Abstract:
The present invention relates to potato virus NIa protease variants or fragments thereof, polynucleotides encoding them, and methods of making and using the foregoing.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. Provisional Application Ser. No. 61/324,972, filed 16 Apr. 2010, the entire contents of which is incorporated herein by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to potato virus NIa protease variants or fragments thereof, polynucleotides encoding them, and methods of making and using the foregoing. 
     BACKGROUND OF THE INVENTION 
     Considerable effort has been employed to engineer enzymes and other proteins to achieve higher selectively and/or specific activity (Matsumura and Ellington, J. Mol. Biol. 305:331-339, 2001; Rothman and Kirsch, J. Mol. Biol. 327:593-608, 2003; Aharoni et al., Nature Genetics, 37:73-76, 2005). Human trypsin-like serine proteases are an appealing target for engineering with the goal to tailor proteases to recognize a specific, predefined primary sequence within a target protein that is normally not recognized, resulting in specific spatial and temporal modulation of target activity. Trypsin-like serine proteases are also valuable research tools in molecular biology. 
     Manufacturing of trypsin-like serine proteases poses challenges due to their structural complexity related to the required appropriate disulfide bond formation and proper processing of the native globular polypeptide chain for activity. Furthermore, trypsin-like serine proteases often have a constricted recognition sequence limiting the absolute specificity that can be engineered into the molecules. (Gosalia et al., Mol. Cell. Proteomics, 4:626-36, 2005, US Pat. Appl. No. US20040072276A1). An alternative to human trypsin-like serine proteases, intracellular plant viral proteases that are easier to manufacture could be used as a starting point to develop therapeutics as well as new research tools. 
     Potyviruses are a class of plant viruses transmitted mainly by aphids, causing significant losses in pasture, agricultural, horticultural and ornamental crops annually. Typical representatives of potyviruses are Potato virus A (PVA), tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV). Potyvirus monopartite genome contains (+) stranded RNA, covalently linked to a viral encoded protein (VPg) at the 5′-end and polyadenylated at the 3′-end (Dougherty et al., The EMBO J. 7:1281-1287, 1988). The genome serves as an mRNA and a template for the synthesis of a complementary (−) stranded RNA by a polymerase translated from the viral genome. Upon entry into the cell, the virus RNA binds to endogenous ribosomes and the genome is translated as a single polypeptide chain. The large single polyprotein is subsequently processed into mature proteins by three virus-encoded proteases (Verchot et al., Virology, 190:298-306, 1992), the first protein (P1), the helper component (HC), and the nuclear inclusion protein (NIa) proteases. The NIa protease is responsible for the majority of the polyprotein processing, including the generation of mature RNA replication-associated proteins and capsid proteins (Verchot et al., Virology, 190:298-306, 1992). 
     The NIa proteases belong to the family of picornavirus 3C cysteine proteases (Parks et al., Virology, 210:194-201, 1995), that exhibit an extended P6-P1′ recognition sequence EXXYXQ*(S/G) (SEQ ID NO: 69) (Dougherty et al., Virology, 171:356-364, 1989). Although there are striking similarities in the recognition sequence for NIa proteases across the potyvirus members, each protease is highly specific for its own target sequence (Tozer et al., The FEBS J. 272:514-523, 2004). Structurally, NIa proteases appear to be related to trypsin-like serine proteases through divergent evolution involving replacement of NIa catalytic cysteine by serine in the trypsin-like proteases (Bazan and Fletterick, Proc. Natl. Acad. Sci. 85:7872-7876, 1988). NIa and trypsin-like serine proteases share a similar overall 3-dimensional protein fold as well as the spatial proximity of their respective catalytic residues. The 3C-like family of cysteine proteases offers several advantages over more complex extracellular proteases. They can be easily produced in the cytosol of bacteria, have no disulfide bonds, and have an extended substrate recognition sequence. The challenge of using the 3C-like proteases is their activity loss in non-reducing conditions due to oxidation of active site and/or surface exposed cysteines, therefore limiting their use (Higaki et al., Cold Spring Harbor Symposia on Quantitative Biology, 615-621, 1987). Therefore, the proteases require reducing agent to sustain their functional activity (Nunn et al., J. Mol. Biol. 350:145-55, 2005; Birch et al., Protein Expression and Purification 6:609-18, 1995). Thus, there is a need for engineered plant viral proteases that remain active in the absence of exogenous reducing agents. 
     SUMMARY OF INVENTION 
     One aspect of the invention is an isolated polypeptide encoding a NIa protease variant, wherein the variant is resistant to oxidation and retains activity. 
     Another aspect of the invention is an isolated polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 1 having amino acid substitutions selected from the group consisting of:
         a. cysteine at position 19 is substituted for serine or valine;   b. cysteine at position 110 is substituted for serine;   c. cysteine at position 151 is substituted for serine or alanine.   d. cysteine at position 181 is substituted for serine; and   e. cysteine at position 211 is substituted for serine.       

