# Patent

## Title
Human SUMO-3 for enhancing protein expression

## Abstract

The inventive subject matter relates to novel compositions, methods, and kits for enhancing the expression, solubility, and isolation of heterologous proteins. Further, the inventive subject matter relates to methods for generating proteins with novel N-terminal amino acids, unlike wild-type proteins which always are translated from mRNA with methionine at the N-terminus.

## Background
This application claims the benefit of U.S. Provisional Application No. 60/640,107, filed Dec. 30, 2004, the contents of which is hereby incorporated by reference in its entirety.


BACKGROUND OF THE INVENTIVE SUBJECT MATTER
1. Field of the Inventive Subject Matter
The inventive subject matter relates to novel compositions, methods, and kits for enhancing the expression, solubility, isolation, and purification of heterologous proteins. Further, inventive subject matter relates to methods for generating proteins with novel N-terminal amino acids, unlike wild-type proteins which always are translated from mRNA with methionine as the N-terminus amino acid.
2. Background
Functional genomic studies have been hampered by the inability to uniformly express and purify biologically active proteins in heterologous expression systems. Despite the use of identical transcriptional and translational signals in a given expression vector, expressed protein levels have been observed to vary dramatically. For this reason, several strategies have been developed to express heterologous proteins in bacteria, yeast, mammalian cells, and insect cells as gene-fusions (see Butt, T. R., S. Jonnalagadda, B. P. Monia, E. J. Sternberg, J. A. Marsh, J. M. Stadel, D. J. Ecker, and S. T. Crooke, 1989, Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli, Proc Natl Acad Sci USA 86:2540-4; Ecker, D. J., J. M. Stadel, T. R. Butt, J. A. Marsh, B. P. Monia, D. A. Powers, J. A. Gorman, P. E. Clark, F. Warren, A. Shatzman, and et al., 1989, Increasing gene expression in yeast by fusion to ubiquitin, J Biol Chem 264:7715-9; Ikonomou, L., Y. J. Schneider, and S. N. Agathos, 2003, Insect cell culture for industrial production of recombinant proteins, Appl Microbiol Biotechnol 62:1-20; and Kapust, R. B., and D. S. Waugh, 1999, Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused, Protein Sci 8:1668-74). Yet such strategies have proved ineffective or insufficient in practice because of poor expression levels, poor solubility, low yields, or a combination thereof.
The expression of heterologous genes in bacteria is by far the simplest and most inexpensive means available for research or commercial purposes. However, some heterologous gene products fail to attain their correct three-dimensional conformation in E. coli, while others become sequestered in large insoluble aggregates or “inclusion bodies” when overproduced (see Georgiou, G., and P. Valax, 1999, Isolating inclusion bodies from bacteria, Methods Enzymol 309:48-58; and Jonasson, P., S. Liljeqvist, P. A. Nygren, and S. Stahl, 2002, Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli, Biotechnol Appl Biochem 35:91-105). Major denaturant-induced solubilization methods followed by removal of the denaturant under conditions that favor refolding are often required to produce a reasonable yield of the recombinant protein. Selection of open reading frames (hereinafter “ORFs”) for structural genomics projects has also shown that only about 20% of the genes expressed in E. coli render proteins that are soluble or correctly folded (see Waldo, G. S., B. M. Standish, J. Berendzen, and T. C. Terwilliger, 1999, Rapid protein-folding assay using green fluorescent protein, Nat Biotechnol 17:691-5). These numbers are startlingly disappointing, especially given that most scientists rely on E. coli for initial attempts to express gene products. Several gene fusion systems ostensibly producing fusion proteins incorporating putative expression enhancers such as NusA, maltose binding protein (MBP), glutathione-S-transferase (GST), and thioredoxin (Trx) have been developed (see Jonasson, P., S. Liljeqvist, P. A. Nygren, and S. Stahl, 2002, Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli, Biotechnol Appl Biochem 35:91-105). All of these systems have clear drawbacks, ranging from inefficient expression to inconsistent cleavage from desired structure.
Thus, there is a need for more effective and efficient protein expression systems. This need is met by the inventive subject matter, using novel compositions and methods which have not heretofore been known. The use of the new SUMO fusions and SUMO proteases disclosed in the inventive subject matter herein circumvents the problems of the prior art, and significantly improves upon previously described expression systems based on Saccharomyces cerevisiae Smt3 and Saccharomyces cerevisiae Ulp1 protease.
SUMMARY OF THE INVENTIVE SUBJECT MATTER
The inventive subject matter relates to a composition for enhancing expression levels of a protein of interest, comprising:
(a) an isolated nucleic acid sequence encoding a SUMO protein;
(b) an isolated nucleic acid sequence encoding said protein of interest; and
(c) an isolated nucleic acid encoding at least one purification tag,
wherein, upon expression in a suitable expression system, said nucleic acid composition produces a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said protein of interest,
and provided that said SUMO protein is not Saccharomyces cerevisiae Smt3.
In addition, the inventive subject matter relates to a composition for enhancing solubility of a protein of interest, comprising:
(a) an isolated nucleic acid sequence encoding a SUMO protein;
(b) an isolated nucleic acid sequence encoding said protein of interest; and
(c) an isolated nucleic acid encoding at least one purification tag,
wherein, upon expression in a suitable expression system, said nucleic acid composition produces a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said protein of interest,
and provided that said SUMO protein is not Saccharomyces cerevisiae Smt3.
Further, the inventive subject matter relates to an expression vector comprising:
(a) an isolated nucleic acid sequence encoding a SUMO protein;
(b) an isolated nucleic acid sequence encoding a protein of interest; and
(c) an isolated nucleic acid encoding at least one purification tag,
wherein, upon expression in a suitable expression system, said expression vector produces a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said protein of interest,
and provided that said SUMO protein is not Saccharomyces cerevisiae Smt3.
The inventive subject matter also relates to a method for enhancing expression and isolation of a protein of interest, comprising the steps of:
(a) transforming a target cell with an expression vector comprising (1) a vector which is suitable for transforming a target cell and (2) a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding a SUMO protein, (ii) an isolated nucleic acid sequence encoding said protein of interest, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked; and
(b) isolating the protein produced by expression of said nucleic acid insert,
wherein upon expression in said target cell, said expression vector encodes a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of the protein of interest,
and provided that said SUMO protein is not Saccharomyces cerevisiae Smt3.
Further, the inventive subject matter relates to a method for enhancing the solubility and isolation of a protein of interest, comprising the steps of:
(a) transforming a target cell with an expression vector comprising (1) a vector which is suitable for transforming a target cell and (2) a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding a SUMO protein, (ii) an isolated nucleic acid sequence encoding said protein of interest, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked; and
(b) isolating the protein produced by expression of said nucleic acid insert;
wherein upon expression in said target cell, said expression vector encodes a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of the protein of interest,
and provided that said SUMO protein is not Saccharomyces cerevisiae Smt3.
More particularly, the inventive subject matter relates to a method for enhancing expression and isolation of a protein of interest, comprising the steps of:]
(a) transforming a target cell with an expression vector comprising (1) a vector which is suitable for transforming a target cell and (2) a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding human SUMO-3 (SEQ ID NO: 4), (ii) an isolated nucleic acid sequence encoding said protein of interest, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked;
(b) isolating the protein produced by expression of said nucleic acid insert;
(c) lysing the cell expressing the fusion protein;
(d) purifying the fusion protein by way of said at least one purification tag;
(e) cleaving the SUMO moiety and purification tag(s) from the protein of interest with human SUMO protease SENP2 (SEQ ID NO: 43) or a catalytic domain thereof; and
(f) isolating the protein of interest,
wherein upon expression in said target cell, said expression vector encodes a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of the protein of interest.
Further, the inventive subject matter particularly relates to a method for enhancing solubility and isolation of a protein of interest, comprising the steps of:
(a) transforming a target cell with an expression vector comprising (1) a vector which is suitable for transforming a target cell and (2) a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding human SUMO-3 (SEQ ID NO: 4), (ii) an isolated nucleic acid sequence encoding said protein of interest, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked;
(b) isolating the protein produced by expression of said nucleic acid insert;
(c) lysing the cell expressing the fusion protein;
(d) purifying the fusion protein by way of said at least one purification tag;
(e) cleaving the SUMO moiety and purification tag(s) from the protein of interest with human SUMO protease SENP2 (SEQ ID NO: 43) or a catalytic domain thereof; and
(f) isolating the protein of interest,
wherein upon expression in said target cell, said expression vector encodes a fusion protein in which said purification tag is N-terminal to said SUMO protein, and said SUMO protein is attached to the amino terminus of the protein of interest.
In addition, the inventive subject matter relates to a method for generating proteins with a novel N-terminal amino acid which is not proline, comprising the steps of:
(a) transforming a target cell with an expression vector comprising (1) a vector which is suitable for transforming a target cell and (2) a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding a SUMO protein, (ii) an isolated nucleic acid sequence encoding a protein of interest having a novel N-terminal amino acid, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked;
(b) isolating the protein produced by expression of said nucleic acid insert,
wherein upon expression in said target cell, said expression vector encodes a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said novel N-terminal amino acid of the protein of interest;
(c) cleaving the SUMO moiety and purification tag(s) from the protein of interest with an appropriate SUMO protease, or a catalytic domain thereof; and
(d) isolating the protein of interest having a novel N-terminal amino acid,
provided that said SUMO protease is not Saccharomyces cerevisiae Ulp1,
and provided that said isolated nucleic acid sequence encoding a SUMO protein is not Saccharomyces cerevisiae Smt3.
In another aspect, the inventive subject matter relates to a method for generating proteins with a proline N-terminal amino acid, comprising the steps of:
(a) transforming a target cell with an expression vector comprising (1) a vector which is suitable for transforming a target cell and (2) a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding a SUMO protein, (ii) an isolated nucleic acid sequence encoding a protein of interest having at least one N-terminal amino acid which is not proline and having a proline adjoining said at least one N-terminal amino acid, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked;
(b) isolating the protein produced by expression of said nucleic acid insert,
wherein upon expression in said target cell, said expression vector encodes a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said at least one N-terminal amino acid of the protein of interest,
and provided that said isolated nucleic acid sequence encoding a SUMO protein is not Saccharomyces cerevisiae Smt3;
(c) cleaving the SUMO moiety and purification tag(s) from the protein of interest with an appropriate SUMO protease, or a catalytic domain thereof,
provided that said SUMO protease is not Saccharomyces cerevisiae Ulp1;
(d) isolating the protein of interest having at least one N-terminal amino acid which is not proline and having a proline adjoining said at least one N-terminal amino acid; and
(e) treating said protein of interest with an aminopeptidase to remove said at least one N-terminal amino acid which is not proline and leaving proline as the amino terminus.
Finally, the inventive subject matter relates to a kit for enhancing expression levels of a protein of interest, comprising:
(a) a vector which is suitable for transforming a target cell and which has at least one cloning site for cloning a nucleic acid encoding a protein of interest, operably linked to a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding a SUMO protein, and (ii) an isolated nucleic acid encoding at least one purification tag,
provided that said isolated nucleic acid sequence encoding a SUMO protein is not Saccharomyces cerevisiae Smt3,
wherein said at least one cloning site is oriented in said vector so that upon cloning in a nucleic acid sequence encoding said protein of interest and expression in a target cell, said vector encodes a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of the protein of interest; and
(b) a cleavage enzyme for cleaving the SUMO moiety and purification tag(s) from the protein of interest.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is an alignment of the polypeptide sequences of the predicted mature forms of known and putative SUMO proteins from various representative organisms. From top to bottom, the sequence are SEQ ID NO: 1, SEQ ID NO: 71, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85.
FIG. 1B is an alignment of the polypeptide sequences of the catalytic domains of known and putative SUMO proteases from various representative organisms. From top to bottom, the sequences are SEQ ID NOs: 86-108.
FIG. 2 is a schematic representation of a representative cloning technique used to generate SUMO fusion proteins. The BsaI digestion site under Vector Cloning Site is SEQ ID NO: 109 (top strand) and SEQ ID NO: 110 (bottom strand). The BsaI recognition site under PCR Product is SEQ ID NO: 111 (top strand) and SEQ ID NO: 112 (bottom strand).
FIG. 3A shows a Coomassie-stained SDS-PAGE gel of soluble (S) and insoluble inclusion body (IB) fractions from pET24d-6xHis-SUMO-eGFP-containing cells induced with IPTG.
FIG. 3B shows fluorescence comparisons of eGFP un-fused or fused with described SUMO tags.
FIG. 4A shows a Coomassie-stained SDS-PAGE gel of a 2-fold dilution series comparing human SENP2 and yeast Ulp1 used to cleave a variety of SUMO-eGFP fusions.
FIG. 4B shows a Coomassie-stained SDS-PAGE gel of a time course comparing cleavage of various SUMO-eGFP fusions by human SENP2 and yeast Ulp1.
FIG. 4C shows Coomassie-stained SDS-PAGE gels comparing human SENP2 and yeast Ulp1 used to cleave hSUMO3-eGFP fusions with each of the 20 amino acids at the N-terminus of eGFP.
FIG. 5 shows a Coomassie-stained SDS-PAGE gel of in vitro cleavage reactions of the SUMO proteases SENP1, SENP6, AtULP1c, and GST-XopD for various fusion proteins.
FIG. 6A is a map of an exemplary plasmid pET24d-6xHis-SUMO expression vector, as used in the inventive subject matter.
FIG. 6B shows the regions that flank the sequences of 6xHis-SUMO in the plasmid, using hSUMO-3 as an example. The nucleotide sequence is SEQ ID NO: 113 and the amino acid sequence is SEQ ID NO: 15.



