Abstract:
A mutated severe acute respiratory syndrome-associated coronavirus 3C-like protease and use thereof for cleaving a protein that includes a cleavage site recognizable by the mutated protease to yield a polypeptide fragment of interest.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    Fusion protein technology, i.e., expressing in a host cell a target protein fused with a protein tag, confers several advantages. For example, many protein tags increase water-solubility and facilitate purification of their fusion partners. Further, several protein tags serve as protein chaperons to guide proper folding of the target proteins fused to them, thereby increasing the yields of the target proteins in native form. 
         [0002]    One the other hand, fusion protein technology is disadvantaged in that protein tags often interfere with the structural or functional properties of the target proteins. Thus, they need to be removed via, e.g., chemical or enzymatical cleavage, from fusion proteins to release the target proteins. Cleavage of a fusion protein to remove the protein tag remains a major challenge in fusion technology as imprecise cleavage would result in failure to recover a structurally intact target protein. 
       SUMMARY OF THE INVENTION 
       [0003]    The present invention is based on the unexpected discovery that replacing T at position 25 (T 25 ) in the severe acute respiratory syndrome-associated coronavirus 3C-like protease (SARS-CoV 3CL pro ) with G alters the substrate specificity of the protease. 
         [0004]    Accordingly, one aspect of this invention relates to a SARS-CoV 3CL pro  mutant, in which the T 25  residue in the wild-type SARS-CoV 3CL pro  is replaced by G. This mutant recognizes the cleavage site P 4 P 3 P 2 QP 1′ , in which each of P 4  and P 3 , independently, is A, V, G, L, or I, P 2  is L, V, or F, and P 1′  is G, A, V, L, I, S, T, M, C, D, E, Q, N, W, Y, or F, and cleaves between Q and P 1′ . In one example, the cleavage site is ALVQM. The SARS-CoV 3CL pro  mutant can have an amino acid sequence at least 90% (e.g., 95%) identical to that of its wild-type counterpart (SEQ ID NO:1). In one example, the mutant has the amino acid sequence of SEQ ID NO:2. Also disclosed herein is a nucleic acid that encodes the SARS-CoV 3CL pro  mutant. 
         [0005]    Another aspect of this invention relates to use of the above-described SARS-CoV 3CL pro  mutant to cleave a polypeptide that contains the cleavage site P 4 P 3 P 2 QP 1′  also described above. Preferably, the polypeptide is a fusion protein containing a protein tag and a target protein. The cleavage site P 4 P 3 P 2 QP 1′  is located at the junction of the protein tag and the target protein, P 1′  being the N-terminal amino acid residue of the target protein. When contacted with the SARS-CoV 3CL pro  mutant, the fusion protein is cleaved by this protease to release the target protein. 
         [0006]    In yet another aspect, the present invention features an expression vector for producing the polypeptide mentioned above. This expression vector includes a first nucleotide sequence that encodes the amino acid sequence of P 4 P 3 P 2 Q, in which each of P 4  and P 3 , independently, is A, V, G, L, or I and P 2  is L, V, or F. The 3′ end of the first nucleotide sequence is a restriction site (e.g., Pst I) for cloning a nucleotide sequence encoding a target protein. In one example, the amino acid sequence P 4 P 3 P 2 Q is AVLQ, which can be encoded by the nucleotide sequence of GCGGTGCTGCAG. The expression vector can further include a second nucleotide sequence encoding a protein tag and optionally, a third nucleotide sequence encoding a target protein. The second nucleotide sequence is located upstream to the first nucleotide sequence while the third nucleotide sequence is located downstream to it. The first and the third nucleotide sequences, taken together, encode a polypeptide including the cleavage site P 4 P 3 P 2 QP 1′  described above, P 1′  being the N-terminus amino acid residue of the target protein encoded by the third nucleotide sequence. 