     Another aspect of the invention is an isolated polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 28. 
     Another aspect of the invention is isolated polynucleotides encoding the polypeptides of the invention. 
     Another aspect of the invention is a vector comprising an isolated polynucleotide encoding a polypeptide of the invention. 
     Another aspect of the invention is an isolated host cell comprising the vector of the invention. 
     Another aspect of the invention is a method for expressing the polypeptides of the invention. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth. 
     As used herein and in the claims, the singular forms “a,” “and,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a polypeptide” is a reference to one or more polypeptides and includes equivalents thereof known to those skilled in the art. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which an invention belongs. Although any compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the invention, exemplary compositions and methods are described herein. 
     The term “NIa protease” as used herein refers to the potato virus A (PVA) NIa protease encoded by amino acids 2032-2264 of the virus proprotein shown in GenBank Acc. No. CAB58238. The polypeptide sequence of the NIa protease is shown in SEQ ID NO: 1. 
     The term “polypeptide” as used herein refers to a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as “peptides”. Polypeptides may also be referred as “proteins.” 
     The term “polynucleotide” as used herein refers to a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. Double and single stranded DNAs and RNAs are typical examples of polynucleotides. 
     The term “complementary sequence” means a second isolated polynucleotide sequence that is antiparallel to a first isolated polynucleotide sequence and that comprises nucleotides complementary to the nucleotides in the first polynucleotide sequence. Typically, such “complementary sequences” are capable of forming a double-stranded polynucleotide molecule such as double-stranded DNA or double-stranded RNA when combined under appropriate conditions with the first isolated polynucleotide sequence. 
     The term “variant” as used herein refers to a polypeptide or a polynucleotide that differs from a reference “wild type” polypeptide or a polynucleotide and may or may not retain essential properties. Generally, differences in sequences of the wild type and the variant are closely similar overall and, in many regions, identical. A variant may differ from the wild type in its sequence by one or more modifications for example, substitutions, insertions or deletions of nucleotides or amino acids. Substitutions or insertions may result in conservative or non-conservative amino acid substitutions, or in the generation of a stop codon. A variant of a polynucleotide may be naturally occurring, and may have 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the wild type polynucleotide. 
     It is possible to modify the structure or function of the polypeptides encoded by variant polynucleotide sequences for such purposes as enhancing activity, specificity, stability, solubility, and the like. A replacement of a codon encoding leucine with codons encoding isoleucine or valine, a codon encoding an aspartate with a codon encoding glutamate, a codon encoding threonine with a codon encoding serine, or a similar replacement of codons encoding structurally related amino acids (i.e., conservative mutations) will, in some instances but not all, not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that share chemically related side chains. Naturally occurring amino acids can be divided into four families based on their side chains: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. Alternatively, naturally occurring amino acids can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co., 1981). Whether a change in the amino acid sequence of a polypeptide or fragment thereof encoded by a variant polynucleotide results in a functional homolog can be readily determined by assessing the ability of the modified polypeptide or fragment to produce a response in a fashion similar to the unmodified polypeptide or fragment using the assays described herein. Peptides, polypeptides or proteins in which more than one replacement has taken place can readily be tested in the same manner. 
     The term “wild type” or “WT” refers to a polypeptide or a polynucleotide that has the characteristics of that polypeptide or polynucleotide when isolated from a naturally occurring source. An exemplary wild type polynucleotide is a polynucleotide encoding a gene that is most frequently observed in a population and is thus arbitrarily designated the “normal” or “reference” or “wild type” form. 
     The term “activity” or “active” as used herein refers to an active NIa protease, e.g., a NIa protease capable of cleaving its substrate. Exemplary substrates are synthetic peptides corresponding to identified recognition sequences, for example SEVVLFQASS (SEQ ID NO: 70), SEAVYTQGSS (SEQ ID NO: 71), or SENVTFQGSS (SEQ ID NO: 72), as described in Table 5. and in Mertis et al., (Mertis et al., J. Gen. Virol. 83:1211-1221, 2002). Partial cleavage of the substrate is sufficient for effective biological activity of the protease, for example cleavage of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of a substrate. Thus, biological activity does not require complete cleavage of the substrate. “Partially active” refers to a NIa protease that partially cleaves its substrate. 
     The term “resistant to oxidation” or “oxidation resistant” as used herein means that the NIa protease variant is active and functionally stable in the absence of a reducing agent that is required for functional stability of the wild type NIa protease. The reducing agent required for the activity of the wild type NIa protease can be dithiotreitol (DTT), 2-mercaptoethanol or tris carboxyethylphosphate (TCEP), typically in the range of 0.1-10 mM. 
     “Heterologous amino acid sequence” as used herein refers to an amino acid sequence not naturally fused to the NIa protease polypeptide. Heterologous amino acid sequences can be attached to either the N- or C-terminus of the NIa protease polypeptide using standard methods. The heterologous sequences can be used to provide a tag for fusion protein purification, such as attachment of polyhistidine or glutamine S-transferase tags, or to increase half life of the NIa protease, such as attachment of a constant domain of an immunoglobulin or albumin, or fragments thereof. Heterologous amino acid sequences can be fused to the polypeptide using well known methods, for example chemical coupling, or via an amide bond. An immunoblogulin hinge or a fragment thereof, a fragment of a variable region of an immunoglobulin, or a linker can also be fused to the NIa protease polypeptide. 
     The term “vector” means a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, bacteria, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The polynucleotides comprising a vector may be DNA or RNA molecules or hybrids of these. 
     The term “expression vector” means a vector that can be utilized in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector. 
     The present invention provides NIa protease variants that are resistant to oxidation, polynucleotides encoding the variants, vectors comprising these polynucleotides, isolated host cells, methods for expressing the polypeptides of the invention, and methods of using the polynucleotides and polypeptides of the invention. The variants of the invention are useful as research tools, and can be used, e.g., to cleave fusion proteins to remove tags. 
     One embodiment of the invention is an isolated polypeptide encoding a NIa protease variant, wherein the variant is resistant to oxidation and retains its activity. In oxidizing conditions, i.e., in the absence of a reductant, the wild type NIa aggregates and becomes inactive (Example 1). 
     In another embodiment, the NIa protease variant resistant to oxidation and retaining its activity has at least one cysteine residue substituted. Other variants may have 2, 3, 4 or 5 cysteine residues substituted. The wild type NIa protease shown in SEQ ID NO: 1 has a total of five cysteines: one active site cysteine at position 151, and four cysteines at positions 19, 110, 181 and 211 which, based on crystal structure predictions are on the surface of the protease and thus susceptible to oxidation. Exemplary substitutions are substitutions for serine, valine or alanine. Sequences of exemplary NIa protease variants are shown in Table 2. 
     Variants of the invention can be made by well known methods, for example site-directed or random mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-492, 1985; Weiner et al., Gene, 151:119-123, 1994; Ishii et al., Methods Enzymol., 293:53-71, 1988), or by chemical synthesis (U.S. Pat. Nos. 6,670,127, 6,521,427). Rational design can be employed to design variants anticipated to have specific effect on structure or activity of the wild type protease. Whether a change in the amino acid sequence of a polypeptide or fragment thereof results in a functional homolog can be readily determined by assessing the ability of the variant polypeptide or fragment to produce a response in a fashion similar to the wild type polypeptide or fragment using the assays described herein. Peptides, polypeptides or proteins in which more than one replacement has taken place can readily be tested in the same manner. Exemplary assays assessing protease activity measure fluorescence released by a fluorophore/quencher substrate peptide such as 4-(4-dimethylaminophenylazo)benzoyl (DABCYL)-YGENVTFQGSK-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS) (SEQ ID NO: 73) upon proteolysis, or evaluate cleavage of a peptide substrate on SDS-PAGE after protease cleavage. 
     The polypeptides of the invention may be produced by chemical synthesis, such as solid phase peptide synthesis on an automated peptide synthesizer. Alternatively, the polypeptides of the invention can be obtained from polynucleotides encoding these polypeptides by the use of cell-free expression systems such as reticulocyte lysate based expression systems or by expression and isolation from cells harboring a nucleic acid sequence of the invention by well known techniques, such as recombinant expression of easily isolated affinity labeled polypeptides. 
     Another embodiment of the invention is an isolated polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 1 having substitutions selected from the group consisting of:
         a. cysteine at position 19 is substituted for serine or valine;   b. cysteine at position 110 is substituted for serine;   c. cysteine at position 151 is substituted for serine or alanine.   d. cysteine at position 181 is substituted for serine; and   e. cysteine at position 211 is substituted for serine.       

