Abstract:
A UBP1 protease mutant and the sequence coding it, their application and products and the methods used to produce them may be used in the production of recombinant proteins, particularly on an industrial scale.

Description:
RELATED APPLICATIONS 
     This is a continuation-in-part of and claims benefit under 35 U.S.C. §120 of International Patent Application No. PCT/PL2004/00003 filed on Apr. 30, 2004, which claims benefit under 35 U.S.C. §119 of Polish Patent Application No. P.359813 filed on May 2, 2003, the content of both applications are incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to a UBP1 protease mutant and the sequence coding it, their application as well as products and methods for their production. The present invention is used in the production of recombinant proteins, particularly on an industrial scale. 
     Ubiquitin is a protein commonly expressed in eukaryotes. It has been shown that it is a useful carrier for heterologous proteins obtained through expression in  Escherichia coli  (R. Baker, Current Opinion in Biotechnology 1996, 7:541-546). Ubiquitin is composed of 76 amino-acid residues, with a combined molecular mass of 8.8 kDa. This protein is an element of the universal protein modification system. Ubiquitination is involved in almost all metabolic processes, from cell division to its death. Ubiquitin is involved in the regulation of gene expression, DNA repair, and it influences chromatin activity. It takes part in oncogenesis. It also plays a pivotal role in the proteolysis of regulatory proteins with short half-lives, and proteins with longer half lives as well, which must be removed from a cell for various reasons. 
     Protein ubiquitination does not occur in bacteria. It has been shown that proteins fused to ubiquitin undergo greater expression in  E. coli , and are more stable. Crystallographic analysis of ubiquitin using nuclear magnetic resonance demonstrated that both in solid state and in aqueous solution ubiquitin maintains a dense, globular structure (S. Vijay-Kumar, C. Bugg, W. Cook, J. Mol. Biol. 1987, 194:531-544). The hydrophobic core of ubiquitin is composed of five parallel lengths of the peptide chain, held together with regularly spaced hydrogen bonds, forming a so-called β-pleated sheet. Its surface edges are joined with short chain lengths, coiled into 3.5 turns of an α-helix. Such a structure gives ubiquitin an uncommon resistance to high temperatures, a wide range of pH and polarity changes in the environment (Harding M M, Williams D H, Woolfson D N Biochemistry 1991, 30:3120-3128). 
     The UBP1 protease is an enzyme isolated from yeast, which severs ubiquitin from a protein located at its C-end. The enzyme was described in 1991 (J. Tobias, A. Varshavsky, J. Biol. Chem. 1991, 266; 12021-12028) and is a subject of patent application WO91/17245 (European Patent EP 531 404). Its activity has been studied, and its culture conditions have been described in  E. coli . In accordance to the contents of the description, it is a cysteine protease, which binds ubiquitin with an ester bond. UBP1 is made up of an 809 amino-acid chain. The enzymatic activity depends on its ability to sever the ubiquitin peptide from the polypeptide found at its C-end, regardless of the amino-acid sequence at the N-end of the ubiquitin. 
     Application WO93/09235 describes other yeast proteins belonging to the same protease family, namely UBP2 and UBP3. This proteins show a 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). 
     No improved mutants of UBP1 have been shown to date. 
     Expression systems are known, which yield fusion proteins composed of ubiquitin or its derivative and a polypeptide of interest. These then apply an enzyme which removes the ubiquitin (ie. UBP1), and recover the protein of interest (for examples see U.S. Pat. No. 5,132,213, U.S. Pat. No. 6,018,102). Such a method contains many advantages, encompassing among others an improvement in the quality and yield efficiency of the protein, and a simplification of the purification process of the protein, a significant feature for the industrial production of recombinant proteins (for an example see WO03/010204). Using an enzyme which removes ubiquitin along with appropriately designed fusion proteins, one may also obtain N-modified polypeptides (for example U.S. Pat. No. 5,847,097). 
     The application of an enzyme which removes ubiquitin in technological processes requires large amounts of this enzyme. Known methods, however, are not conducive to the efficient expression of said enzyme, and significantly limit the possibilities of its application, particularly in industrial processes. 
     SUMMARY OF THE INVENTION 
     The goal of the present invention is to obtain an efficient method for the production of a protein for severing ubiquitin and the means for its realisation. A particular goal of the present invention is to obtain the means to produce a protein exhibiting UBP1 activity more easily. 
     Thus, the goal of this invention is to also obtain a nucleotide sequence facilitating the efficient expression of an enzyme with UBP1 activity. Thus, the goal of the present invention is to also obtain a new, improved polypeptide comprising a protein with UBP1 activity. 
     Unexpectedly, the above goals were met thanks to the present invention. 
     A subject of the present invention is a mutant of the UBP1 protease, which contains an amino-acid sequence containing at least one of the following modifications:
         a substitution of proline at position 415 of the UBP 1 sequence with leucine,   a substitution of phenyloalanine at position 739 of the UBP1 sequence with leucine,   a substitution of glutamine at position 754 of the UBPI sequence with leucine,   fusion of the polypeptide sequence of ubiquitin to an N-terminal amino-acid with a peptide bond,   deletion of at least a portion of the amino-acids in positions from 1 through 54 of the UBP1 sequence.       