DETAILED DESCRIPTION OF THE INVENTIVE SUBJECT MATTER
Definitions
For clarity, the term “vector” as used herein refers to a plasmid or other structure which is capable of receiving a heterologous nucleic acid sequence by standard cloning techniques, while the term “expression vector” as used herein refers to a plasmid or other vector into which a heterologous nucleic acid sequence has been cloned.
The term “purification tag” as used herein refers to a moiety attached to a protein of interest which enhances the ability to specifically isolate the protein of interest from other compositions, or from a solution or suspension. An exemplary purification tag is poly (6x) histidine. Purification tags are well known in the art, and are referred to herein without limitation.
The term “catalytic domain” as used herein in relation to proteases, refers to a fragment of a protease which retains the catalytic activity of the wild-type protease.
The term “novel N-terminal amino acid” as used herein refers to an N-terminal amino acid of a polypeptide which is not methionine. As is well known in the art, methionine is the first amino acid in the natural translation of mRNA to a polypeptide, and it is considered novel to produce proteins with an amino acid other than methionine at the N-terminal without resort to use of an aminopeptidase.
The term “mature” as used herein in relation to certain proteins refers to polypeptide sequences remaining after post-translational processing.
The term “equivalent” as used herein in relation to the inventive compositions refers to a composition that performs substantially the same function as another composition in substantially the same way.
The term “homologue” as used herein in relation to the inventive compositions refers to a composition having an amino acid or nucleic acid sequence which corresponds or is similar to another composition in position, structure, and/or function. Homologous proteins should have at least 70% sequence homology, preferably at least 80% sequence homology, and most preferably at least 90% sequence homology. It is now well known to one of ordinary skill in the art that many proteins have identifiable functional domains, such as a substrate binding domain, a recognition sequence, or an active domain having, for example, a protease function. It is also well known, as discussed below, that substantially greater sequence variation is permitted in non-functional domains than functional domains, and thus sequence homology for purposes of this definition may be established over less than all of a protein sequence. Unless otherwise specifically described herein, Applicants hereby incorporate by reference the characteristics which are known in the art relating to particular proteins.
Further, the concept of sequence homology is tied to the distinction between conservative and non-conservative substitutions. In this regard, “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences thereof. In the art, “identity” also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between sequences of two or more nucleotides or two or more amino acids. “Identity” measures the percentage of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model, computer program, or algorithm.
The term “similarity” is used with regard to a related concept; in contrast to “identity,” “similarity” refers to a measure of relatedness that includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, 10/20 identical amino acids, and the remainder are all non-conservative substitutions, then the percentage identity and similarity would both be 50%. If in the same example, there are five more positions where there are conservative substitutions, then the percentage identity remains 50%, but the percentage similarity would be 75% (15/20). Therefore, in cases where there are conservative substitutions, the percentage similarity between two polypeptides will be higher than the percentage identity between those two polypeptides. For purposes of this application, Applicants consider conservative substitutions to be a “match” for purposes of determining percent homology.
Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.
Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced, for example, into non-homologous regions of a molecule.
In making such changes, according to certain embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, e.g., Kyte, et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within ±2 is included. In certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where a biologically functional protein is intended for use in embodiments involving protein-protein interactions, as disclosed herein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5) and tryptophan (−3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. Such regions are referred to as “epitopic core regions, and in some embodiments correlate with a biological property of the protein.”
Exemplary amino acid substitutions are set forth in the following Table A.