     The polypeptides of the invention may comprise fusion polypeptides comprising a polypeptide of the invention fused with a heterologous polypeptide. Such heterologous polypeptides may be leader or secretory signal sequences, a pre- or pro- or prepro-protein sequence, a Histidine tag (His-tag) (Gentz et al.,  Proc. Natl. Acad. Sci . (USA) 86:821-284, 1989), the HA peptide tag (Wilson et al.,  Cell  37:767-778, 1984), glutathione-S-transferase, fluorescent tags such as green fluorescent protein (GFP), and the like. Exemplary NIa protease variant—His-tag fusion proteins have amino acid sequences shown in SEQ ID NOs: 37, 38 or 39. In one aspect, the NIa protease variant polypeptide is fused to an immunoglobulin constant domain or a fragment thereof. Such constructs are well known and are described in e.g. U.S. Pat. Nos. 5,116,964, 5,709,859, 6,018,026; WO 04/002417; WO 04/002424; WO 05/081687; and WO 05/032460. Immunoglobulin constant domain may be a CH1, CH2, or a CH3 domain, or a hinge region, and can be derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, or IgA. The NIa protease variant polypeptide can be fused to an immunoglobulin constant domain or a fragment thereof via a linker, for example a glycine-rich linker, or via a fragment of an immunoglobulin variable region. Such linkers and variable region fragments are described in e.g. WO08/011,446 and U.S. Pat. No. 5,908,626. Exemplary fusion proteins can be formed by conjugating together a NIa protease variant having an amino acid sequence shown in SEQ ID NO: 28 and one or more domains derived from or similar to an immunoglobulin domain, such as CH1, CH2, and CH3 domain. 
     Another embodiment of an invention is an isolated polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 28. 
     In another embodiment, the invention provides for an isolated polypeptide comprising a polypeptide having the sequence shown in SEQ ID NO: 28. 
     The polypeptides of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. An exemplary carrier is phosphate buffered saline. This technique has been shown to be effective with conventional protein preparations. Lyophilization and reconstitution techniques are well known in the art, see e.g., Rey and May, Drugs and the Pharmaceutical Sciences Vol. 137, 1999; Wang, Int. J. Pharm. 203:1-60, 2000. These techniques allow for the development of protein formulations with increased long term stability, including storage at room temperature, as well as easier geographical distribution. This process also affords the protein to be used at higher concentrations by adjusting the reconstitution procedure. 
     Another aspect of the invention is isolated polynucleotides encoding any of the polypeptides of the invention or their complement. Certain exemplary polynucleotides are disclosed herein, however, other polynucleotides which, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the NIa protease variants of the invention are also within the scope of the invention. Exemplary polynucleotides are polynucleotides comprising the nucleic acid sequence shown in SEQ ID NOs: 41-43 and 46-48. 
     The polynucleotides of the invention may be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer. Alternatively, the polynucleotides of the invention may be produced by other techniques such a PCR based duplication, vector based duplication, or restriction enzyme based DNA manipulation techniques. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art. 
     The polynucleotides of the invention may also comprise at least one non-coding sequence, such as transcribed but not translated sequences, termination signals, ribosome binding sites, mRNA stabilizing sequences, introns and polyadenylation signals. 
     Another embodiment of the invention is a vector comprising an isolated polynucleotide encoding polypeptides of the invention. 
     Another embodiment of the invention is a vector comprising an isolated polynucleotide having a sequence shown in SEQ ID NO: 42 or 47. The vectors of the invention are useful for maintaining polynucleotides, duplicating polynucleotides, or driving expression of a polypeptide encoded by a vector of the invention in a biological system, including reconstituted biological systems. Vectors may be chromosomal-, episomal- and virus-derived such as vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses and vectors derived from combinations thereof, such as cosmids and phagemids. 
     The vectors of the invention can be formulated in microparticles, with adjuvants, lipid, buffer or other excipients as appropriate for a particular application. 
     In one embodiment of the invention the vector is an expression vector. Expression vectors typically comprise nucleic acid sequence elements that can control, regulate, cause or permit expression of a polypeptide encoded by such a vector. Such elements may comprise transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate the expression of encoded polypeptides in a given expression system. Such expression systems may be cell-based, or cell-free systems well known in the art. Nucleic acid sequence elements and parent vector sequences suitable for use in the expression of encoded polypeptides are also well known in the art. 