         [0007]    Also within the scope of this invention is a kit for producing a recombinant target protein, including the expression vector described above and the SARS-CoV 3CL pro  mutant also described above. 
         [0008]    The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0009]      FIG. 1  is a schematic diagram showing a protein expression system. In this system, a fusion protein, including one or more protein tags, a protease cleavage site AVLQM, and a target protein, is expressed and then treated with a SARS-CoV 3CL pro  mutant, i.e., T25G, to release the target protein. 
           [0010]      FIG. 2  is a schematic diagram showing the structures of fusion proteins produced in the protein expression system described herein. A: A fusion protein containing a number of protein tags, a cleavage site recognizable by the T25G mutant, and the  E. coli  undecaprenyl diphosphate synthase (UPPs). B: A fusion protein containing two protein tags, a cleavage site recognizable by the T25G mutant, and an enhanced green fluorescent protein (EGFP). 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0011]    Described herein is a protein expression system, in which a target protein is initially expressed as a fragment of a fusion protein and then released from the fusion protein via cleavage by a mutated SARS-CoV 3CL pro . See  FIG. 1 . 
         [0012]    In this system, a fusion protein containing a target protein can be produced in suitable host cells (e.g.,  E. coli  cells, yeast cells, insect cells, and mammalian cells) by conventional recombinant technology. More specifically, a nucleotide sequence encoding the target protein can be isolated from its natural source via, e.g., polymerase chain reaction, and then cloned into an expression vector for protein production. The term “expression vector” used herein refers to a DNA plasmid that includes a promoter sequence operably linked to an encoding nucleotide sequence. A promoter sequence is a nucleotide sequence containing elements that initiates the transcription of an operably linked nucleic acid sequence. At a minimum, a promoter contains an RNA polymerase binding site. It can further contain one or more enhancer elements which, by definition, enhances transcription, or one or more regulatory elements that control the on/off status of the promoter. 
         [0013]    The expression vector used in the protein expression system described herein is designed for expressing a fusion protein, in which a target protein is linked to one or more suitable protein tags (e.g., hexa-His, the starch-binding domain of fungi glycomylase, maltose binding protein, N-utilizing substance A, thioredoxin, calmodulin-binding protein, glutathione S-transferase, and α-factor). The junction region of a protein tag and the target protein has the amino acid sequence of P 4 P 3 P 2 Q↓P 1′  described above, P 1′  being the N-terminal residue of the target protein. Preferably, the nucleotide sequence encoding residues P 2 Q is also a restriction site for cloning a nucleotide sequence encoding the target protein. In one example, the junction region has the amino acid sequence of AVLQM, in which LQ is encoded by the nucleotide sequence of CTGCAG, a Pst I site. See  FIG. 1 . The fusion protein described above is then treated with the mutated SARS-CoV 3CL pro  described herein, which recognizes the cleavage site P 4 P 3 P 2 QP 1′  and cut between Q and P 1′ , to release the target protein. 
         [0014]    The mutated SARS-CoV 3CL pro  is derived from the wild-type SARS-CoV 3CL pro  having the amino acid sequence of SEQ ID NO:1 shown below: 
         [0000]    
       