TABLE A







Amino Acid Substitution










Exemplary
Preferred


Original Residues
Substitutions
Substitutions





Ala
Val, Leu, Ile
Val


Arg
Lys, Gln, Asn
Lys


Asn
Gln
Gln


Asp
Glu
Glu


Cys
Ser, Ala
Ser


Gln
Asn
Asn


Glu
Asp
Asp


Gly
Pro, Ala
Ala


His
Asn, Gln, Lys, Arg
Arg


Ile
Leu, Val, Met, Ala, Phe,
Leu



Norleucine



Leu
Norleucine, Ile, Val, Met, Ala, Phe
Ile


Lys
Arg, 1,4 Diamino-butyricAcid,
Arg



Gln, Asn



Met
Leu, Phe, Ile
Leu


Phe
Leu, Val, Ile, Ala, Tyr
Leu


Pro
Ala
Gly


Ser
Thr, Ala, Cys
Thr


Thr
Ser
Ser


Trp
Tyr, Phe
Tyr


Tyr
Trp, Phe, Thr, Ser
Phe


Val
Ile, Met, Leu, Phe, Ala, Norleucine
Leu









A skilled artisan will be able to determine suitable variants of polypeptides as set forth herein using well-known techniques. In certain embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In other embodiments, the skilled artisan can identify residues and portions of the molecules that are conserved among similar polypeptides. In further embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
Additionally, one skilled in the art can review structure-function studies identifying residues, in specific proteins or in similar proteins, that are important for activity or structure. In view of such a comparison, the skilled artisan can readily determine or predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may then opt for amino acid substitutions for such predicted important amino acid residues in order to maintain function.
One skilled in the art can also analyze the three-dimensional structure of a protein, and the amino acid sequence of the protein in relation to that structure. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an protein with respect to its three-dimensional structure, or may determine three-dimensional structure experimentally. In certain embodiments, one skilled in the art may choose to not make radical changes to amino acid residues on the surface of the protein, since such residues may be involved in interactions with other molecules. Moreover, one skilled in the art may generate test variants, containing a single amino acid substitution at each desired amino acid residue which can be screened using activity assays known to those skilled in the art. Thus, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change can be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided, either alone or in combination with other mutations.
The term “operably linked” means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions. For example, a control sequence “operably linked” to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.
The Inventive Subject Matter
The expression of heterologous genes in bacteria is by far the simplest and most inexpensive means available for research or commercial purposes. However, some heterologous gene products fail to attain their correct three-dimensional conformation in E. coli, while others become sequestered in large insoluble aggregates or “inclusion bodies” when overproduced. Major denaturant-induced solubilization methods followed by removal of the denaturant under conditions that favor refolding are then required to produce a reasonable yield of the recombinant protein.
For purposes of evaluating and selecting expression systems, selection of open reading frames for structural genomics projects has shown that only about 20% of the genes expressed in E. coli render proteins that are soluble or correctly folded. These numbers are startlingly disappointing, especially given that most scientists rely on E. coli for initial attempts to express gene products. Several gene fusion systems, which ostensibly produce fusion proteins, incorporate putative expression enhancers such as NusA, MBP, GST, and Trx. All of these systems have clear drawbacks, ranging from inefficient expression to inconsistent cleavage from desired structure.
Recent work shows that the use of yeast SUMO (Smt3) as an N-terminal tag can increase the expression and solubility of various proteins. Further, yeast SUMO protease Ulp1 has been used to process fusion proteins the fusions in vitro to produce an un-fused protein product of interest. Using these yeast components or variations thereof, expression systems have been developed (see Applicants' work in Malakhov, et al., 2004, SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins, J Struct Funct Genomics 5:75-86), but SUMO proteins and proteases other than Saccharomyces cerevisiae Smt3 and Ulp1 have not previously been employed in expression systems.
Fusion proteins made with other enhancers, such as NusA, promote solubility of partner proteins, presumably due to their large size. In addition, protein fusions with glutathione-S-transferase (GST) or maltose binding protein (MBP) have been proposed to enhance expression and yield of fusion partners. However, enhanced expression is not always observed when GST is used, as GST forms dimers and can retard protein solubility. Another problem with GST, and other fusion systems, is that the desired protein may have to be removed from the fusion (See, e.g., Marblestone, et al., (2006) Comparison of SUMO Fusion Technology with Traditional Gene Fusion System: Effects of Recombinant Protein Expression and Purification. Protein Science 15(1):182-9; Butt, et al., (2005) SUMO Fusion Technology for Difficult to Express Proteins. Protein Expr. Purif. 43:1-9; Zuo, et al., (2005) Expression and Purification of SARS Coronavirus Proteins Using SUMO-Fusions. Protein Expr. Purif. 42:100-110; and Zuo, et al., (2005) Enhanced expression and purification of membrane proteins by SUMO fusion in Echerichia coli. J Struct. Funct. Genomics 6(2-3):103-11.). To circumvent this problem, protease sites, such as Factor Xa, thrombin, enterokinase or Tev protease sites have been engineered downstream of the fusion partner. Often in these cases, however, incomplete cleavage, and inappropriate cleavage within the fusion protein, or both, are observed (see Jonasson, et al. supra).
Inventive Compositions
The inventive subject matter relates to a composition for enhancing expression levels of a protein of interest, comprising:
(a) an isolated nucleic acid sequence encoding a SUMO protein;
(b) an isolated nucleic acid sequence encoding said protein of interest; and
(c) an isolated nucleic acid encoding at least one purification tag,
wherein, upon expression in a suitable expression system, said nucleic acid composition produces a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said protein of interest,
and provided that said SUMO protein is not Saccharomyces cerevisiae Smt3.
In one aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein is derived from an organism selected from the group consisting of human, mouse, insect, plant, yeast, and other eukaryotic organisms.
In another aspect of the inventive subject matter, said organism comprises Homo sapiens, Arabidopsis thalania, tomato, Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe, Plasmodium falciparum, or Aspergillus nidulans. 
In a preferred embodiment, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, an equivalent thereof, a homologue thereof, or a combination thereof.
In another preferred embodiment, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, or a combination thereof.
In a more preferred embodiment, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-3 (SEQ ID NO: 4).
In a further aspect of the inventive subject matter, said nucleic acid composition for enhancing expression levels of a protein of interest, comprises:
(a) an isolated enhancer nucleic acid sequence comprising human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, or a combination thereof;
(b) an isolated nucleic acid sequence encoding said protein of interest; and
(c) an isolated nucleic acid encoding at least one purification tag,
wherein, upon expression in a suitable expression system, said nucleic acid composition produces a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said protein of interest.
In a preferred embodiment, said enhancer nucleic acid sequence comprises human SUMO-3 (SEQ ID NO: 4).
In addition, the inventive subject matter relates to a composition for enhancing solubility of a protein of interest, comprising:
(a) an isolated nucleic acid sequence encoding a SUMO protein;
(b) an isolated nucleic acid sequence encoding said protein of interest; and
(c) an isolated nucleic acid encoding at least one purification tag,
wherein, upon expression in a suitable expression system, said nucleic acid composition produces a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said protein of interest,
and provided that said SUMO protein is not Saccharomyces cerevisiae Smt3.
In one aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein is derived from an organism selected from the group consisting of human, mouse, insect, plant, yeast, and other eukaryotic organisms.
In another aspect of the inventive subject matter, said organism comprises Homo sapiens, Arabidopsis thalania, tomato, Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe, Plasmodium falciparum, or Aspergillus nidulans. 
In a further aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, an equivalent thereof, a homologue thereof, or a combination thereof.
In an alternate aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, or a combination thereof.
In a preferred embodiment, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-3 (SEQ ID NO: 4).
In yet another aspect of the inventive subject matter, said nucleic acid composition for enhancing solubility of a protein of interest, comprising:
(a) an isolated enhancer nucleic acid sequence comprising human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, or a combination thereof;
(b) an isolated nucleic acid sequence encoding said protein of interest; and
(c) an isolated nucleic acid encoding at least one purification tag,
wherein, upon expression in a suitable expression system, said nucleic acid composition produces a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said protein of interest.
In a preferred embodiment, said enhancer nucleic acid sequence comprises human SUMO-3 (SEQ ID NO: 4).
Further, the inventive subject matter relates to an expression vector comprises:
(a) an isolated nucleic acid sequence encoding a SUMO protein;
(b) an isolated nucleic acid sequence encoding a protein of interest; and
(c) an isolated nucleic acid encoding at least one purification tag,
wherein, upon expression in a suitable expression system, said expression vector produces a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said protein of interest,
and provided that said SUMO protein is not Saccharomyces cerevisiae Smt3.
In one aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein is derived from an organism selected from the group consisting of human, mouse, insect, plant, yeast, and other eukaryotic organisms.
In another aspect of the inventive subject matter, said organism comprises Homo sapiens, Arabidopsis thalania, tomato, Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe, Plasmodium falciparum, or Aspergillus nidulans. 
In a further aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein is selected from the group consisting of human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, an equivalent thereof, a homologue thereof, or a combination thereof.
In an alternate aspect of the inventive subject matter, said organism comprises Homo sapiens, Arabidopsis thalania, tomato, Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe, Plasmodium falciparum, or Aspergillus nidulans. 
In a preferred embodiment, said nucleic acid sequence encoding a SUMO protein is human SUMO-3 (SEQ ID NO: 4).
In yet another aspect of the inventive subject matter, said expression vector comprises:
(a) an isolated enhancer nucleic acid sequence comprising human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, or a combination thereof;
(b) an isolated nucleic acid sequence encoding said protein of interest; and
(c) an isolated nucleic acid encoding at least one purification tag,
wherein, upon expression in a suitable expression system, said nucleic acid composition produces a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said protein of interest.
In a preferred embodiment, said enhancer nucleic acid sequence comprises human SUMO-3 (SEQ ID NO: 4).
Inventive Methods
The inventive subject matter also relates to a method for enhancing expression and isolation of a protein of interest, comprising the steps of:
(a) transforming a target cell with an expression vector comprising (1) a vector which is suitable for transforming a target cell and (2) a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding a SUMO protein, (ii) an isolated nucleic acid sequence encoding said protein of interest, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked; and
(b) isolating the protein produced by expression of said nucleic acid insert,
wherein upon expression in said target cell, said expression vector encodes a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of the protein of interest,
and provided that said SUMO protein is not Saccharomyces cerevisiae Smt3.
In one aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein is derived from an organism selected from the group consisting of human, mouse, insect, plant, yeast, and other eukaryotic organisms.
In another aspect of the inventive subject matter, said organism comprises Homo sapiens, Arabidopsis thalania, tomato, Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe, Plasmodium falciparum, or Aspergillus nidulans. 
In a further aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein is selected from the group consisting human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through sumo-3, Drosophila melanogaster SUMO, Caenorhabditis elegans SUMO, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, an equivalent thereof, a homologue thereof, or a combination thereof.
In an alternate aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, or a combination thereof.
In a preferred embodiment, said nucleic acid sequence encoding a SUMO protein is human SUMO-3 (SEQ ID NO: 4).
In yet another aspect of the inventive subject matter, said method for enhancing expression and isolation of a protein of interest, comprising the steps of:
(a) transforming a target cell with an expression vector comprising an isolated enhancer nucleic acid sequence comprising human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, or a combination thereof;
(b) an isolated nucleic acid sequence encoding said protein of interest; and
(c) an isolated nucleic acid encoding at least one purification tag,
wherein, upon expression in a suitable expression system, said nucleic acid composition produces a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said protein of interest.
In a preferred embodiment, said enhancer nucleic acid sequence comprises human SUMO-3 (SEQ ID NO: 4).
In another aspect of the inventive subject matter, said method optionally comprises one or more additional step(s) of:
(d) lysing the target cell expressing the fusion protein;
(e) purifying the fusion protein by way of said at least one purification tag;
(f) cleaving the SUMO moiety and purification tag(s) from the protein of interest with an appropriate SUMO protease or a catalytic domain thereof,
provided that said SUMO protease is not Saccharomyces cerevisiae Ulp1; and
(g) isolating the protein of interest.
In a further aspect of the inventive subject matter, said SUMO protease is derived from an organism selected from the group consisting of human, mouse, insect, plant, yeast, other eukaryotes, and prokaryotic plant and animal pathogens.
In an alternate aspect of the inventive subject matter, said organism comprises Homo sapiens, Mus musculus, Saccharomyces cerevisiae, Arabidopsis thalania, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Aspergillus nidulans, Xenopus laevis, Xanthomonas campestris, Kluyveromyces lactis, or Plasmodium falciparum. 
In yet another aspect of the inventive subject matter, said SUMO protease comprises Saccharomyces cerevisiae Ulp2, human SENP1, human SENP2, human SENP3, human SENP5, human SENP6, human SENP7, mouse SENP1, mouse SENP2, mouse SENP3, mouse SENP5, mouse SENP6, mouse SENP7, any one of Arabidopsis thalania Ulp1a through Ulp1d, any one of Arabidopsis thalania Ulp2a through Ulp2h, Arabidopsis thalania ESD4, Caenorhabditis elegans Ulp-1, Caenorhabditis elegans Ulp-2, Drosophila melanogaster ULP1, Schizosaccharomyces pombe Ulp1, Schizosaccharomyces pombe ULP2, Aspergillus nidulans Ulp, Xenopus laevis XSENP1a, Xenopus laevis XSENP1b, Xanthomonas campestris XopD, Kluyveromyces lactis Ulp1, Plasmodium falciparum Ulp, an equivalent thereof, a homologue thereof, a catalytic domain thereof, or a combination thereof.
In a preferred embodiment, said SUMO protease comprises human SUMO protease SENP2 (SEQ ID NO: 43) or a catalytic domain thereof.
In another aspect of the inventive subject matter, said SUMO protease comprises Saccharomyces cerevisiae Ulp2, human SENP1, human SENP2, human SENP3, human SENP5, human SENP6, human SENP7, mouse SENP1, mouse SENP2, mouse SENP3, mouse SENP5, mouse SENP6, mouse SENP7, any one of Arabidopsis thalania Ulp1a through Ulp1d, any one of Arabidopsis thalania Ulp2a through Ulp2h, Arabidopsis thalania ESD4, Caenorhabditis elegans Ulp-1, Caenorhabditis elegans Ulp-2, Drosophila melanogaster ULP1, Schizosaccharomyces pombe Ulp1, Schizosaccharomyces pombe ULP2, Aspergillus nidulans Ulp, Xenopus laevis XSENP1a, Xenopus laevis XSENP1b, Xanthomonas campestris XopD, Kluyveromyces lactis Ulp1, Plasmodium falciparum Ulp, a catalytic domain thereof, or a combination thereof.
In a preferred embodiment, said SUMO protease comprises human SUMO protease SENP2 (SEQ ID NO: 43) or a catalytic domain thereof.
Further, the inventive subject matter relates to a method for enhancing the solubility and isolation of a protein of interest, comprising the steps of:
(a) transforming a target cell with an expression vector comprising (1) a vector which is suitable for transforming a target cell and (2) a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding a SUMO protein, (ii) an isolated nucleic acid sequence encoding said protein of interest, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked; and
(b) isolating the protein produced by expression of said nucleic acid insert;
wherein upon expression in said target cell, said expression vector encodes a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of the protein of interest,
and provided that said SUMO protein is not Saccharomyces cerevisiae Smt3.
In one aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein is derived from an organism selected from the group consisting of human, mouse, insect, plant, yeast, and other eukaryotic organisms.
In another aspect of the inventive subject matter, said organism comprises Homo sapiens, Arabidopsis thalania, tomato, Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe, Plasmodium falciparum, or Aspergillus nidulans. 
In a further aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein is selected from the group consisting selected from the group consisting of human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, an equivalent thereof, a homologue thereof, or a combination thereof.
In a preferred embodiment, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-3 (SEQ ID NO: 4).
In an alternate aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, or a combination thereof.
In a preferred embodiment, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-3 (SEQ ID NO: 4).
In yet another aspect of the inventive subject matter, said method optionally comprises one or more additional step(s) of:
(d) lysing the target cell expressing the fusion protein;
(e) purifying the fusion protein by way of said at least one purification tag;
(f) cleaving the SUMO moiety and purification tag(s) from the protein of interest with an appropriate SUMO protease, or a catalytic domain thereof; and
(g) isolating the protein of interest,
provided that said SUMO protease is not Saccharomyces cerevisiae Ulp1.
In another aspect of the inventive subject matter, said SUMO protease is derived from an organism selected from the group consisting of human, mouse, insect, plant, yeast, other eukaryotes, and prokaryotic plant and animal pathogens.
In a further aspect of the inventive subject matter, said organism comprises Homo sapiens, Mus musculus, Saccharomyces cerevisiae, Arabidopsis thalania, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Aspergillus nidulans, Xenopus laevis, Xanthomonas campestris, Kluyveromyces lactis, or Plasmodium falciparum. 
In an alternate aspect of the inventive subject matter, said SUMO protease comprises Saccharomyces cerevisiae Ulp2, human SENP1, human SENP2, human SENP3, human SENP5, human SENP6, human SENP7, mouse SENP1, mouse SENP2, mouse SENP3, mouse SENP5, mouse SENP6, mouse SENP7, any one of Arabidopsis thalania Ulp1a through Ulp1d, any one of Arabidopsis thalania Ulp2a through Ulp2h, Arabidopsis thalania ESD4, Caenorhabditis elegans Ulp-1, Caenorhabditis elegans Ulp-2, Drosophila melanogaster ULP1, Schizosaccharomyces pombe Ulp1, Schizosaccharomyces pombe ULP2, Aspergillus nidulans Ulp, Xenopus laevis XSENP1a, Xenopus laevis XSENP1b, Xanthomonas campestris XopD, Kluyveromyces lactis Ulp1, and Plasmodium falciparum Ulp, an equivalent thereof, a homologue thereof, a catalytic domain thereof, or a combination thereof.
In a preferred embodiment, said SUMO protease comprises human SUMO protease SENP2 (SEQ ID NO: 43) or a catalytic domain thereof.
In yet another aspect of the inventive subject matter, said SUMO protease comprises Saccharomyces cerevisiae Ulp2, human SENP1, human SENP2, human SENP3, human SENP5, human SENP6, human SENP7, mouse SENP1, mouse SENP2, mouse SENP3, mouse SENP5, mouse SENP6, mouse SENP7, any one of Arabidopsis thalania Ulp1a through Ulp1d, any one of Arabidopsis thalania Ulp2a through Ulp2h, Arabidopsis thalania ESD4, Caenorhabditis elegans Ulp-1, Caenorhabditis elegans Ulp-2, Drosophila melanogaster ULP1, Schizosaccharomyces pombe Ulp1, Schizosaccharomyces pombe ULP2, Aspergillus nidulans Ulp, Xenopus laevis XSENP1a, Xenopus laevis XSENP1b, Xanthomonas campestris XopD, Kluyveromyces lactis Ulp1, Plasmodium falciparum Ulp, a catalytic domain thereof, or a combination thereof.
In a preferred embodiment, said SUMO protease comprises human SUMO protease SENP2 (SEQ ID NO: 43) or a catalytic domain thereof.
More particularly, the inventive subject matter relates to a method for enhancing expression and isolation of a protein of interest, comprising the steps of:
(a) transforming a target cell with an expression vector comprising (1) a vector which is suitable for transforming a target cell and (2) a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding human SUMO-3 (SEQ ID NO: 4), (ii) an isolated nucleic acid sequence encoding said protein of interest, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked;
(b) isolating the protein produced by expression of said nucleic acid insert;
(c) lysing the cell expressing the fusion protein;
(d) purifying the fusion protein by way of said at least one purification tag;
(e) cleaving the SUMO moiety and purification tag(s) from the protein of interest with human SUMO protease SENP2 (SEQ ID NO: 43) or a catalytic domain thereof; and
(f) isolating the protein of interest,
wherein upon expression in said target cell, said expression vector encodes a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of the protein of interest.
Further, the inventive subject matter particularly relates to a method for enhancing solubility and isolation of a protein of interest, comprising the steps of:
(a) transforming a target cell with an expression vector comprising (1) a vector which is suitable for transforming a target cell and (2) a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding human SUMO-3 (SEQ ID NO: 4), (ii) an isolated nucleic acid sequence encoding said protein of interest, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked;
(b) isolating the protein produced by expression of said nucleic acid insert;
(c) lysing the cell expressing the fusion protein;
(d) purifying the fusion protein by way of said at least one purification tag;
(e) cleaving the SUMO moiety and purification tag(s) from the protein of interest with human SUMO protease SENP2 (SEQ ID NO: 43) or a catalytic domain thereof; and
(f) isolating the protein of interest,
wherein upon expression in said target cell, said expression vector encodes a fusion protein in which said purification tag is N-terminal to said SUMO protein, and said SUMO protein is attached to the amino terminus of the protein of interest.
In addition, the inventive subject matter relates to a method for generating proteins with a novel N-terminal amino acid which is not proline, comprising the steps of:
(a) transforming a target cell with an expression vector comprising (1) a vector which is suitable for transforming a target cell and (2) a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding a SUMO protein, (ii) an isolated nucleic acid sequence encoding a protein of interest having a novel N-terminal amino acid, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked;
(b) isolating the protein produced by expression of said nucleic acid insert,
wherein upon expression in said target cell, said expression vector encodes a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said novel N-terminal amino acid of the protein of interest;
(c) cleaving the SUMO moiety and purification tag(s) from the protein of interest with an appropriate SUMO protease, or a catalytic domain thereof; and
(d) isolating the protein of interest having a novel N-terminal amino acid,
provided that said SUMO protease is not Saccharomyces cerevisiae Ulp1,
and provided that said isolated nucleic acid sequence encoding a SUMO protein is not Saccharomyces cerevisiae Smt3.
In one aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein is derived from an organism selected from the group consisting of human, mouse, insect, plant, yeast, and other eukaryotic organisms.
In another aspect of the inventive subject matter, said organism comprises Homo sapiens, Arabidopsis thalania, tomato, Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe, Plasmodium falciparum, or Aspergillus nidulans. 
In a further aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, an equivalent thereof, a homologue thereof, or a combination thereof.
In a preferred embodiment, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-3 (SEQ ID NO: 4).
In an alternate aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, or a combination thereof.
In a preferred embodiment, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-3 (SEQ ID NO: 4).
In yet another aspect of the inventive subject matter, said expression vector comprises
(a) human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, or a combination thereof;
(b) an isolated nucleic acid sequence encoding said protein of interest having a novel N-terminal amino acid; and
(c) an isolated nucleic acid encoding at least one purification tag,
wherein, upon expression in a suitable expression system, said nucleic acid composition produces a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said protein of interest having a novel N-terminal amino acid.
In a preferred embodiment, said enhancer nucleic acid sequence comprises human SUMO-3 (SEQ ID NO: 4).
In another aspect of the inventive subject matter, said cleavage enzyme comprises a SUMO protease, or a catalytic domain thereof.
In a further aspect of the inventive subject matter, said SUMO protease is derived from an organism selected from the group consisting of human, mouse, insect, plant, yeast, other eukaryotes, and prokaryotic plant and animal pathogens.
In an alternate aspect of the inventive subject matter, said SUMO protease comprises Saccharomyces cerevisiae Ulp2, human SENP1, human SENP2, human SENP3, human SENP5, human SENP6, human SENP7, mouse SENP1, mouse SENP2, mouse SENP3, mouse SENP5, mouse SENP6, mouse SENP7, any one of Arabidopsis thalania Ulp1a through Ulp1d, any one of Arabidopsis thalania Ulp2a through Ulp2h, Arabidopsis thalania ESD4, Caenorhabditis elegans Ulp-1, Caenorhabditis elegans Ulp-2, Drosophila melanogaster ULP1, Schizosaccharomyces pombe Ulp1, Schizosaccharomyces pombe ULP2, Aspergillus nidulans Ulp, Xenopus laevis XSENP1a, Xenopus laevis XSENP1b, Xanthomonas campestris XopD, Kluyveromyces lactis Ulp1, or Plasmodium falciparum Ulp, an equivalent thereof, a homologue thereof, a catalytic domain thereof, or a combination thereof.
In a preferred embodiment, said SUMO protease comprises human SUMO protease SENP2 (SEQ ID NO: 43) or a catalytic domain thereof.
In yet another aspect of the inventive subject matter, said SUMO protease comprises Saccharomyces cerevisiae Ulp2, human SENP1, human SENP2, human SENP3, human SENP5, human SENP6, human SENP7, mouse SENP1, mouse SENP2, mouse SENP3, mouse SENP5, mouse SENP6, mouse SENP7, any one of Arabidopsis thalania Ulp1a through Ulp1d, any one of Arabidopsis thalania Ulp2a through Ulp2h, Arabidopsis thalania ESD4, Caenorhabditis elegans Ulp-1, Caenorhabditis elegans Ulp-2, Drosophila melanogaster ULP1, Schizosaccharomyces pombe Ulp1, Schizosaccharomyces pombe ULP2, Aspergillus nidulans Ulp, Xenopus laevis XSENP1a, Xenopus laevis XSENP1b, Xanthomonas campestris XopD, Kluyveromyces lactis Ulp1, Plasmodium falciparum Ulp, a catalytic domain thereof, or a combination thereof.
In a preferred embodiment, said SUMO protease comprises human SUMO protease SENP2 (SEQ ID NO: 43) or a catalytic domain thereof.
In another aspect, the inventive subject matter relates to a method for generating proteins with a proline N-terminal amino acid, comprising the steps of:
(a) transforming a target cell with an expression vector comprising (1) a vector which is suitable for transforming a target cell and (2) a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding a SUMO protein, (ii) an isolated nucleic acid sequence encoding a protein of interest having at least one N-terminal amino acid which is not proline and having a proline adjoining said at least one N-terminal amino acid, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked;
(b) isolating the protein produced by expression of said nucleic acid insert,
wherein upon expression in said target cell, said expression vector encodes a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of said at least one N-terminal amino acid of the protein of interest,
and provided that said isolated nucleic acid sequence encoding a SUMO protein is not Saccharomyces cerevisiae Smt3;
(c) cleaving the SUMO moiety and purification tag(s) from the protein of interest with an appropriate SUMO protease, or a catalytic domain thereof,
provided that said SUMO protease is not Saccharomyces cerevisiae Ulp1;
(d) isolating the protein of interest having at least one N-terminal amino acid which is not proline and having a proline adjoining said at least one N-terminal amino acid; and
(e) treating said protein of interest with an aminopeptidase to remove said at least one N-terminal amino acid which is not proline and leaving proline as the amino terminus.
In one aspect of the inventive subject matter, said at least one N-terminal amino acid which is not proline comprises methionine and said aminopeptidase comprises methionine aminopeptidase or (b) said at least one N-terminal amino acid which is not proline comprises alanine and said aminopeptidase comprises alanine aminopeptidase.
Inventive Kits
Finally, the inventive subject matter relates to a kit for enhancing expression levels of a protein of interest, comprising:
(a) a vector which is suitable for transforming a target cell and which has at least one cloning site for cloning a nucleic acid encoding a protein of interest, operably linked to a nucleic acid insert comprising (i) an isolated nucleic acid sequence encoding a SUMO protein, and (ii) an isolated nucleic acid encoding at least one purification tag,
provided that said isolated nucleic acid sequence encoding a SUMO protein is not Saccharomyces cerevisiae Smt3,
wherein said at least one cloning site is oriented in said vector so that upon cloning in a nucleic acid sequence encoding said protein of interest and expression in a target cell, said vector encodes a fusion protein in which said purification tag is N-terminus to said SUMO protein, and said SUMO protein is attached to the amino terminus of the protein of interest; and
(b) a cleavage enzyme for cleaving the SUMO moiety and purification tag(s) from the protein of interest.
In one aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein is derived from an organism selected from the group consisting of human, mouse, insect, plant, yeast, and other eukaryotic organisms.
In another aspect of the inventive subject matter, said organism comprises Homo sapiens, Arabidopsis thalania, tomato, Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe, Plasmodium falciparum, or Aspergillus nidulans. 
In a further aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, an equivalent thereof, a homologue thereof, or a combination thereof.
In an alternate aspect of the inventive subject matter, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-1, human SUMO-2, human SUMO-3, any one of Arabidopsis thalania SUMO-1 through SUMO-8, tomato SUMO, any one of Xenopus laevis SUMO-1 through SUMO-3, Drosophila melanogaster Smt3, Caenorhabditis elegans SMO-1, Schizosaccharomyces pombe Pmt3, malarial parasite Plasmodium falciparum SUMO, mold Aspergillus nidulans SUMO, or a combination thereof.
In a preferred embodiment, said nucleic acid sequence encoding a SUMO protein comprises human SUMO-3 (SEQ ID NO: 4).
In yet another aspect of the inventive subject matter, said cleavage enzyme is a SUMO protease, or a catalytic domain thereof.
In another aspect of the inventive subject matter, said SUMO protease is derived from an organism selected from the group consisting of human, mouse, insect, plant, yeast, other eukaryotes, and prokaryotic plant and animal pathogens.
In a further aspect of the inventive subject matter, said organism comprises Homo sapiens, Mus musculus, Saccharomyces cerevisiae, Arabidopsis thalania, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Aspergillus nidulans, Xenopus laevis, Xanthomonas campestris, Kluyveromyces lactis, or Plasmodium falciparum. 
In an alternate aspect of the inventive subject matter, said SUMO protease comprises Saccharomyces cerevisiae Ulp2, human SENP1, human SENP2, human SENP3, human SENP5, human SENP6, human SENP7, mouse SENP1, mouse SENP2, mouse SENP3, mouse SENP5, mouse SENP6, mouse SENP7, any one of Arabidopsis thalania Ulp1a through Ulp1d, any one of Arabidopsis thalania Ulp2a through Ulp2h, Arabidopsis thalania ESD4, Caenorhabditis elegans Ulp-1, Caenorhabditis elegans Ulp-2, Drosophila melanogaster ULP1, Schizosaccharomyces pombe Ulp1, Schizosaccharomyces pombe ULP2, Aspergillus nidulans Ulp, Xenopus laevis XSENP1a, Xenopus laevis XSENP1b, Xanthomonas campestris XopD, Kluyveromyces lactis Ulp1, and Plasmodium falciparum Ulp, an equivalent thereof, a homologue thereof, a catalytic domain thereof, or a combination thereof.
In a preferred embodiment, said SUMO protease comprises human SUMO protease SENP2 (SEQ ID NO: 43) or a catalytic domain thereof.
In yet another aspect of the inventive subject matter, said SUMO protease comprises Saccharomyces cerevisiae Ulp2, human SENP1, human SENP2, human SENP3, human SENP5, human SENP6, human SENP7, mouse SENP1, mouse SENP2, mouse SENP3, mouse SENP5, mouse SENP6, mouse SENP7, any one of Arabidopsis thalania Ulp1a through Ulp1d, any one of Arabidopsis thalania Ulp2a through Ulp2h, Arabidopsis thalania ESD4, Caenorhabditis elegans Ulp-1, Caenorhabditis elegans Ulp-2, Drosophila melanogaster ULP1, Schizosaccharomyces pombe Ulp1, Schizosaccharomyces pombe ULP2, Aspergillus nidulans Ulp, Xenopus laevis XSENP1a, Xenopus laevis XSENP1b, Xanthomonas campestris XopD, Kluyveromyces lactis Ulp1, Plasmodium falciparum Ulp, a catalytic domain thereof, or a combination thereof.
In a preferred embodiment, said SUMO protease comprises human SUMO protease SENP2 (SEQ ID NO: 43) or a catalytic domain thereof.
Thus, some embodiments of the inventive subject matter may be understood to encompass compositions and methods for enhancing expression levels and solubility of a protein of interest, and for isolating said protein. Optionally, the protein of interest may be produced with a desired N-terminus, other than methionine as produced in nature.
In accordance with an embodiment of the inventive subject matter, a method for improving the expression of a protein, particularly an unexpressed or poorly expressed protein, is provided. In particular, the inventive subject matter relates to a method which comprises attaching a SUMO protein from human, mouse, insect, plant, yeast, or other eukaryote to the amino-terminus of the protein to be expressed, also referred to herein as the protein of interest. Applicants have found that SUMO proteins share a similar, common 3-D structure with one another and with ubiquitin. A hallmark of SUMO and most other ubiquitin-related proteins is that they are synthesized as precursors and processed by a hydrolase or protease at the carboxy-terminus, at a di-glycine motif, to generate a mature sequence. Another feature of the inventive subject matter is the use of such a protease from human, mouse, insect, plant, yeast, or other eukaryote or prokaryotic plant or animal pathogen to process expressed SUMO fusion proteins to generate proteins of interest with desired N-termini.