     Another embodiment of the invention is an isolated host cell comprising a vector of the invention. Representative host cell examples include Archaea cells; bacterial cells such as Streptococci, Staphylococci, Enterococci,  E. coli, Streptomyces , cyanobacteria,  B. subtilis  and  S. aureus ; fungal cells such as  Kluveromyces, Saccharomyces , Basidomycete,  Candida albicans  or  Aspergillus ; insect cells such as  Drosophila  S2 and  Spodoptera  Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1, Bowes melanoma and myeloma; and plant cells, such as gymnosperm or angiosperm cells. The host cells in the methods of the invention may be provided as individual cells, or populations of cells. 
     Introduction of a polynucleotide, such as a vector, into a host cell can be effected by methods well known to those skilled in the art (Davis et al., Basic Methods in Molecular Biology, 2 nd  ed., Appleton &amp; Lange, Norwalk, Conn., 1994; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd  ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). These methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection. 
     Another embodiment of the invention is a method for expressing a polypeptide comprising the steps of providing a host cell of the invention and culturing the host cell under conditions sufficient for the expression of at least one polypeptide of the invention. The polypeptides of the invention comprise polypeptides having an amino acid sequence shown in SEQ ID NOs: 2-34 and 37-39. 
     Host cells can be cultured under any conditions suitable for maintaining or propagating a given type of host cell and sufficient for expressing a polypeptide. Culture conditions, media, and related methods sufficient for the expression of polypeptides are well known in the art. For example, many mammalian cell types can be aerobically cultured at 37° C. using appropriately buffered DMEM media while bacterial, yeast and other cell types may be cultured at 37° C. under appropriate atmospheric conditions in LB media. 
     In the methods of the invention the expression of a polypeptide can be confirmed using a variety of different techniques well known in the art. For example, expression of a polypeptide can be confirmed using SDS page, detection reagents, such as antibodies or receptor ligands specific for an expressed polypeptide, or using for example FACS or immunofluorescent techniques. 
     Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof. 
     Example 1 
     Generation and Characterization of NIa Variants 
     Cloning and Mutagenesis 
     The amino acid sequence of potato virus A NIa protease (Genbank Acc. No. CAB58238, amino acids residues 2032-2263), shown in SEQ ID NO: 1, including an N-terminal poly-histidine tag for affinity purification was back translated into a cDNA sequence optimizing codon usage. The full-length cDNA was generated by parsing the sequence into smaller fragments and synthesizing these as oligonucleotides using GENEWRITER™ technology and purified by RP HPLC (Dionex, Germany). The purified oligonucleotides were then assembled into a full-length, double stranded cDNA fragment as described in U.S. Pat. No. 6,670,127 and U.S. Pat. No. 6,521,427. 
     The cDNA from the gene assembly process was cloned into the pET9d vector (Novagen, Madison, Wis.) into NcoI/XhoI sites using standard protocols. Mutagenesis targeting active site cysteine and surface sulfydryl changes was done using the QuikChange site-directed mutatgenesis kit (Stratagene, La Jolla, Calif.) using oligonucleotides shown in Table 1. Protein sequence alignments and the solved crystal structures of TEV NIa protease ((Allison et al., Virology 154:9-20, 1986; Phan et al., J. Biol. Chem. 277:50564-72, 2002) were used to estimate whether the unpaired cysteine residues in NIa protease were surface exposed. As they all appeared to be surface exposed, all were targeted for point mutations. As a first pass, all except the active site cysteine were changed to serine residues. 
     The cysteine residue at position 19 did not tolerate the serine substitution, as indicated by a lack of protein expression (see below). Consequently, position 19 was randomized using an NNK oligo in a QuikChange site-directed mutagenesis reaction using standard protocols. Variants with tolerated substitutions at residue 19 were identified by protein expression (see below). C151S active site substitutions were introduced into these variants as described above, to assess the differences in catalytic activity. Generated variants and their amino acid sequences are shown in Table 2. Exemplary cDNA sequences are shown for the wild type NIa (SEQ ID NO: 40) and for the following NIa variants: C151S (SEQ ID NO: 41), C19V/C110S/C181S/C211S (SEQ ID NO: 42), C19V/C110S/C151S/C181S/C211S (SEQ ID NO: 43), His6-WT (WEQ ID NO: 44), WT-His6 (SEQ ID NO: 45), C151S-His6 (SEQ ID NO: 46), C19V/C110S/C181S/C211S-His6 (SEQ ID NO: 47), and C19V/C110S/C151S/C181S/C211S-His6 (SEQ ID NO: 48). 
     