         
               
               
             
               
               
             
               
               
               
             
           
               
                 Amino acid sequence of wild-type SARS-CoV 3CL pro   
                   
               
             
          
           
               
                 (SEQ ID NO: 1) 
                   
               
             
          
           
               
                 sgfrkmafps gkvegcmvqv tcgt T tlngl wlddtvycpr hvictaedml 
                 50 
                   
               
               
                   
               
               
                 npnyedllir ksnhsflvqa gnvqlrvigh smqncllrlk vdtsnpktpk 
                 100 
               
               
                   
               
               
                 ykfvriqpgq tfsvlacyng spsgvyqcam rpnhtikgsf lngscgsvgf 
                 150 
               
               
                   
               
               
                 nidydcvsfc ymhhmelptg vhagtdlegk fygpfvdrqt aqaagtdtti 
                 200 
               
               
                   
               
               
                 tlnvlawlya avingdrwfl nrftttlndf nlvamkynye pltqdhvdil 
                 250 
               
               
                   
               
               
                 gplsaqtgia vldmcaalke llqngmngrt ilgstilede ftpfdvvrqc 
                 300 
               
               
                   
               
               
                 sgvtfq 
               
             
          
         
       
     
         [0015]    The wild-type SARS-CoV 3CL pro  m is a protease that specifically recognizes the cleavage site of AVLQS and cleaves between Q and S. We have discovered that the T residue at position 25 (T 25 ; capitalized and boldfaced) in SEQ ID NO:1 is critical in determining substrate specificity of the protease. The SARS-CoV 3CL pro  mutant disclosed herein has a G residue, instead of T, at the position corresponding to position 25 in SEQ ID NO:1. This mutant has an amino acid sequence at least 80% (e.g., 85%) identical to SEQ ID NO:1 and recognizes the cleavage site P 4 P 3 P 2 QP 1′ , described above. In one example, the SARS-CoV 3CL pro  mutant has the following amino acid sequence: 
         [0000]                                                Amino acid sequence of SARS-CoV 3CL pro  mutant                (SEQ ID NO: 2)                sgfrkmafps gkvegcmvqv tcgt G tlngl wlddtvycpr hvictaedml   50                   npnyedllir ksnhsflvqa gnvqlrvigh smqncllrlk vdtsnpktpk   100               ykfvriqpgq tfsvlacyng spsgvyqcam rpnhtikgsf lngscgsvgf   150               nidydcvsfc ymhhmelptg vhagtdlegk fygpfvdrqt aqaagtdtti   200               tlnvlawlya avingdrwfl nrftttlndf nlvamkynye pltqdhvdil   250               gplsaqtgia vldmcaalke llqngmngrt ilgstilede ftpfdvvrqc   300               sgvtfq            
While the wild-type SARS-CoV 3CL pro  does not recognize a cleavage site of P 4 P 3 P 2 QP 1′ , where P 1′  is an amino acid residue with a bulky side chain (e.g., M and L), the mutated protease recognizes such a cleavage site and cut precisely between Q and P 1′ .
 
         [0016]    The SARS-CoV 3CL pro  mutant can be prepared by conventional methods, e.g., mutagenesis technology. For example, mutations can be introduced into a nucleotide sequence encoding the wild-type SARS-CoV 3CL pro  so that the codon encoding T 25  in the wild-type protease is replaced with a codon encoding G. The nucleotide sequence carrying the mutations can then be inserted into an expression vector and its encoding SARS-CoV 3CL pro  mutant can be expressed in a suitable host cell. Upon purification, the mutant can be analyzed to confirm the protease activity and substrate specificity by conventional methods, some of which are described in Examples 1 and 2 below. 
         [0017]    Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference. 
       Example 1 
     Preparation of 3CL pro  Mutant T25G 
       [0018]    A DNA fragment encoding the wild-type SARS-CoV 3CL pro  was cloned into pET32Xa/Lic vector and expressed in  E. coli ; the protease thus obtained was purified from the host cell following the methods described in Kuo et al.,  Biochem. Biophys. Res. Comm.  318:862-867 (2004). 
         [0019]    The nucleotide sequence encoding the wild-type SARS 3CL pro  was subjected to mutagenesis to produce a nucleotide sequence encoding the mutated SARS-CoV 3CL pro  T25G using the QuickChange site-directed mutagenesis kit (Invitrogen). More specifically, a DNA fragment encoding the T25G mutant was obtained by polymerase chain amplification (PCR) using the nucleotide sequence encoding the wild-type SARS-CoV 3CL pro  as a template and the primers shown below: 
         [0020]    Forward primer: 5′-GGTGCATGGTACAAGTAACCTGTGGAACTGGAACTCTTAA TGGAT TGTGGTTGG-3′; (the underlined nucleotides referring to the mutated codon). 
         [0021]    Reverse primer: 5′-CCAACCACAATCCATTAAGAGTTCCAGTTCCACAGGTTACT TGTACCATGCACC-3′ (the underlined nucleotides referring to the mutated codon). After being treated with DpnI to remove those that contain the wild-type SARS-CoV 3CL pro  gene, the PCR products were introduced into  E. coli  BL21 host cells and positive transformants were selected. Plasmids, isolated from those transformants, were analyzed by DNA sequencing to confirm that they included the nucleotide sequence encoding the T25G mutant. After confirmation, the transformants were cultured under suitable conditions for expression of the T25G mutant. 
         [0022]    The DNA fragment encoding the T25G mutant was used as a template for producing a DNA fragment encoding His-tagged T25G, using the primers: 
         [0000]                    Forward primer:       5′-CATG CCATGG CCAGTGGTTTTAGGAAAATGGCATTCCCG-3′;       and               Reverse primer:       5′-CCG CTCGAG CGGTCAATGATGATGATGATGATGTTGGAAGGTAACAC               CAGAGCA-3′.            
The underlined regions refer to the restriction sites of Nco I and Xho I. The PCR product was cloned into the pET16b vector (Novagen) via the Nco I and Xho I cloning sites. The resultant pET16b-His-T25G plasmid was introduced into  E. coli  BL21 (DE3) for expression of His-tagged T25G.
 
