAspLysAspGlyAspGlyTyrlleSerAlaAlaGluAlaMetAla) (SEQ ID No. 19) as well as the substrate His6UBI::ElisaC (SEQ ID No: 8).
The reactions were performed at a volume of 50 μl at 37° C., for min. in a buffer with the following composition: 20 mM phosphate pH 7.5, 2 mM DDT, 1 mM EDTA. 40 μl of the substrate UB1::KALA were digested with a protein concentration of 0.1 mg/ml and 20 μl of substrate His6UBI::ElisaC (SEQ ID No. 8) with a protein concentration of 1 mg/ml, and 3 μl of proteases UBP1ΔC (SEQ ID No. 16) and UBP1ΔC2 (SEQ ID No. 6) with concentrations of 0.5 mg/ml (1.5 μg of enzyme).
The digestion reactions were stopped by heating to 100° C., and the samples were put on SDS-PAGE gels. SDS-Polyacrylamide gel image analysis showed that the enzyme UBP1ΔC2 (SEQ ID No. 6) is 5-6 times more active than mutant UBP1ΔC (SEQ ID No. 16). The results are shown in FIGS. 4 and 5.
It should be shown that for following truncation, the UBP1 protease gene with mutation C, the time of bacterial cell growth to OD=1 was also shortened in E. coli BLD21 (table No. 1).









TABLE NO. 1









Protease-mutants











UBP1Δ
UBP1ΔC
UBP1ΔC2



SEQ ID No: 14
SEQ ID No: 16
SEQ ID No: 6














Time to OD = 1 (h)
48
12
9









The level of expression of this gene grew significantly in comparison to the UBP1ΔC protease gene, whereas the UBP1Δ protease gene (SEQ ID No. 13) is expressed at trace levels, (undetectable in SDS-PAGE gel images). Thus the application of unmodified UBP1 protease in laboratory practice and the production of recombinant medicinal proteins is essentially impossible.
EXAMPLE 3
Heterologous Protein Expression System and its Application
UBP1 mutants described in the present invention were used to design an expression system used to efficiently produce heterologous proteins in bacterial cells. According to the present invention, application of the system is based on the production of heterologous proteins in bacterial cells transformed with DNA containing an expression cassette coding a hybrid protein composed of a peptide containing ubiquitin and a heterologous protein sequence. It may be expected that this form of hybrid protein will be produced more efficiently than a heterologous protein. Furthermore, ubiquitin or its derivative contained in the hybrid protein performs the role of a carrier protein, which additionally increases the stability and facilitates the purification of the protein produced. The purification is particularly simplified when the composition of the expressed hybrid protein contains a ubiquitin derivative containing an amino-acid marker sequence. A heterologous protein is freed from the hybrid protein using a UBP1 protease mutant according to the present invention, which is a part of the expression system.
Because earlier examples showed sample variants of UBP1 mutants which may be incorporated into the proposed expression system, the sections below concentrate on describing examples of the remaining means for the production and application of the proposed system according to the present invention.
To perfect the system described in WO03/010204, based on the fusion of the proteins UBI::protein, the gene coding the UB1::protein hybrid protein was modified through the addition of a marker sequence to the ubiquitin sequence, which marker sequence is easy to isolate using affinity chromatography. In the example described, the hybrid protein produced is in the form His6UBI::protein, in which 6 histidine residues have been added to the N-end of ubiquitin. Following digestion with the UBP1ΔC2 protease (SEQ ID No. 6), such a hybrid protein is easily purified using affinity chromatography with a chelating column, such as NI-NTA. If the removal of the modified ubiquitin containing the marker sequence is performed using a UBP1 protease mutant according to the present invention, which contains the same marker sequence, it is possible to separate both the protease as well as the carrier protein (modified ubiquitin) in the same chromatography column. This greatly simplifies the purification of the heterologous protein produced, and limiting the number of purification stages greatly enhances the efficiency of the whole process. In accordance to the present invention, analogous systems containing marker sequences may be proposed, which can be separated from the reaction mixture using affinity chromatography.
In order to obtain a sequence coding the hybrid protein His6UBI::ElisaC (SEQ ID No. 8) the following synthetic nucleotides were designed:









6HisG



    Nde I                        EcoRI



5′ TATGGCACATCATCACCATCACCACTCTGGTTCTG 3′



(SEQ ID No. 20)


 



6HisD



    EcoRI                          Nde I



5′ AATTCAGAACCAGAGTGGTGATGGTGATGATGTGCCA 3′



(SEQ ID No. 21)






The synthetic fragments were kinased and ligated with the pT7-7ArgStop plasmid digested with the EcoRI and NdeI restrictases. The vector pT7-7ArgStop is a derivative of plasmid pT7-7 (Tabor, S. and Richardson, C. C. (1985) Proc. Nat. Sci. USA, 262, 1074-1078).
The ligation mixture was used to transform competent DH5α cells. Plasmid DNA was isolated using the alkaline method. The vector pT7-7RSH was obtained in this fashion.
To amplify the ubiquitin-coding sequence using PCR, the following primers were designed:









HisUbiK



      BamHI



5′ GGGGATCCTTAACCACCGCGGAGACGTAA 3′



(SEQ ID No. 22)


 



HisUbiP



       EcoRI



5′ GGGGAATTCCAAATTTTTGTTAAAACTTTAACTGG 3′



(SEQ ID No. 23)






The plasmid pUC19UBI was used as a template, which contained a cloned sequence coding ubiquitin composed of synthetic oligonucleotide fragments. The PCR product was isolated in a standard fashion in a polyacrylamide gel and digested with the EcoRI and BamHI restrictases, whose recognition sequences were planned in the primers (in bold). The insert prepared was ligated with the pT7-7RSH vector digested with the same enzymes. The ligation mixture was used to transform competent DH5α cells. Plasmid DNA was isolated using the alkaline method, and following restriction analysis it was sequenced. Plasmid pT7-7RSHU was obtained, which contains the ubiquitin gene which contains an N-terminal DNA fragment coding 6 histidine residues, shown in FIG. 6. This is an example sequence coding a peptide containing ubiquitin also encompassing the marker sequence, which may be used to obtain sequences coding hybrid proteins according to the present invention.
The Construction of the Example Hybrid Protein His6UBI::ElisaC
The DNA fragment coding the C-terminal end of the UBP1ΔC protease (SEQ ID No. 4) 360 bp long was amplified. PCR with ubi::UBP1ΔC (SEQ ID No. 3) as a template was performed using the following primers:









ELISA P



        SacII



5′ GACTCCGCGGTGGTACCCATAATTATGGTCATTAC



(SEQ ID No. 24)


 



ELISA K



      BamHI



5′ GGGGATCCTTAGTTTACATCTTTACCAGAAATA



(SEQ ID No. 25)






The 360 bp. (ElisaC) fragment was digested with restriction enzymes SacII and BamHI and ligated with the expression plasmid pT7-7RSH digested with the same enzymes. The ligation mixture was used to transform competent DH5α cells. Plasmid DNA was isolated using the alkaline method and sequenced following restriction analysis. The plasmid pT7-7RSHUC was obtained (FIG. 7). The plasmid DNA was used to transform competent E. coli BLD21 cells. The culture was maintained until OD(λ600)=1 in LB medium with ampicillin and a very high expression of 6Hisubi::ElisaC was achieved. The bacteria were sonicated following centrifugation. The supernatant was purified using affinity chromatography on a column of Ni-NTA medium (Qiagen). High purity of the isolated 6HisUBI::ElisaC protein (SEQ ID No. 8) was achieved (the sequence is shown in FIG. 8). The sample was dialised for 16 h against 50 mM phosphate buffer. The hybrid protein was digested with the UBP1ΔC2 protease, which contains 6 histidine residues at its C-end.
The digestion was performed in a volume of 5 ml at 35° C. for 1 h. The reaction was performed in 20 mM phosphate buffer pH 7.5, 2 mM DDT, 1 mM EDTA.
The sample of protein produced was then again applied to a Ni-NTA column and chromatography was performed. High purity ElisaC protein was obtained, cleaved from 6HisUBI. The results are shown in FIG. 9.
The presented hybrid protein expression system facilitates the production of large quantities of hybrid proteins. Using this system of expression-digestion-purification, one may obtain protein of very high purity due to the application of UBP 1 AC2 protease, which contains histidine residues, and due to the fusion of these residues to ubiquitin. Both proteins are retained in a chromatography column containing Ni-NTA medium. The protein digested away from 6His-ubiquitin on the other hand, does not bind to Ni-NTA.