       
         
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Oligo 
                 Sequence 
                 SEQ ID NO: 
               
               
                   
               
             
             
               
                 PVAH6-5′ 
                 CTAACCATGGGCTCTACCTCTATGTTCCGTGGTGTTCGTGACTACAA 
                 49 
               
               
                   
               
               
                 PVAH6-3′ 
                 GTTACTCGAGTTATTAATGGTGATGGTGATGGTGGGTAACCAGTTTAACGG 
                 50 
               
               
                   
               
               
                 C151S-5′ 
                 CTACCAAAGACGGTCAGAGCGGTTCTCCGATCGTTTC 
                 51 
               
               
                   
               
               
                 C151S-3′ 
                 GAAACGATCGGAGAACCGCTCTGACCGTCTTTGGTAG 
                 52 
               
               
                   
               
               
                 C151A-5′ 
                 CTCTACCAAAGAAGGTCACGCCGGTTCTCCGATCGTTTC 
                 53 
               
               
                   
               
               
                 C151A-3′ 
                 GAAACGATCGGAGAACCGGCGTGACCTTCTTTGGTAGAG 
                 54 
               
               
                   
               
               
                 C19S-5′ 
                 CCCGATCTCTTCTGTTATCAGCCAGCTGGAAAACGAATCTGAAGG 
                 55 
               
               
                   
               
               
                 C19S-3′ 
                 CCTTCAGATTCGTTTTCCAGCTGGCTGATAACAGAAGAGATCGGG 
                 56 
               
               
                   
               
               
                 C110S-5′ 
                 CGACCCACTCTGAAAAAGTTAGCCTGATCCTGACCAACTTCCAG 
                 57 
               
               
                   
               
               
                 C110S-3′ 
                 CTGGAAGTTGGTCAGGATCAGGCTAACTTTTTCAGAGTGGGTCG 
                 58 
               
               
                   
               
               
                 C181S-5′ 
                 CACCTCTAACTACTTCGCGAGCTTCCCGAAAGGTTTCACCG 
                 59 
               
               
                   
               
               
                 C181S-3′ 
                 CGGTGAAACCTTTCGGGAAGCTCGCGAAGTAGTTAGAGGTG 
                 60 
               
               
                   
               
               
                 C211S-5′ 
                 CAACGCGTCTAACGTTAGCTGGGGTTCTTTCCACCTG 
                 61 
               
               
                   
               
               
                 C211S-3′ 
                 CAGGTGGAAAGAACCCCAGCTAACGTTAGACGCGTTG 
                 62 
               
               
                   
               
               
                 C19NNK-5′ 
                 ACCCGATCTCTTCTGTTATCNNKCAGCTGGAAAACGAATCTGAAG 
                 63 
               
               
                   
               
               
                 C19NNK-3′ 
                 CTTCAGATTCGTTTTCCAGCTGMNNGATAACAGAAGAGATCGGGT 
                 64 
               
               
                   
               
             
          
         
       
     
                                                   TABLE 2                   NIa variant   SEQ ID NO:   DNA                                    WT   1   40           C151S   2   41           C110S   3               C181S   4               C211S   5               C19S/C110S/C181S   6               C19S/C110S/C211S   7               C19S/C181S/C211S   8               C19S/C110S/C181S/C211S   9               C110S/C181S   10               C110S/C211S   11               C19A/C110S/C181S/C211S   12               C110S/C181S/C211S   13               C19D/C110S/C181S/C211S   14               C19E/C110S/C181S/C211S   15               C19F/C110S/C181S/C211S   16               C110S/C181S/C211S   17               C19H/C110S/C181S/C211S   18               C19I/C110S/C181S/C211S   19               C19K/C110S/C181S/C211S   20               C19L/C110S/C181S/C211S   21               C19M/C110S/C181S/C211S   22               C19N/C110S/C181S/C211S   23               C19P/C110S/C181S/C211S   24               C19Q/C110S/C181S/C211S   25               C19R/C110S/C181S/C211S   26               C19T/C110S/C181S/C211S   27               C19V/C110S/C181S/C211S   38*   42           C19W/C110S/C181S/C211S   29               C19Y/C110S/C181S/C211S   30               C110S/C151S/C181S/C211S   31               C181S/C211S   32               C19V/C110S/C151S/C181S/C211S   33   43           C151A   34               His6-WT   35   44           WT-His6   36   45           C151S-His6   37   46           C19V/C110S/C181S/C211S-His6   38   47           C19V/C110S/C151S/C181S/C211S-His6   39   48               *NIa variant has an amino acid sequence of residues 1-233 of SEQ ID NO: 38            
Protein Expression
 