Methods employing fusion proteins with full-length or partial SUMO and other Ubls to obtain expression and purification have been previously described by Applicants in U.S. application Ser. Nos. 10/338,411 and 10/389,640. These previous SUMO-fusion expression systems have been based on the SUMO protein Smt3 from the yeast Saccharomyces cerevisiae and have employed the S. cerevisiae SUMO protease Ulp1 for cleavage of the fusion. Cleavage of SUMO fusions was possible except when proline was the amino-terminal residue of the protein of interest.
An exemplary method comprises i) generating a construct encoding a fusion protein by operably linking a nucleic acid sequence encoding a SUMO protein from human, mouse, insect, plant, yeast, or other eukaryotic organism, except not Saccharomyces cerevisiae Smt3, to a nucleic acid sequence encoding the protein of interest and to a nucleic acid sequence encoding at least one purification tag, whereby the purification tag is added to the amino terminus of the SUMO protein, which in turn is attached to the amino-terminus of the protein of interest, and ii) introducing the nucleic acid encoding the fusion protein into a host cell. To purify the protein of interest and generate a non-methionine N-terminus, the method may further comprise: iii) lysing host cells expressing the fusion protein, iv) purifying the fusion protein by way of the affinity tag, and v) cleaving off the SUMO moiety with an appropriate recombinant SUMO protease, which may be derived from human, mouse, insect, plant, yeast, or other eukaryote, or prokaryotic plant or animal pathogen to release the protein of interest from the fusion protein.
A more specific example of the inventive subject matter is the use of the human SUMO-3 protein (SEQ ID NO: 4) as an N-terminal fusion to increase the expression of a protein of interest in E. coli, followed by purification and in vitro cleavage by the catalytic domain of the human SUMO protease SENP2 (SEQ ID NO: 43). Applicants have found that, surprisingly, human SUMO-3 is a more effective expression tag than yeast SUMO (Smt3), and the human SUMO protease SENP2 cleaves an hSUMO-3 fusion protein in vitro much more effectively than does the yeast SUMO protease Ulp1.
There are a number of reasons for the lack of efficient recombinant protein expression in a host, including, for example, short half-life, improper folding, compartmentalization and codon bias. While the Human Genome project has successfully created a DNA “map” of the human genome, the development of protein expression technologies that function uniformly in different expression platforms and for all the protein motifs has not yet been achieved.
Provided herein are methods which allow for the expression of fusion proteins with SUMO protein species other than ySmt3, such as human SUMO-3, to improve expression. Purification can be based on an optionally SUMO-attached affinity tag (i.e. purification tag) such as 6xHis, and cleavage is then obtained in vitro with a specific SUMO hydrolase other than S. cerevisiae Ulp1, such as human SENP2, that recognizes the particular SUMO protein used. The SUMO protein and protease pair may potentially be derived from any organism(s), and may be from the same species or different species.
The inventive subject matter may also be utilized to generate proteins with novel N-termini in eukaryotic cells.
The identity of the N-terminus of a protein has been proposed to control its half-life, also called the N-end Rule (Varshavsky, A., 1996, The N-end rule: functions, mysteries, uses, Proc Natl Acad Sci USA 93:12142-9). Many important biopharmaceuticals, such as growth factors, chemokines, and other cellular proteins, require desired N-termini for therapeutic activity. It has not been previously possible to generate desired N-termini in vivo, as nature initiates translation from methionine. In previously existing N-terminal modification systems, one or more amino acids may be removed from the N-terminus, other amino acids may be added, and various covalent modifications may be introduced to the exposed N-termini (Link, A. J., K. Robison, and G. M. Church, 1997, Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12, Electrophoresis 18:1259-313). These modification systems are complex and vary to some degree in specificity and performance (Varshavsky, A., 2003, The N-end rule and regulation of apoptosis, Nat Cell Biol 5:373-6). This means that recombinantly produced proteins that do not undergo in vitro processing by a protease of known specificity may have unexpected and/or heterogeneous N-termini.
According to the inventive subject matter, any amino acid can be incorporated as the N-terminal residue of a protein of interest by altering the appropriate codon in the nucleic acid encoding for the protein of interest. The altered protein of interest is then attached to a SUMO protein, expressed in a desired cell, and purified as described herein and then cleaved with a specific protease.
Alternatively, the fusion protein may be expressed in vivo to produce the protein of interest so long as there is specific protease activity in the cell (i.e. native or provided exogenously). Every amino acid except for proline may be employed as the amino-terminal residue by these methods. To employ proline as the amino-terminal residue of the protein of interest, a fusion protein in which the cleavage site of the Ubl protease is followed by the amino acid sequence Met-Pro. Following reconstitution and cleavage with the appropriate protease, the Met-Pro amino-terminus of the protein of interest is further treated with an aminopeptidase such as alanine aminopeptidase or methionine aminopeptidase (see Ben-Bassat, A., 1991, Methods for removing N-terminal methionine from recombinant proteins, Bioprocess Technol 12:147-59) to remove the N-terminal amino acid and leave proline as the amino-terminus.
Any of the known or putative SUMO proteins (see FIG. 1A for representative sets of sequences of their mature forms) may be utilized in the compositions and methods of the inventive subject matter to (1) enhance expression of heterologous fusion proteins of interest, (2) enhance solubility of heterologous fusion proteins of interest, (3) allow affinity purification of the fusion protein by way of an additional N-terminal tag such as 6xHis, and (4) allow recognition for cleavage. These SUMO proteins include but are not limited to those presented in the alignment in FIG. 1A. FIG. 1A is an alignment of the published polypeptide sequences of the predicted mature forms of known and putative SUMO proteins from various organisms. Only a representative set of previously described SUMO sequences are provided; it is expected that all eukaryotes have one or more forms of SUMO. There is only one SUMO protein in the yeast Saccharomyces cerevisiae (ySmt3), but there are 3 in mammals such as human (hSUMO-1, -2, and -3) and mouse, and there are as many as 8 in the plant Arabidopsis thalania (see Kurepa, et al., 2003, The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis, Accumulation of SUMO1 and -2 conjugates is increased by stress, J Biol Chem 278:6862-72; and Lois, et al., 2003, Small ubiquitin-like modifier modulates abscisic acid signaling in Arabidopsis, Plant Cell 15:1347-59); examples of other diverse eukaryotes for which SUMO proteins have been identified are also known: the tomato (Hanania, et al., 1999, Isolation of a novel SUMO protein from tomato that suppresses EIX-induced cell death, Plant J 19:533-41), the frog Xenopus laevis (Saitoh, et al., 1998, Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2, Curr. Biol. 8:121-124), the fruit fly Drosophila melanogaster (Lehembre, et al., 2000, Covalent modification of the transcriptional repressor tramtrack by the ubiquitin-related protein Smt3 in Drosophila flies, Mol. Cell. Biol. 20:1072-1082), the nematode Caenorhabditis elegans, the yeast Schizosaccharomyces pombe (Tanaka, et al., 1999, Characterization of a fission yeast SUMO-1 homologue, pmt3p, required for multiple nuclear events, including the control of telomere length and chromosome segregation, Mol. Cell. Biol. 19:8660-8672), the malarial parasite Plasmodium falciparum, and the mold Aspergillus nidulans, among many others.
Any of the known or putative SUMO proteases (see FIG. 1B and the Sequence Listing for the sequences of their catalytic domains) may be utilized in the compositions and methods of the inventive subject matter to remove SUMO from the proteins of interest, provided that said SUMO protease is not Saccharomyces cerevisiae Ulp1. These SUMO proteases include but are not limited to those presented in an alignment in FIG. 1B. FIG. 1B is an alignment of the published polypeptide sequences of the catalytic domains of known and putative SUMO proteases from various organisms. The catalytic triad residues of the protease active site are indicated in bold. Only a representative set of previously described SUMO protease sequences are provided, and it is expected that all eukaryotes may have multiple SUMO proteases. There are two SUMO proteases in S. cerevisiae (Ulp1 and Ulp2), at least 6 in human (SENP1, 2, 3, 5, 6, and 7), and possibly more than a dozen in Arabidopsis. 
The inventive SUMO fusion system has been successfully applied to express the enhanced green fluorescent protein (eGFP) in E. coli, purify the fusion, and generate the final product by cleavage with proteases in vitro. The inventive subject matter allows for: (1) the enhancement of the expression of under-expressed proteins; (2) increasing of the solubility of proteins that are insoluble or of low solubility; (3) protecting a protein of interest from degradation by intracellular N-terminus proteases by fusing SUMO sequences to the N-terminus; (4) cleaving the fusion protein in vitro to efficiently generate authentic proteins; and (5) generating proteins with novel N-termini amino acids.
Most known proteases, including the ones that are commonly used in recombinant protein processing, recognize small, 4-8 residues, and degenerate stretches of amino acid sequence and, as a consequence, often cleave within a protein of interest. Importantly, SUMO proteases are structure-specific enzymes that recognize the entire structure of a respective SUMO protein and not just several amino acid residues. The combination of properties of SUMO fusion proteins, along with the specificity and robustness of the respective proteases, creates a superior system for recombinant protein expression, purification and processing. Since SUMO fusion can both enhance recombinant protein yield and generate new N-termini, this technology provides an important tool for post-genomic biotechnology analyses.
In another embodiment of the inventive subject matter, kits are provided for performing the methods described herein, for use in effecting enhanced expression, solubility, secretion, purification, localization, and alteration of the amino terminus of a protein of interest. Such kits comprise a recombinant vector containing a nucleic acid sequence encoding a SUMO protein from human, mouse, insect, plant, yeast, or other eukaryotic organism, except not Saccharomyces cerevisiae Smt3, operably linked to a promoter suitable for expression in the desired host cell and a multiple cloning site suitable for cloning a nucleic acid encoding the protein of interest. The recombinant vector may also contain a nucleic acid sequence encoding for at least one purification tag.
Such kits comprise a recombinant vector containing a nucleic acid sequence encoding a SUMO molecule operably linked to a promoter suitable for expression in the desired host cell and a multiple cloning site suitable for cloning a nucleic acid encoding the protein of interest in the reading frame (“in-frame”) with the nucleic acid sequence encoding the SUMO molecule. The promoter is preferably a strong promoter and may be constitutive or regulated. Such promoters are well known in the art and include, but are not limited to, the examples of T7 in E. coli, CUP1 or ADH1 in yeast, or the polyhedrin promoter in the insect baculovirus system.