     Preferentially, the deletion encompasses all amino-acids in positions from 1 through 54 of the UBPI sequence. In accordance with the particularly preferential embodiment of the present invention mutant, a protease according to the present invention possess one of the amino-acid mutant sequences presented in  FIGS. 6 ,  8 - 10 . 
     A subject of the present invention is also the nucleotide sequence coding a UBP1 protease mutant, characterised in that it contains at least one of the following mutations:
         a substitution of the proline codon at position 415 of the UBP 1 amino-acid sequence with a leucine codon,   a substitution of the phenyloalanine codon at position 739 of the UBP1 amino-acid sequence with a leucine codon,   a substitution of the glutamine codon at position 754 of the UBP1 amino-acid sequence with a leucine codon,   fusion of the sequence coding ubiquitin, preferentially in the starting region of the open reading frame,   deletion of at least a portion of the first 54 codons of the sequence coding UBP1. Preferentially, the deletion encompasses the initial 132 nuclotides.       

     Preferentially, the nucleotide sequence according to the present invention also contains codon changes accounting for the requirements of the planned expression system. In a particularly preferential embodiment the expressing host is  E. coli , and the codon changes encompass the substitution of at least one of the arginine codons in positions 96, 476, 482, 487, 702, 705, 710, 796, 801 of the UBP1 amino-acid sequence with the the CGT or CGC codon. 
     In one preferential embodiment, the nucleotide sequence according to the present invention contains one of the nucleotide sequences presented in  FIGS. 1 ,  5 ,  8 - 10 . 
     Another subject of the present invention is the application of the UBP1 protease mutant in the production of the enzyme which severs ubiquitin, where the mutant contains the characteristics defined above. Preferentially, the obtained enzyme which severs ubiquitin is used to produce a protein of interest from a hybrid protein composed of ubiquitin and the protein of interest. The protein of interest is a medicinal protein, preferentially interleukin, interferon, growth hormone, insulin or erythropoetin. 
     The next subject of the present invention is the application of a nucleotide sequence coding the UBP1 protease mutant to obtain an enzyme which severs ubiquitin, where a sequence is used according to the present invention, as defined above. 
     A subject of the present invention is also the expression vector which contains the nucleotide sequence coding the UBP1 protease mutant according to the present invention, as defined above. Preferentially, the nucleotide sequence coding the UBP1 protease mutant is contained in the pT7-7ArgStop plasmid. 
     A subject of the present invention is also a host cell transformed with an expression vector containing a nucleotide sequence coding the UBP1 protease mutant according to the present invention, as defined above. 
     A subject of the present invention is also a method for the production of a protein which severs ubiquitin, characterised in that the host cells which have been transformed with the expression vector containing the nucleotide sequence coding the UBP1 protease mutant are cultured, and the desired enzyme or fraction containing it is isolated, where the nucleotide sequence coding the UBP1 protease mutant is a sequence according to the present invention, as defined above. 
     Unexpectedly, it turned out that the new UBP1 mutants proposed in the present invention retain the basic enzymatic activity of severing ubiquitin, and are easier to produce. The presented means facilitate the easy and efficient expression of an enzyme with UBP1 activity, for example in the well understood system based on  E. coli  cells. Thanks to this, mutants according to the present invention are suitable for industrial application, for example in the process of synthesis of recombinant proteins, encompassing the expression of fusion proteins containing ubiquitin. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       To better illustrate the nature of the present invention, the description includes the following figures: 
         FIG. 1  (SEQ ID NO.: 1) represents the sequence coding the hybrid protein Ubiquitin::UBP1 (lower case letters describe the ubiquitin sequence, the UBP1 sequence is in upper case, bold print marks the SacII recognition sequence, the stop codon and the sites of primers UBP1MG and UBP1MD); 
         FIG. 2  represents a map of the pT7-7ArgStop expression vector, where Amp is the ampicillin resistance gene, ArgU is a gene coding tRNA complementary to the AGA codon, Stop Transkrypcji is a transcription stop nucleotide sequence from the φ10 gene of the T7 phage; 