     Plasmids encoding cDNAs for the NIa protease variants in Table 1 were transformed in BL21 cells and single colonies from the transformants cultured in LB media with 100 μg/ml kanamycin at +37° C. overnight. Induction took place when the cultures reached an OD600 of 0.6-0.8 with 1 mM IPTG, or by culturing the cells in TB auto-induction media (Overnight Express Autoinduction Media, EMD Biosciences, Gibbstown, N.J.). The cells were further cultured overnight at 25° C. or 18° C., centrifuged and stored at −80° C. All NIa protease variants with a wild-type C19 residue expressed very well in all surface sulfhydryl change combinations explored (Table 3). 
     For the NNK library, the constructs were screened for soluble protein expression in TB auto induction media, as described above. A Western blot was run to analyze the expression of the NNK variants. 
     
       
         
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
             
             
               
                   
                   
               
               
                   
                 Substitutions 
                 Plasmid 
                   
               
             
          
           
               
                 Variant 
                 C19 
                 C110 
                 C181 
                 C211 
                 C151 
                 Number 
                 Expression 
                 Activity 
               
               
                   
               
               
                 His 6 -WT 
                   
                   
                   
                   
                   
                 pDR1706 
                 + 
                 + 
               
               
                 WT 
                   
                   
                   
                   
                   
                 pDR2090 
                 + 
                 + 
               
               
                 C151S 
                   
                   
                   
                   
                 S 
                 pDR2092 
                 + 
                 + 
               
               
                 C151A 
                   
                   
                   
                   
                 A 
                 pDR2091 
                 + 
               
               
                 C110S 
                   
                 S 
                   
                   
                   
                 pDR3385 
                 + 
               
               
                 C181S 
                   
                   
                 S 
                   
                   
                 pDR3388 
                 + 
               
               
                 C211S 
                   
                   
                   
                 S 
                   
                 pDR3390 
                 + 
               
               
                 C19S/C110S/C181S 
                 S 
                 S 
                 S 
                   
                   
                 pDR3384 
                 − 
               
               
                 C19S/C110S/C211S 
                 S 
                 S 
                   
                 S 
                   
                 pDR3383 
                 − 
               
               
                 C19S/C181S/C211S 
                 S 
                   
                 S 
                 S 
                   
                 pDR3382 
                 − 
               
               
                 C19S/C110S/C181S/C211S 
                 S 
                 S 
                 S 
                 S 
                   
                 pDR2371 
                 − 
               
               
                 C110S/C181S 
                   
                 S 
                 S 
                   
                   
                 pDR3386 
                 + 
               
               
                 C110S/C211S 
                   
                   
                   
                   
                   
                 pDR3387 
                 + 
               
               
                 C110S/C181S/C211S 
                   
                 S 
                 S 
                 S 
                   
                 pDR3202 
                 + 
                 + 
               
               
                 C110S/C151S/C181S/C211S 
                   
                 S 
                 S 
                 S 
                 S 
                 pDR3467 
                 + 
                 + 
               
               
                 C181S/C211S 
                   
                   
                 S 
                 S 
                   
                 pDR3389 
                 + 
               
               
                 C19V/C110S/C181S/C211S 
                 V 
                 S 
                 S 
                 S 
                   
                 pDR3217 
                 + 
                 + 
               
               
                 C19V/C110S/C151S/C181S/C211S 
                 V 
                 S 
                 S 
                 S 
                 S 
                 pDR3466 
                 + 
                 + 
               
               
                   
               
             
          
         
       
     