         [0023]    The wild-type SARS-CoV 3CL pro , T25G mutant, and His-tagged T25G, expressed in  E. coli  host cells, were purified and then analyzed for their protease activity and substrate specificity as follows. 
         [0024]    The protease activity of the three proteins were determined using a fluorogenic substrate Dabcyl-KTS         GFRKME-Edans. See Kuo et al., 2004. This substrate includes the native cleavage site of SARS-CoV 3CL pro  (highlighted), which cleaves between Q (position P 1 ) and S (position P 1′ ). Both T25G and His-T25G exhibited similar protease activity in cleaving the just-noted substrate as compared to the wild-type SARS-CoV 3CL pro . This result indicated that the T →G mutation at position 25 in the wild-type SARS-CoV 3CL pro  does not affect the protease activity. 
         [0025]    To determine substrate specificity of the wild-type SARS-CoV 3CL pro  and the T25G mutant, either protein was mixed with each of the ten substrates listed below: 
         [0000]    
       
         
               
             
           
               
                   
               
             
             
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
         [0026]    These peptide substrates were synthesized using a 433A peptide synthesizer (Applied Biosystems, USA) as follows. Starting with 0.10 mmol (0.101 g) of p-hydroxymethyl phenoxymethyl polystyrene resin (1.01 mmol/g), the synthesis of the peptides was performed using a stepwise FastMoc protocol (Applied Biosystems, USA). The amino acids were introduced using the manufacturer&#39;s prepacked cartridges (1 mmol each). 
         [0027]    To analyze the substrate specificity of the wild-type SARS-CoV 3CL pro  and the T25G mutant, each of the peptide substrates (100 μM) was incubated with 0.1 μM protease for 1, 2, and 6 h, and the reaction products were analyzed by HPLC using a C-18 reverse-phase analytic column (Vydac) to determine whether the substrate was cleaved. 
         [0028]    The results obtained from this study indicate that both the wild-type SARS-CoV 3CL pro  and the T25G mutant recognized the cleavage sites where position P 1′  is a small amino acid residue, i.e., G and S, and both proteases did not recognize the cleave sites where position P 1′  is H, K, or P. The results also indicated that T25G cleaved substrates 1, 2, 6, and 7, which contain the cleave sites where position P 1′  is E, F, L, and M, while the wild-type SARS-CoV 3CL pro  did not cleave these substrates. 
         [0029]    Next, the kinetics of the protease activity of both the wild-type SARS-CoV 3CL pro  and the T25G mutant were determined as follows, using peptide SAVLQMGFRK as the substrate, i.e., Substrate (7). The cleavage products were resolved using a 30 min, 2-90% liner gradient of acetonitrile supplemented with 0.1% TFA. The areas of the product peaks, determined by HPLC analysis, were integrated to calculate the reaction rate of either wild-type SARS-CoV 3CL pro  (0.1 μM) or the T25G mutant (0.1 μM) for cleaving each substrate at various concentrations (10-200 μM). A reaction curve for each protein was drawn based on the reaction rates versus substrate concentrations and the kinetic parameters (i.e., k cat  and K m ) were determined based on the reaction curve using Michaelis-Menten equation fitted with the KaleidaGraph computer program. The results are shown below: 
         [0030]    Wild-type SARS-CoV 3CL pro : k cat  is 1.6±0.2 min −1  and K m  is 76.6±3.5 μM (k cat /K m =0.02 μM −1 min −1 ). 
         [0031]    T25G mutant: k cat  is 16.2±0.5 min −1  and K m  is 18.6±2.4 μM (k cat /K m =0.87 μM −1 min −1 ). The catalytic efficiency of the T25G mutant in cleaving SAVLQ↓MGFRK was 43.5-fold higher than that of the wild type. 
       Example 2 
     Preparation of Target Proteins with Protein Expression System Involving SARS-CoV 3CL pro  Mutant T25G 