         FIG. 3  represents a probability curve for the existence of a transmembrane domain in UBP1, obtained using the TMHMM Prediction of transmembrane helices in proteins (CBS; Denmark) package; a 51 amino-acid transmembrane domain was noted. 
         FIG. 4  represents a micrograph of Gramm-stained BLD21  E. coli  cells containing the plasmid pT7-7ArgStop UBI+UBP1. 
         FIG. 5  (SEQ ID NO: 3) The sequence coding the hybrid protein UBI::UBP1 ΔC with the transmembrane domain removed (lower case denotes the UBI sequence, upper case denotes the UBP1 ΔC protease coding sequence) 
         FIG. 6  (SEQ ID NO: 5) The amino-acid sequence of the UBP1 protease, and the proposed changes. The sequence of the removed transmembrane fragment is in bold. The active centre is in underlined italics, whereas amino-acids substituted with leucine, called by us mutations A, B and C are in bold underlined type. 
         FIG. 7  A micrograph representing an in vivo preparation of a BLD21  E. coli  culture with the plasmid pT7-7ArgStopUBI+UBP1ΔC with exchanged ariginine codons. 
         FIG. 8  (SEQ ID NO: 6 and SEQ ID NO: 7) The nucleotide and amino-acid sequence of UBI+UBP 1ABC, the mutations are substitutions of: proline for leucine (position 493), phenyloalanine for leucine (position 814) and glutamine for leucine (position 829); amino-acid residues are marked with bold, underlined type. 
         FIG. 9  (SEQ ID NO: 8 and SEQ ID NO: 9) The nucleotide and amino-acid sequence of UBI+UBP1BC, the mutations are substitutions of: phenyloalanine for leucine (position 814) and glutamine for leucine (position 829); amino-acid residues are marked with bold, underlined type. 
         FIG. 10  (SEQ ID NO: 10 and SEQ ID NO: 11) The nucleotide and amino-acid sequence of UBI+UBP1C, the mutation is a substitution of glutamine for leucine (position 829); the amino-acid residue is marked with bold, underlined type. 
         FIG. 11  Examination of the activity of the mutants obtained, digestion products: electrophoretic separation in polyacrylamide gel with SDS. From the left, the lanes are: lane No. 1, molecular mass marker (from the top 97, 66, 45, 30, 20.1, 14.4 [kDa]); lanes 2 and 3, digestion of UBI::INF with the UBP1ΔC protease over 2 h at 37° C.; lane 4, undigested UBI::INF; lanes 5 and 6, UBI::INF digested with the UBP1ΔC protease over 1 h at 37° C. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     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 
     Mutants of the UBP1 Protein 
     Example UBP1 mutants containing point mutations: 
     I UBP1ABC with the A, B, C mutation, a substitution of proline, phenyloalanine and glutamine with leucine at positions 415, 739 and 754 respectively. 
     II UBP1BC with mutations at positions 739 and 754, substitutions phenyloalanine and glutamine respectively with leucine, 
     III UBP1C a mutation at position 754, a substitution of glutamine with leucine. Using these mutants, hybrid proteins were designed which additionally contain the ubiquitin amino-acid sequence at the N-end (proteins: UBI+UBP1ABC, UBI+UBP1BC and UBI+UBP1C). 
     The next group of mutants was produced by deleting the transmembrane domain of UBP1 or a portion thereof from the above proteins (example proteins: UBP1ΔC, UBI+UBP1ΔC and their mutants containing at least one of the mutations A, B or C). 
     A1 mutations are located outside of the active centre, composed of cysteine (100-117 aa) and histidine (681-725 aa) residues, marked with underlined italics in  FIG. 6  which represents the amino-acid sequence of UBP1 with the modified portions indicated. 
     The above mentioned protease variants were used as an enzyme severing ubiquitin from proteins fused to its C-end. In our case it was the hybrid UBI: Interferon α. 
     Example 2 
     Construction of a Plasmid with the UBP1 Protease Gene and its Mutants 
     The UBP1 protease gene, 2430 base pairs long, was obtained using PCR. The template used was genomic DNA of  Saccharomyces cerevisiae , strain W303 (ade2-1, leu2-3, 112, trp1-1, his3-11, ura3-1, mit+, rho+). For amplification, the following primers were designed: 
     
       
         
               
               
             
           
               
                 UBP1P 
                   
               
               
                     SacII 
                   
               
               
                 5′ AGACT   CCGCGG   TGGTGATTTGTTTATTGAAA 
                 (SEQ ID NO: 12) 
               
               
                 GCAAGATA 
                   
               
               
                   
               
               
                 UBP1K 
                   
               
               
                    BamHI 
                   
               
               
                 5′ GG   GGATCC   TTAGTTTACATCTTTACCAGAAA 
                 (SEQ ID NO: 13) 
               
               
                 TA 
                   
               
             
          
         
       
     