     Although several of the position 19 NNK variants were detectable at low levels (variants I, K, L, M, R, S, T, W, Y, F, G and H substitutions) (1-2% of the wild-type NIa), the variant C19V was expressed at significantly higher level than any other variant, and at a level equivalent to the wild type NIa. Based on the information, the following variants were selected for further studies: WT, C151S, C110S/C181S/C211S, C110S/C151S/C181S/C211S, C19V/C110S/C181S/C211S and C19V/C110S/C151S/C181S/C211S. 
     Protein Purification 
     Protein purification was done using standard methods in the presence of a reducing agent, 2 mM TCEP. Briefly, cell pellets were resuspended in Buffer A (20 mM tris-HCl, pH 7.5, 500 mM NaCl, 2 mM TCEP) supplemented with 0.1 U/ml benzonase and 0.3 mg/ml lysozyme, soincated on ice, filtered, and the cleared lysates were loaded onto a 5 ml HisTrap HP (GE Biosciences, Piscataway, N.J.) column pre-equilibrated with buffer A using an AKTA Explorer purification system (GE Lifesciences, Piscataway, N.J.). Proteins were eluted using an imidazole step gradient of 50-500 mM imidazole in buffer A. Fractions were analyzed by SDS-PAGE and the fractions containing the protein of interest were pooled and concentrated and filtered, followed by further purification by size exclusion chromatography (SEC). Concentrated and clarified samples were loaded directly onto a Superdex75 SEC matrix (GE Lifesciences, Piscataway, N.J.) pre-equilibrated with buffer A and separated isocratically at a flow rate of 1 ml/min. Fractions were analyzed by SDS-PAGE and the fractions containing protein were pooled and tested for enzymatic activity. All purified variants expressed well and were purified to over 95% purity. 
     Some of the variants (C151A, C19V/C110S/C181S/C211S and C19V/C110S/C151S/C181S/C211S) were also purified in the absence of the reducing agent, 2 mM TCEP, in order to evaluate the effect of oxidizing environment to protein expression, stability, and activity. Only under reducing conditions does the C151A variant with 4 free surface sulfhydryls collapse to a predominantly single, monomeric species. However, the proteases with all surface sulfhydryls changes behave as monomeric proteins in the complete absence of reducing agent. This suggests that these changes provide a clear physical benefit while retaining catalytic activity (see below). 
     Substrate and Activity Determination 
     A wild-card recognition sequence, EXVXXQX (SEQ ID NO: 74), was used to search the polyprotein sequence of PVA to determine a consensus recognition sequence for the NIa protease. This was done independently of published work identifying the processing junction points within the PVA polyprotein (Mertis et al., J. General Virol., 83:1211-1221, 2002). Published and potential recognition sequences, as well as the consensus sequence determined in this study listed in Table 4. Synthetic peptides corresponding to select recognition sequences were synthesized using solid-phase peptide chemistry (Anaspec, San Jose, Calif.) and tested for cleavage by the wild type NIa protease. Reactions were performed in 20mM tris-HCl, pH 8.0, 150mM NaCl and 1mM dithiothreitol (DTT) containing 5 μM PVA NIa protease and 500 μM peptide and were analyzed by reverse-phase HPLC and LC-MS. 
     Enzyme activity was also determined for each variant using a fusion substrate protein containing the NIa protease consensus recognition sequence, ENVTFQG (SEQ ID NO:65). The consensus sequence was engineered into a fusion protein and used as a substrate to assess the enzymatic activity for all PVA NIa protease variants. Since the sequence contained a consensus site for N-linked glycosylation (NVT), another sequence was explored, EAVTFQG (SEQ ID NO: 66), with equal success. These fusion proteins contained an N-terminal poly-histidine tag to facilitate purification, the PNIa protease consensus recognition sequence, an S-tag for sensitive detection of proteolytic cleavage and a highly soluble “filler” protein to facilitate soluble expression of the fusion substrate protein. This cassette was generated by amplifying the region between the 3′ end of the thrombin cleavage site and the XhoI site in pET41 (Novagen), adding the recognition sequence and NdeI cloning site in the 5′ primer and inserting into the NdeI and XhoI restriction sites of pET28 (Novagen). The “filler” proteins could then be inserted into the multiple cloning site pulled over from pET41. Polypeptide sequence of the fusion proteins with the ENVTFQG and the EAVTFQG consensus recognition sequences are shown in SEQ ID NO:s 67 and 68, respectively. 
     As fusion substrate controls, analogous constructs were generated with both TEV (Dougherty et al., Virology, 171:356-364, 1989) and TVMV NIa protease recognition sequences (Nallamsetty et al., Protein Expr. and Purific. 38:108-115, 2004) (Table 4). Analogous to human rhinovirus 3C(HRV3C) recognition sequence, a fusion protein with a P2′ proline was also generated for the consensus sequence and tested as a substrate (Table 4). All recognition sequences were inserted into the fusion substrate protein, described above, including the published recognition sequences for TEV and TVMV proteases listed. Reactions were performed in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl and 1 mM DTT and allowed to run overnight at 37° C. 
     Although it has been shown that the substrate specificity of 3C-like proteases is very high (Tozer et al., The FEBS J. 272:514-523, 2004), NIa wild type protease was able to cleave the fusion substrate with the TVMV NIa protease recognition sequence, although at a much lower rate than the PVA NIa protease consensus sequence. However, the NIa wild type protease was unable to cleave either the TEV NIa protease recognition sequence or the PVA NIa protease consensus sequence with a P2′ proline residue in this format, the latter suggesting some level of P2′ specificity (Table 4). 
     
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                   
                   
                 Recognition 
                 SEQ ID 
                 Synthetic 
                 SEQ ID 
                 Cleaved by 
               
               
                   
                 Junction* 
                 Sequence 
                 NO: 
                 Peptide** 
                 NO: 
                 NIa 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 P3/6K1 
                 EVVLFQA{circumflex over ( )} 
                 75 
                 SEVVLFQASS 
                 70 
                 Yes 
               
               
                   
                 6K1/CI 
                 NTVQFQS 
                 76 
                   
                   
                   
               
               
                   
                 CI/6K2 
                 EAVQFQS{circumflex over ( )} 
                 77 
                   
                   
                   
               
               
                   
                 6K2/VPg 
                 GVVAFQG 
                 78 
                   
                   
                   
               
               
                   
                 VPg/Pro 
                 ESVEFES 
                 79 
                   
                   
                   
               
               
                   
                 NIa/NIb 
                 EAVYTQG{circumflex over ( )} 
                 80 
                 SEAVYTQGSS 
                 71 
                 Yes 
               