## Claims

1. A ubiquitin specific protease 1 (UBP1) mutant comprising the following modifications relative to a Saccharomyces cerevisiae UBP1 protein:
a substitution of the glutamine residue corresponding to position 754 of the amino-acid sequence of Saccharomyces cerevisiae UBP1, and
a deletion of the amino-acids at positions corresponding to positions 1 to 98 of the amino-acid sequence of Saccharomyces cerevisiae UBP1,
wherein position numbers of the amino acid sequence of Saccharomyces cerevisiae UBP1 are the same as positions of the amino-acid sequence set forth in SEQ ID NO: 26.

2. The mutant according to claim 1, wherein the mutant additionally comprises at least one of the following modifications:
a replacement of the proline at a position corresponding to position 415 of the amino-acid sequence of Saccharomyces cerevisiae UBP1 with leucine,
a replacement of the phenylalanine at a position corresponding to position 739 of the amino-acid sequence of Saccharomyces cerevisiae UBP1 with leucine,
fusion of a ubiquitin polypeptide to the N-terminal amino-acid of the mutant with a peptide bond,
fusion of a marker polypeptide to the C-terminal amino-acid of the mutant with a peptide bond, and/or
fusion of a fusion polypeptide to the N-terminal amino-acid of the mutant with a peptide bond, wherein the fusion polypeptide comprises a marker polypeptide and a ubiquitin polypeptide fused to the C-terminal amino acid of the marker polypeptide.

3. The mutant according to claim 2, wherein the marker polypeptide comprises six consecutive histidines (His6).

4. The mutant according to claim 2, wherein the mutant is UBP1ΔC2 (SEQ ID NO:6).

5. The mutant according to claim 2, wherein the fusion polypeptide is 6HisUbi of SEQ ID NO:2.

6. The mutant according to claim 2, wherein the mutant comprises the following modifications:
a replacement of the proline at a position corresponding to position 415 of the amino-acid sequence of Saccharomyces cerevisiae UBP1 with leucine,
a replacement of the phenylalanine at a position corresponding to position 739 of the amino-acid sequence of Saccharomyces cerevisiae UBP1 with leucine,
fusion of a ubiquitin polypeptide to the N-terminal amino-acid of the mutant with a peptide bond, and
fusion of a marker polypeptide to the C-terminal amino-acid of the mutant with a peptide bond.

7. A nucleic acid molecule coding a UBP1 mutant comprising the following modifications relative to a nucleic acid molecule coding a Saccharomyces cerevisiae UBP1:
a substitution of the glutamine codon at a position corresponding to position 754 of the amino-acid sequence of Saccharomyces cerevisiae UBP1 for another amino-acid codon,
a deletion of the codons at positions corresponding to positions 1 to 98 of the amino acid sequence of Saccharomyces cerevisiae UBP1,
wherein position numbers of the amino acid sequence of Saccharomyces cerevisiae UBP1 are the same as positions of the amino-acid sequence set forth in SEQ ID NO: 26.