The recombinant vector may also contain a nucleic acid sequence encoding at least one purification tag in-frame with the sequence encoding the SUMO molecule. Purification tags are well known in the art (see Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; and Terpe, K., 2003, Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems, Appl Microbiol Biotechnol 60:523-33) and include, but are not limited to: polyhistidine tags such as 6xHis, polyarginine tags, glutathione-S-transferase (GST), maltose binding protein (MBP), S-tag, influenza virus HA tag, thioredoxin, staphylococcal protein A tag, the FLAGTM epitope, AviTag epitope, which also permits subsequent biotinylation, and the c-myc epitope. Materials and methods for the purification of fusion proteins via purification tags are also well known in the art (see Sambrook et al., supra, and such commercial sources as the Novagen catalog, 2004, as well as the Examples below). Reagents, including but not limited to at least one solid support capable of binding the purification tag, lysis buffers, wash buffers, and elution buffers may also be included in the inventive kits.
Such kits may further comprise a protease composition capable of cleaving a SUMO molecule from the fusion protein and optional purification tag. A more complete kit embodiment includes at least one solid phase capable of binding at least one purification tag; appropriate buffers including wash and cleavage buffers; instruction material; and frozen stocks of appropriate host cells. The kits may also further comprise reagents for altering the nucleic acid encoding a protein of interest to generate amino termini which are different from those native to the wild-type protein. Methods for altering the nucleic acid are well known in the art and include, but are not limited to, site-directed mutagenesis and oligonucleotide-based site-directed mutagenesis (see commercial sources such as the BD Biosciences, Qiagen, and Stratagene catalogs, 2004; Ausubel et al., eds., 1995, Current Protocols in Molecular Biology, John Wiley and Sons, Inc.).
FIG. 2 is a schematic representation of a cloning strategy used to generate various SUMO fusion proteins. In this cloning strategy, the nucleic acid sequence encoding the protein to be expressed as a fusion with SUMO is amplified by PCR with primers that introduce a Bsa I site (or other type II restriction enzyme capable of generating an ACCT overhang) at the 5′ end and BamHI site (or other site in the multiple cloning sequence of pET24) at the 3′ end. The vector encoding SUMO contains, in this example, a BsaI site and a BamHI site at the 3′ end of the SUMO encoding region. The PCR product and vector are cleaved by BsaI and BamHI (or other appropriate restriction enzyme) for insertion of the cleaved PCR product into the vector. The ligation reconstitutes the codons encoding the Gly-Gly motif, the cleavage site of the resulting fusion protein.
FIG. 3A shows a Coomassie-stained gel that demonstrates that the attachment of various SUMO proteins to the amino-terminus of target protein eGFP increases the expression and/or enhances the solubility of the protein in E. coli. Shown are soluble (S) and insoluble inclusion body (IB) fractions from pET24d-6xHis-SUMO-eGFP-containing cells that were induced with IPTG and grown at 37° C. or 20° C.
FIG. 3B shows a quantitative comparison of yields of different SUMO-eGFP fusions in soluble fractions, by way of eGFP fluorescence. The data are presented in arbitrary units and the values were from three fluorimetric measurements (mean with standard deviations) under the same conditions.
FIG. 4A shows Coomassie-stained gels of in vitro reactions in which 2-fold dilution series of human SENP2 or yeast Ulp1 were used to cleave a variety of SUMO-eGFP fusions, demonstrating the different efficiencies and specificities of cleavage.
FIG. 4B shows Coomassie-stained gels of time courses of cleavage of various SUMO-eGFP fusions by human SENP2 or yeast Ulp1, demonstrating the different efficiencies and specificities of cleavage.
FIG. 4C shows Coomassie-stained SDS-PAGE gels comparing human SENP2 and yeast Ulp1 used to cleave hSUMO3-eGFP fusions with each of the 20 amino acids at the N-terminus of eGFP, demonstrating the improved efficiencies and specificities of cleavage by SENP2.
FIG. 5 shows Coomassie-stained gels of in vitro reactions of four additional SUMO proteases from several species against various SUMO-eGFP fusions, demonstrating that various SUMO protease catalytic domains are capable of cleaving different SUMO proteins from GFP with differing specificities and efficiencies.
FIG. 6A is a map of an exemplary plasmid pET24d-6xHis-SUMO expression vector, as used in the inventive subject matter; it can contain any of the SUMO proteins described in this application or from any eukaryote. The parental vector used for the present data was pET24d, but alternative versions of pET vectors or other expression plasmids may be used. FIG. 6B shows the regions that flank the sequences of 6xHis-SUMO in the plasmid (using hSUMO-3 as an example).
The Sequence Listing contains the amino acid and nucleotide sequence sequences of the various SUMO proteins used in these studies.
SEQ ID NO: 1 and SEQ ID NO: 2 depict the amino acid sequence and nucleotide sequence of human SUMO-1 (hSUMO-1).
SEQ ID NO: 4 and SEQ ID NO: 4 depict the amino acid sequence and nucleotide sequence of human SUMO-3 (hSUMO-3).
SEQ ID NO: 5 and SEQ ID NO: 6 depict the amino acid sequence and nucleotide sequence of Arabidopsis thalania SUMO1 (AtSUMO1).
SEQ ID. NO: 7 and SEQ ID NO: 8 depict the amino acid sequence and nucleotide sequence of Arabidopsis thalania SUMO2 (AtSUMO2).
SEQ ID NO: 9 and SEQ ID NO: 10 depict the amino acid sequence and nucleotide sequence of tomato (Lycopersicum esculentum) T-SUMO.
SEQ ID NO: 11 and SEQ ID NO: 12 depict the amino acid sequence and nucleotide sequence of Saccharomyces cerevisiae Smt3 (ySmt3, ySUMO).
SEQ ID NO: 13 and SEQ ID NO: 14 depict the amino acid sequence and nucleotide sequence of 6xHis-tagged human SUMO-1.
SEQ ID NO: 15 and SEQ ID NO: 16 depict the amino acid sequence and nucleotide sequence of 6xHis-tagged human SUMO-3.
SEQ ID NO: 17 and SEQ ID NO: 18 depict the amino acid sequence and nucleotide sequence of 6xHis-tagged Arabidopsis thalania SUMO1.
SEQ ID NO: 19 and SEQ ID NO: 20 depict the amino acid sequence and nucleotide sequence of 6xHis-tagged Arabidopsis thalania SUMO2.
SEQ ID NO: 21 and SEQ ID NO: 22 depict the amino acid sequence and nucleotide sequence of 6xHis-tagged tomato T-SUMO.
SEQ ID NO: 23 and SEQ ID NO: 24 depict the amino acid sequence and nucleotide sequence of 6xHis-tagged Saccharomyces cerevisiae Smt3.
SEQ ID NO: 25 and SEQ ID NO: 26 depict the amino acid sequence and nucleotide sequence of eGFP (enhanced green fluorescent protein).
SEQ ID NO: 27 and SEQ ID NO: 28 depict the amino acid sequence and nucleotide sequence of the N-terminally tagged 6xHis-eGFP protein as it appears in pET-6xHis-eGFP.
SEQ ID NO: 29 and SEQ ID NO: 40 depict the amino acid sequence and nucleotide sequence of 6xHis-hSUMO-1-eGFP.
SEQ ID NO: 41 and SEQ ID NO: 42 depict the amino acid sequence and nucleotide sequence of 6xHis-hSUMO-3-eGFP.
SEQ ID NO: 43 and SEQ ID NO: 44 depict the amino acid sequence and nucleotide sequence of 6xHis-AtSUMO1-eGFP.
SEQ ID NO: 45 and SEQ ID NO: 46 depict the amino acid sequence and nucleotide sequence of 6xHis-AtSUMO2-eGFP.
SEQ ID NO: 47 and SEQ ID NO: 48 depict the amino acid sequence and nucleotide sequence of 6xHis-T-SUMO-eGFP.
SEQ ID NO: 49 and SEQ ID NO: 40 depict the amino acid sequence and nucleotide sequence of 6xHis-ySmt3-eGFP.
SEQ ID NO: 41 and SEQ ID NO: 4 depict the amino acid sequence and nucleotide sequence of human SENP1 (hSENP1).
SEQ ID NO: 43 and SEQ ID NO: 44 depict the amino acid sequence and nucleotide sequence of human SENP2 (hSENP2).
SEQ ID NO: 45 and SEQ ID NO: 46 depict the amino acid sequence and nucleotide sequence of human SENP6 (hSENP6).
SEQ ID NO: 47 and SEQ ID NO: 48 depict the amino acid sequence and nucleotide sequence of Arabidopsis thalania ULP1c (AtULP1c).
SEQ ID NO: 49 and SEQ ID NO: 50 depict the amino acid sequence and nucleotide sequence of Saccharomyces cerevisiae Ulp1 (yeast Ulp1, yUlp1).
SEQ ID NO: 51 and SEQ ID NO: 52 depict the amino acid sequence and nucleotide sequence of a GST fusion of the catalytic domain of the Xanthomonas campestris pathovar vesicatoria protease XopD (GST-XopD).
SEQ ID NO: 53-68 depict the sequence of PCR primers.
SEQ ID NO: 69 AND SEQ ID NO: 70 depict the amino acid sequence and nucleotide sequence of 6xHis-hSUMO-3-X-eGFP, where “X” represents each of the 20 naturally occurring amino acids tested to demonstrate the ability to make proteins with a novel, i.e. non-methionine, N-terminal amino acid.
EXAMPLES
The following examples are illustrative of the inventive subject matter and are not intended to be limitations thereon. Unless otherwise indicated, all percentages are based upon 100% by weight of the final composition. All starting materials, reagents, and solvents were commercially available and were used either as obtained from commercial suppliers, synthesized according to known literature procedures, and/or washed, dried, distilled, recrystallized, and/or purified before use.
Example 1
Design and Construction of E. coli Expression Vectors
The original vector backbone was developed using pET 24d vector from Novagen. pET24d uses a T7 promoter system that is inducible with IPTG. The vector has a kanamycin selection marker and does not contain any translation terminator. This vector was modified by PCR to encode an N-terminal 6xHis tag followed by a BsaI site, becoming the plasmid pET24d-6xHis.
Example 2
Construction of 6xHis-SUMO and 6xHis-SUMO-eGFP Expression Plasmids
The amino-terminal 6xHis-tagged SUMO fusion vectors were constructed as follows. For human SUMO-1, a PCR product was generated with the primers 5′-TTTTTTCCATGGGTCATCACCATCATCATCACTCTGACCAGGAGGCAAAAC-3′ (SEQ ID NO: 53) and 5′-TTTTTTGGATCCGGTCTCAACCTCCCGTTTGTTCCTGATAAAC-3′ (SEQ ID NO: 54) using a SUMO-1 gene as a template (kind gift of K. I. Kim). For Saccharomyces cerevisiae Smt3, a PCR product was generated with the primers 5′-CCATGGGTCATCACCATCATCATCACGGGTCGGACTCAGAAGTCAATCAA-3′ (SEQ ID NO: 55) and 5′-GGATCCGGTCTCAACCTCCAATCTGTTCGCGGTGAG-3′ (SEQ ID NO: 56) using a modified SUMO (Smt3) gene as a template (kind gift of Erica Johnson). Specifically, the SUMO (SEQ ID NO: 12) used throughout the examples provided herein contains an adenine nucleotide at position 229 in place of a guanine nucleotide reported in the reported SUMO sequence (GenBank accession number U27233). This alteration produces an alanine instead of a threonine in the encoded amino acid sequence (compare SEQ ID NO: 11 with GenBank Accession No. Q12306). Although the following examples employ the altered version of SUMO, the instant inventive subject matter includes the utilization of the “wild-type” SUMO as provided in the above listed GenBank Accession Nos. For both hSUMO-1 and ySmt3, the PCR fragment was double-digested with NcoI and BamHI and then ligated into pET24d that had been similarly digested, resulting in 6xHis-hSUMO-1 and 6xHis-ySmt3 plasmids.
To make 6xHis-SUMO-1 and 6xHis-Smt3 fusions with eGFP, the eGFP sequence (46) was amplified with the primers 5′-GGTCTCAAGGTATGGTGAGCAAGGGCGAGGAGC-3′ (SEQ ID NO: 57) and 5′-AAGCTTATTACTTGTACAGCTCGTCCATGCC-3′ (SEQ ID NO: 58). The PCR product was purified and double-digested with BsaI and HindIII and then ligated into pET24d-6xHis-SUMO vectors that had been similarly digested.
For human SUMO-3, a PCR product was generated with the primers 5′-GATCGAACCTGCATATAGGTATGTCCGAGGAGAAGCCCAAG-3′ (SEQ ID NO: 59) and 5′-GATCGGATCCGGTCTCAACCTCCCGTCTGCTGCTGGAACACG-3′ (SEQ ID NO: 60) using SUMO-3-containing MGC cDNA clone #3505840 (Open Biosystems). For Arabidopsis thalania SUMO1, a PCR product was generated with the primers 5′-GATCGAACCTGCATATAGGTATGTCTGCAAACCAGGAGGA-3′ (SEQ ID NO: 61) and 5′-GATCGGATCCGGTCTCAACCTCCAGTCTGATGGAGCATCGC-3′ (SEQ ID NO: 62) using cDNA clone U17495 from the Arabidopsis Biological Resource Center. For Arabidopsis thalania SUMO2, a PCR product was generated with the primers 5′-GATCGACGTCTCAAGGTATGTCTGCTACTCCGGAAGAAG-3′ (SEQ ID NO: 63) and 5′-GATCGGATCCGGTCTCAACCTCCAGTCTGATGAAGCATTGCA-3′ (SEQ ID NO: 64) using cDNA clone U21609 from the Arabidopsis Biological Resource Center. For tomato T-SUMO, a PCR product was generated with the primers 5′-GATCGAACCTGCATATAGGTATGTCTGCTAGCGGCGGCACC-3′ (SEQ ID NO: 65) and 5′-GATCGGATCCGGTCTCAACCTCCAGTTTGATGTAGCATTG-3′ (SEQ ID NO: 66) a T-SUMO plasmid kindly provided by M. B. Mudgett.