               
                   
                 NIb/cap 
                 DMVYFQA 
                 81 
                   
                   
                   
               
               
                   
                 NA 
                 ENVTKQL{circumflex over ( )} 
                 82 
                 SENVTKQLSS 
                 87 
                 No 
               
               
                   
                 NA 
                 EMVTNQS{circumflex over ( )} 
                 83 
                 SEMVTNQSSS 
                 88 
                 No 
               
               
                   
                 Consensus 
                 ENVTFQG 
                 65 
                 SENVTFQGSS 
                 72 
                 Yes 
               
               
                   
                   
                 ENVTFQGP 
                 84 
                   
                   
                 No 
               
               
                   
                 TEV 
                 ENLYGQGS 
                 85 
                   
                   
                 No 
               
               
                   
                 TVMV 
                 ETVRFQGS 
                 86 
                   
                   
                 Yes 
               
               
                   
               
               
                 *As determined in Mertis et. al., 2002. 
               
               
                 {circumflex over ( )}Sequences that met the EXVXXQX search criteria and from which the consensus sequence peptide was generated 
               
               
                 *Synthetic peptide used in the assays 
               
             
          
         
       
     
     The wild type NIa protease and variants C151S, C110S/C181S/C211S, C110S/C151S/C181S/C211S, C19V/C110S/C181S/C211S and C19V/C110S/C151S/C181S/C211S were screened for activity against the fusion substrate protein with the ENVTFQG (SEQ ID NO: 66) consensus recognition site. Reaction conditions were identical to those described above. Proteolytic cleavage of the substrate was monitored by SDS-PAGE. Each NIa protease with an active site cysteine residue (WT, C110S/C181S/C211S, C19V/C110S/C181S/C211S) cleaved the substrate to completion under these conditions. The NIa protease active site variants (C151S, C110S/C151S/C181S/C211S, C19V/C110S/C151S/C181S/C211S) also cleaved the substrate, albeit with less efficiency (1-5% of substrate cleaved) when compared to the wild type NIa (data not shown). 
     Enzyme Kinetics 
     Wild-type NIa protease and active site and surface cysteine variants were tested for activity against the fluorophore/quencher substrate peptide 4-(4-dimethylaminophenylazo)benzoyl (DABCYL)-YGENVTFQGSK-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS) (Anaspec, San Jose, Calif.). Kinetic measurements were performed on a Spectramax M2 microplate reader (Molecular Devices) using an excitation wavelength of 340 nm and emission wavelength of 490 nm. The reactions were performed in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT with 2 mM enzyme and 0.1-300 μM substrate and followed for 30 minutes at 37° C. Enzyme concentrations were determined from the calculated theoretical extinction coefficient. Initial velocities were determined for each and are shown in Table 5. 
     
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                   
                   
                   
                 V max   
                   
                   
               
               
                   
                 Plasmid 
                   
                 (RFU/ 
                 K m   
                 Relative ° 
               
               
                 Variant 
                 Number 
                 DTT 
                 min) 
                 (uM) 
                 K cat /K m   
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 WT 
                 pDR2090 
                 + 
                 78466 
                 177.6 
                 100 
               
               
                 C151S 
                 pDR2092 
                 + 
                 3236 
                 164.9 
                 4.3 
               
               
                 C110S/C181S/C211S 
                 pDR3202 
                 + 
                 51110 
                 251.5 
                 45.9 
               
               
                 C110S/C151S/C181S/ 
                 pDR3467 
                 + 
                 2346 
                 71.8 
                 7.6 
               
               
                 C211S 
                   
                   
                   
                   
                   
               
               
                 C19V/C110S/C181S/ 
                 pDR3217 
                 + 
                 75184 
                 275.4 
                 59.5 
               
               
                 C211S 
                   
                   
                   
                   
                   
               
               
                 C19V/C110S/C151S/ 
                 pDR3466 
                 + 
                 1888 
                 39.6 
                 10.8 
               
               
                 C181S/C211S 
                   
                   
                   
                   
                   
               
               
                 C19V/C110S/C181S/ 
                 pDR3217 
                 − 
                 53847 
                 175.4 
                 69.1 
               
               
                 C211S 
                   
                   
                   
                   
                   
               
               
                 C19V/C110S/C151S/ 
                 pDR3466 
                 − 
                 1358 
                 43.1 
                 7.1 
               
               
                 C181S/C211S 
               
               
                   
               
             
          
         
       
     
     The substitutions to the surface exposed cysteine residues had a minor effect on catalytic activity of NIa protease, whereas substitutions at the active site cysteine (C151) reduced activity significantly. This can be explained by the inability of the substituted serine to donate its hydroxyl proton required for catalysis in the micro-environment within the active site, whereas deprotonation of cysteine readily occurs at physiological pH. 
     However, to determine whether having a reducing agent present during the purification process as well as during activity measurements impacted only molecules with an active site cysteine; two PVA NIa protease variants (C19V/C110S/C181S/C211S and C19V/C110S/C151S/C181S/C211S) were purified and assayed in the absence of reductant. The absence of reductant had little effect on the activity of either variant (Table 5). This suggests that the active site cysteine in these proteins may not be overly sensitive to an oxidizing environment and liability is predominantly due to the non-active site cysteine residues.