     The oligonucleotides contained recognitions sites for the restriction endonucleases SacII and BamHI. The amplified DNA fragment was ligated with the pBluescript SK(−) vector, digested with the same enzymes. The ligation mixture was used to transform competent  E. coli  cells, strain NM522. Plasmid DNA was isolated using the alkaline method. Next, the 2430 bp UBP1 gene was excised from the recombinant using the restriction enzymes SacII and BamHI. DNA obtained in this way was ligated with the expression vector pT7-7ArgStopUBI, which was created by ligating the 240 bp ubiquitin gene sequence into the pT7-7ArgStop plasmid ( FIG. 2 ) into NdeI and EcoRI restriction sites. The PT7-7ArgStop plasmid was created in the laboratory of Prof. Dr. hab. Andrzej Plucienniczak, based on the pT7-7 plasmid (S. Tabor, C. Richardson,  Proc. Nat. Acad. Sci.  1985,262:1074-1078). 
     The pT7-7ArgStopUBI vector was digested with the SacII and BamHI enzymes, and then ligated with the DNA fragment coding UBP1. The ligation mixture was used to transform the competent  E. coli  strain DH5α. The DNA was then isolated, and the sequence was determined, shown in  FIG. 1 . 
     The protease gene was included into the expression vector pT7-7ArgStopUBI. The obtained plasmids with the hybrid gene UBI::UBP1 were used to transform BLD21  E. coli  bacteria. The protein UBP1 was synthesized (produced) while these bacteria were cultyred. The culture was maintained at 25° C. in LB medium with an addition of 50 mg/ml of ampicillin. 30 hours were required for the culture to reach an OD 600 =1. Gramm-stained slides were made. It turned out that the  E. coli  bacteria were several dozen times longer than usual ( FIG. 4 ). 
     The UBP1 sequence was examined using the TMHMM software package, which determines the likelihood of the existence of a transmembrane domain ( FIG. 3 ). The domain discovered could retard bacterial growth and cell division. This might have been the cause of the long time it took for the growth to reach OD 600 =1. 
     PCR was used to remove this domain from the UBP1 gene. The modification was based on inserting an additional SacII restriction site into the sequence coding UBP1. 
     Primers were designed for this reason, which were used in for point mutagenesis using the “QuikChange Site-Directed Mutagenesis Kit” from Stratagene: 
     
       
         
               
               
             
           
               
                 UBP1MG 
                   
               
               
                        SacII 
                   
               
               
                 5′ GGCATAGTAGTATTTTTTTA   CCGCGG   TGGTG 
                 (SEQ ID NO: 14) 
               
               
                 ACCATCTAAACTACATTGT 
                   
               
               
                   
               
               
                 UBP1MD 
                   
               
               
                           SacII 
                   
               
               
                 5′ ACAATGTAGTTTAGATGGTCACCA   CCGCGG   T 
                 (SEQ ID NO: 15) 
               
               
                 AAAAAAATACTACTATGCC 
                   
               
             
          
         
       
     
     Using the UBP1MG and UBP1MD primers (marked in bold in  FIG. 1 ), recognition sequences for the SacII enzyme (underlined) were inserted into the interior of the UBP1 coding sequence. Thanks to this a 169 bp fragment was removed during the digestion of the pT7-7ArgStopUBI+UBP1 plasmid with the SacII restrictase. This resulted in a new plasmid, which we designated pT7-7ArgStopUBI+UBP1ΔC. It contains the coding sequence shown in  FIG. 5 . Other plasmids coding alternate hybrid mutants according to the present invention containing the UBI sequence were produced in an analogous fashion. 
     Example 3 
     Expression of the UBP1ΔC Protease and Enzyme Purification 
     BLD21  E. coli  bacteria were transformed with a plasmid containing the UBP1 protease gene, or one of its mutants. During the culturing it was determined that the removal of the transmembrane domain facilitated the culturing, and shortened the time from 30 to about 12 hours. It was also observed that the cells producing the mutant according to the present invention returned to the original shape ( FIG. 7 ). 
     BLD21  E. coli  bacteria containing the appropriate plasmid were cultured on LB medium containing ampicillin (50 mg/ml) at 25° C. over 12 h until OD 600 =1, and subsequently induced with the addition of IPTG (isopropylthiogalactoside). After 2.5 h, the bacteria were centrifuged. The cell pellet was suspended in lysis buffer, and incubated for 30 min. at 20° C. Triton X-100 was added to a final concentration of 1%. The mixture was sonificated and centrifuged. The supernatant was applied to an SP column (the strong cationite Sepharose FF) and subsequently to a hydrophobic Phenylo Sepharose FF column. The protease activity was measured via the digestion of UBI::Interferonoc with purified enzyme fractions. The results are presented in  FIG. 11 . 
     Additionally, the UBP1 protease gene was modified through the exchange of certain arginine codons unfavourable to  E. coli  (AGA or AGG) for codons which occur in these bacteria  E. coli  (CGT or CGC). In the ultimate version, the arginine codons at positions 96, 476, 482, 487, 702, 705, 710, 796 and 801 were replaced, marked in bold in  FIG. 6 .