       [0032]    A yeast expression vector, pHTPY6, for expression a fusion protein, was constructed as follows. Two oligonucleotides: 5′-TCGAAAAAAGAGAGGCTGAAGCTGAATTCTGCA GCTCGAGCGTGGCCCAGCCGGCCGTCTCGGATCGGTACG-3′ and 5′-TCGACGTACCG ATCCGAGACGGCCGGCTGGGCCACGCTCGAGCTGCAGAATTCAGCTTCAGCCTCTCT TTTT-3′ were annealed to form a double-stranded fragment including an EcoR I and an Xho I sites, as well as two mutated Xho I sites (underlined). The fragment was then phosphorylated and inserted into pPICZαA (Invitrogen) via the two Xho I cloning sites contained therein to form the pHTPY1 vector. In this vector, the two Xho I sites (CTCGAG) contained in pPICZαA were replaced by the nucleotide sequences CTCGAA and CTCGAC, both of which could no longer be digested by Xho I. 
         [0033]    Next, the pHTPY6 vector was further modified to insert a nucleotide sequence that encodes a His-tag and the SARS-CoV 3CL pro  recognition sequence Ala-Val-Leu-Gln. Two oligonucleotides: 5′-AATTCACGGGTACCGCCCAGCCGGCCCACCACCACCACCACC ACGGAGGAGGAACTAGTGCGGTGCTGCAGC-3′ and 5′-TCGAGCTGCAGCACCGCA CTAGTTCCTCCTCCGT GGTGGTGGTGGTGGTGGGCCGGCTGGGCGGTACCCGTG-3′ were annealed to form a double-stranded fragment, the fragment being phosphorylated and then inserted into the pHTPY6 vector via the EcoR I and Xho I cloning sites. Subsequently, the following primers, i.e., forward primers 5′-AATTCGCAAGTATTCCTAGCAGTGCT-3′, 5′-CGCAAGTATTCCTAGCAGTGCT-3′, and backward primers 5′-GTACCTGTAGATACTT GGTAATTGGC-3′ and 5′-CTGTAGATACTTGGTAATTGGC-3′ were used to generate a DNA fragment (containing the EcoR I and Kpn I cloning sites) that encodes the starch-binding domain (SBD) of glucomylase derived from fungi  Rhizopus  ssp, using the SBD-encoding gene as a template. See US Patent Publication 20060198792. The PCR product was then ligated into the pHTPY6 vector via the EcoR I and Kpn I cloning sites to form the pHTPY7 vector. 
         [0034]    A DNA fragment encoding enhanced green fluorescent protein (EGFP), prepared by the sticky-end PCR method described in Zeng, Biotechniques 25:206-208 (1998) and Shih et al., Protein Sci. 11:1714-1719 (2002), was cloned into the pHTPY7 vector via cloning sites Pst I and Xho Ito generate pHTPY7-EGFP expression plasmid. 
         [0035]    An  E. coli  expression plasmid was constructed as follows. The following two primers were used to amplify a DNA fragment encoding the undecaprenyl diphosphate synthase (UPPs, see Pan et al.,  Biochemistry  39:10936-10942, 2000) and the cleavage site of SARS-CoV 3CL pro  (Ala-Val-Leu-Gln, encoded by the underlined nucleotide sequence in the primers): 5′-GGTATT GAGGGTCGCGCGGTGCTGCAGATGTTGTCTGCTACTCAACC-3′ and reverse primer 5′-AGAGGAGAGTTAGAGCCTCAGGCTGTTTCATCACC-3′. The PCR product was cloned into the pET32Xa/Lic vector to produce the expression plasmid pET32Xa/Lic-UPPs. 
         [0036]    The expression plasmids pET32Xa/Lic-UPPs and pHTPY7-EGFP mentioned above were introduced into an  E. coli  host cell and a yeast  Pichia  host cell, respectively, for expression of fusion proteins His-Thioredoxin (Trx)-AVLQM-UPPs (see  FIG. 2 , Panel A) and His-SBD-AVLQM-EGFP (see  FIG. 2 , Panel B), M being the N-terminal amino acid residue of both and UPPs and EGFP. The fusion proteins were purified using a NiNTA column. 5 μg of each purified fusion protein was treated by 0.1 μM the wild-type SARS-CoV 3CL pro  or 0.1 μM the T25G mutant at 37° C. for 90 minutes to release free EGFP and UPPs. Only the T25G mutant cleaved both fusion proteins between the SBD tag and EGFP and between the His-Trx tag and UPPs. These results indicate that this protease mutant recognizes the cleavage site AVLQM and cut precisely between Q and M in the cleavage site. 
       Other Embodiments 
       [0037]    All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. 
         [0038]    From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.