8. The nucleic acid molecule according to claim 7, wherein the nucleic acid molecule also comprises at least one of the following modifications:
a replacement of the proline codon at a position corresponding to position 415 of the amino-acid sequence of Saccharomyces cerevisiae UBP1 with a leucine codon,
a replacement of the phenylalanine codon at a position corresponding to position 739 of the amino-acid sequence of Saccharomyces cerevisiae UBP1 with a leucine codon,
fusion of a polynucleotide coding ubiquitin,
fusion of a polynucleotide coding a marker polypeptide, and/or
fusion of a polynucleotide coding a fusion polypeptide, wherein the fusion polypeptide comprises a marker polypeptide and a ubiquitin polypeptide.

9. The nucleic acid molecule according to claim 8, wherein the polynucleotide coding the marker polypeptide comprises six consecutive histidine codons.

10. The nucleic acid molecule according to claim 7, wherein it further comprises codon changes which account for the preferences of a planned expression system.

11. The nucleic acid molecule according to claim 10, wherein the planned expression system is an E. coli expression system, and the codon changes comprise:
a replacement of at least one of the arginine codons at positions corresponding to positions 96, 334, 425, 476, 482, 487, 613, 702, 705, 710, 796, 801 of the amino-acid sequence of Saccharomyces cerevisiae UBP1 with either codon CGT or CGC, and/or
a replacement of at least one of the leucine codons at positions corresponding to positions 354, 356, 258, 367, 369, 372, 373, 469, 470 of the amino-acid sequence of Saccharomyces cerevisiae UBP1 with codon CTG.

12. The nucleic acid molecule according to claim 7, wherein the nucleic acid molecule comprises a nucleic acid molecule coding UBP1ΔC2 (SEQ ID NO:6).

13. The nucleic acid molecule according to claim 8, wherein the polynucleotide coding ubiquitin is at the 5′ end of the reading frame.

14. The nucleic acid molecule according to claim 8, wherein the polynucleotide coding a marker polypeptide is at the 3′ end of the reading frame.

15. The nucleic acid molecule according to claim 8, wherein the fusion polypeptide is 6HisUbi of SEQ ID NO:2.

16. A method of enzymatically cleaving a ubiquitin polypeptide comprising contacting the ubiquitin polypeptide with the UBP1 mutant according to claim 1.

17. A method for obtaining a protein of interest from a hybrid protein comprising contacting the hybrid protein with the UBP1 mutant according to claim 1, wherein the hybrid protein comprises a ubiquitin polypeptide and the protein of interest.

18. The method of claim 17, wherein the protein of interest is selected from the group consisting of interleukin, interferon, growth hormone, insulin, or erythropoietin.

19. A kit comprising:
an expression cassette coding a hybrid protein, wherein the hybrid protein comprises a ubiquitin polypeptide and a heterologous protein, and
the UBP1 mutant according to claim 1.

20. An expression vector comprising the nucleic acid molecule according to claim 7.

21. A host cell transformed with the expression vector according to claim 20.

22. A method for obtaining a UBP1 mutant, comprising:
a. culturing a host cell,
b. transforming the host cell with an expression vector, wherein the expression vector comprises the nucleic acid molecule according to claim 7, and
c. isolating the UBP1 mutant or a fraction containing it.

23. The method according to claim 22, wherein the UBP1 mutant comprises six consecutive histindines (His6), and wherein the UBP1 mutant is isolated using affinity chromatography.

24. A method of obtaining a heterologous protein, comprising:
a. transforming a host cell with an expression vector comprising an expression cassette coding a hybrid protein, wherein the hybrid protein comprises a ubiquitin polypeptide and a heterologous protein,
b. culturing the transformed host cell under conditions facilitating production of the hybrid protein,
c. isolating the hybrid protein or a fraction containing it,
d. digesting said hybrid protein with the UBP1 mutant according to claim 1, and
e. removing the UBP1 mutant and ubiquitin polypeptide from the reaction mixture,
thereby obtaining the heterologous protein.