The PCR fragments were double-digested with BamHI and BfuAI (hSUMO-3, AtSUMO1, and T-SUMO) or BsmBI (AtSUMO2), and then ligated into pET24d-6xHis that had been digested with BamHI and BsaI.
The eGFP sequence was amplified with the primers 5′-TTTTTTGGTCTCAAGGTATGGTGAGCAAGGGCGAGGGCGAGGAGC-3′ (SEQ ID NO: 67) and 5′-TTTTTTCTCGAGTTACTTGTACAGCTCGTCCATG-3′ (SEQ ID NO: 68). The PCR product was purified and double digested with BsaI and XhoI and then ligated into the pET24d-6xHis-SUMO vectors which had been similarly digested.
Plasmids were sequenced to confirm the presence of correct sequence in each.
Example 3
Expression and Purification of 6xHis-eGFP and 6xHis-SUMO-eGFP Proteins
To test and compare expression of eGFP and the SUMO-eGFP fusion proteins, a single colony of the E. coli Rosetta strain (Novagen) containing each of the SUMO-eGFP plasmids was inoculated into 5 ml of Luria-Bertani (LB) media containing 100 μg/ml Kanamycin and 30 μg/ml chloramphenicol. This strain is derived from the lambda DE3 lysogen strain and carries a chromosomal copy of the IPTG-inducible T7 RNA polymerase along with tRNAs on a pACYC-based plasmid. The cells were grown at 37° C. overnight with shaking at 250 rpm. The next morning the overnight culture was transferred into 100 ml fresh medium to permit exponential growth. When the OD600 value reached  ̃0.6-0.7, protein expression was induced by addition of 1 mm IPTG (isopropropyl-β-D-thiogalactopyranoside), followed by prolonged cultivation at either 37° C. for 3 hours or 20° C. overnight ( ̃15 hours).
After the E. coli cells were harvested from LB medium (100 ml) by centrifugation (8,000×g for 10 min at 4° C.), the cell pellets were suspended in 6 ml of lysis buffer (PBS containing 300 mM NaCl, 10 mM imidazole, 0.1% Triton X100 and 1 mM PMSF, pH 8.0). The cells were lysed by sonication (at 50% output for 5×30 second pulses). The sonication was conducted with the tube jacketed in wet ice and 1 min intervals between the pulse cycles to prevent heating. After the lysates were incubated with DNase and RNase (each at 40 μg/ml) for 15 min to digest nucleic acids, they were centrifuged at 20,000 g for 30 min at 4° C., and the supernatant (soluble protein fractions) was collected. The pellets was washed once with 6 ml of the lysis buffer to further extract the soluble fraction; the wash (6 ml) was combined with previous extract (6 ml) to make final volume of 12 ml for the soluble protein sample.
Insoluble protein samples were prepared from E. coli inclusion bodies. Briefly, after the extract containing soluble proteins were removed, the pellets containing inclusion bodies were suspended in the denaturing solubilization buffer (Novagen) that contained 50 mM CAPS (pH 11.0), 0.3% N-laurylsarcosine, and 1 mM DTT and incubated for 20 min at room temperature with shaking. The extract (insoluble protein fraction) was obtained by high-speed centrifugation (80,000×g for 20 min at 4° C.).
For detection of expressed proteins using SDS-PAGE, 5 μl of the samples prepared above were mixed with 3 μl of SDS-PAGE sample buffer containing SDS and β-mercaptoethanol and were heated at 95° C. for 5 min to facilitate denaturation and reduction of proteins. Proteins were visualized using 15% SDS-polyacrylamide gels with Tris-Glycine running buffer and Coomassie blue staining.
Example 4
Purification of 6xHis-SUMO-eGFP Proteins
All of the SUMO-eGFP fusions were expressed in E. coli as described above. Since all have 6xHis-tags, the soluble proteins extracted from E. coli cells were purified using Ni-NTA affinity chromatography under native conditions and a BioLogic Duo-Flow FPLC system (Bio-Rad) was used for high-throughput fractionations. Briefly, the cell lysate (typically, 20-40 ml containing 0.2-0.5 g proteins) was loaded onto a column containing  ̃10 ml Ni-NTA superflow resin (Qiagen, Valencia, Calif.) and the unbounded proteins (flow-through) were eluted out. The resin was extensively washed with  ̃50-100 ml of Wash Buffer (PBS containing 20 mM imidazole and 300 mM NaCl, pH 8.0) until UV280 reached or fell below the base line (UV value=0). Finally, the 6xHis tagged SUMO-fusion proteins were eluted by Elution Buffer (PBS containing 300 mM imidazole and 300 mM NaCl, pH 8.0). The purified SUMO-fused proteins eluted as a single isolated UV peak. The proteins with high UV280 values were collected in 4 ml fractions that were checked on SDS gels and pooled. The purified SUMO-eGFP fusion proteins were dialyzed with 3.5 kDa cutoff membranes against PBS pH  ̃7.5 overnight at 4° C. to remove high salt and imidazole.
Example 5
Cleavage of SUMO-eGFP Fusion Proteins by SUMO Proteases
To evaluate and compare effectiveness of different SUMO proteases on cleavage of different SUMO-GFP fusions, Applicants either used serially diluted amounts of enzymes to digest substrates (different ratios of enzyme to substrate) for a given time or used the same amount of enzymes to digest substrates at a fixed ratio for over a period of time (time-course assays). For all cleavage reactions, Applicants used PBS buffer (pH  ̃7.5) containing either β-mercaptoethanol or DTT and incubated at 30° C.
For FIG. 4A, serially diluted amounts of yULP1 or hSENP2, starting at 1 μg (0.5 μg for the ySmt3 experiment) and successively decreasing 2-fold by titration, were used to cleave 10 μg of SUMO-GFP in PBS (pH 7.5) containing 5 mM β-mercaptoethanol at 30° C. for 1 hour. M=Molecular weight markers; C−=Negative controls (SUMO-eGFP without enzyme). Arrows highlight expected/observed migration positions of respective expressed proteins.
For the time course in FIG. 4B, cleavage reactions of different SUMO-GFP fusions by yULP1 and hSENP1 were performed enzyme to substrates ratio of 1:1000 (10 μM enzyme to 10 mM SUMO-GFP) and incubated in PBS (pH 7.5) containing 5 mM β-mercaptoethanol at 30° C. over a period of time (15 min., 30 min., and 1, 2, 3, and 4 hrs.). M=Molecular weight markers; C−=Negative controls (SUMO-GFP without enzyme). Arrows highlight expected/observed migration positions of respective expressed proteins.
For cleavage of different SUMO-eGFP fusions by hSENP1, hSENP6, AtULP1c and GST-XopD in FIG. 5, 10 mM of each substrate was added to the enzymes at the amount indicated and incubated in PBS (pH 7.5) containing 5 mM β-Mercaptoethanol at 30° C. for 1 h. Arrows highlight expected/observed migration positions of respective proteins.
Example 6
Attachment of Various SUMO Proteins to the N-Terminus of eGFP Enhances the Expression of the Protein in E. coli 
The design and construction of all the pET vectors expressing eGFP and SUMO-eGFP fusions has been described above. The DNA sequences of the SUMO proteins, eGFP, the fusion constructs, and translation frames are shown in the Sequence Listing. FIG. 3 shows the expression pattern of 6xHis-eGFP, 6xHis-hSUMO-1-eGFP, 6xHis-hSUMO-3-eGFP, 6xHis-AtSUMO1-eGFP, 6xHis-AtSUMO2-eGFP, 6xHis-T-SUMO-eGFP, and 6xHis-ySmt3-eGFP. Induced E. coli cells grown at 37° C. or 20° C. were ruptured by sonication, and soluble fractions and insoluble inclusion bodies were prepared and analyzed on SDS-polyacrylamide gels. The stained gel (FIG. 3) shows that un-fused eGFP is generally poorly expressed in E. coli, while all of the SUMO tags from diverse species increase the overall expression of eGFP. Notably, certain SUMO tags increased expression and solubility more effectively than others. At 37° C., hSUMO-3 and AtSUMO1 produced the most overall eGFP protein and the largest amount of soluble eGFP, while yeast Smt3, AtSUMO2, and T-SUMO resulted in less eGFP expression and solubility. At 20° C., all SUMO fusions demonstrated improved expression and/or solubility. The most effective tags under these conditions were hSUMO-1 and hSUMO-3, which produced a large amount of overall expression and soluble eGFP, with a minor amount of insoluble eGFP. Yeast Smt3 and AtSUMO1 resulted in very low amounts of insoluble eGFP fusion, but their amounts of overall and soluble eGFP were considerably lower than those from the human SUMO fusion strains. Importantly, human SUMO-3 was one of the best expression tags under the two conditions used, while yeast Smt3 was one of the least effective under the same conditions, indicating that hSUMO-3 is a superior agent for expression enhancement in E. coli than is yeast Smt3, the SUMO protein employed in previously described expression systems. The superiority of hSUMO-3 over ySmt3 as an expression tag is surprising and could not be predicted from sequence analysis or other previously published data.
Example 7
Quantitation of Soluble eGFP and SUMO-eGFP by Fluorescence
Because properly folded, active eGFP has fluorescent properties, the expressed eGFP fusions in this study could be quantified by using fluorimetry. The quantitation was only performed on the soluble fractions, since the insoluble proteins from bacterial inclusion bodies are usually incorrectly folded. Specifically, the eGFP fluorescence in soluble fractions (approximately 100 μg of proteins in a final volume of 40 μl) was measured using a Fluoroskan Ascent FL fluorimeter (LabSystems, Helsinki, Finland) with an excitation 485 nm/emission 510 nm filter set with the exposure set to 0.4 s. The data are presented in arbitrary units. A graphic representation of the results of the fluorescence analyses was drawn using Microsoft Excel.
FIG. 3B shows comparisons of fluorescence from eGFP un-fused or fused with different SUMO tags. From either 37° C. or 20° C. culture, the levels of soluble hSUMO3-eGFP and AtSUMO1-eGFP were significantly higher than that of ySmt3-eGFP (p<0.01); especially, hSUMO3-eGFP was more than 50% higher compared with ySmt3-eGFP. The yield of soluble AtSUMO2-eGFP was higher than that of ySmt3 from 37° C. culture, but lower from 20° C. eGFP without fusion was poorly expressed, since its fluorescence was significantly lower than that of any SUMO-fused eGFP (p<0.01). The hSUMO1-eGFP level was very similar to that of ySmt3-eGFP (p>0.05%) and T-SUMO-eGFP was significantly lower than ySmt3-eGFP at 37° C. (p<0.05).
Quantitation of SUMO-eGFP by measurement of the eGFP fluorescence of the soluble fractions (FIG. 3B) confirm that, compared to ySmt3, hSUMO-3 yields significantly higher levels of soluble protein and overall expression (soluble+insoluble) at both 37° C. and 20° C. The soluble fractions demonstrated a 1.6-fold advantage of hSUMO-3 over ySmt3. The generation of larger quantities of soluble protein of interest is particularly important, because it has the potential to impact both the quality of the protein and its ease of purification. A soluble protein is more likely to be properly folded and will typically not require further treatment before purification, while insoluble protein must be rendered soluble by certain methods (such as detergents or chaotropic agents) and often subjected to lengthy refolding/dialysis, adding time and effort to the purification, as well as increasing the chances that a protein will be or become damaged and/or inactive. Obtaining a larger yield of soluble protein by way of hSUMO-3 is expected to obviate the need to work with insoluble protein; although an insoluble fraction of hSUMO-3 fusion is generated, it can be discarded, and yield will still be better than that of ySmt3 fusion.
The higher yield of overall and soluble expressed protein of interest from hSUMO-3 fusion as compared to ySmt3 or most other fusions tested is an unexpected and surprising finding that could not be predicted based on their amino acid sequences or general classification as SUMO proteins.
Example 8
Direct Comparison of Human SENP2 and Yeast Ulp1 Proteases Demonstrates Different Levels of Activity and Preferences for Certain SUMO Proteins In Vitro
The design and construction of the pET vectors expressing SUMO protease catalytic domains of human SENP2 and yeast Ulp1 has been described above. The DNA sequences and translation frames of the proteases are shown in the Sequence Listing. These proteases and each of the six SUMO-eGFP fusions described above were expressed in E. coli and purified on nickel columns by way of the 6xHis tags on the C-termini (proteases) or N-termini (SUMO-eGFP's) of the proteins. In vitro reactions were performed in which each purified protease was used in reactions with each of the purified SUMO-eGFP proteins, and the cleavage results were analyzed by Coomassie-stained SDS-PAGE gels (FIG. 4).
For FIG. 4A, dilution series of the proteases were performed, where a fixed amount of each SUMO-eGFP was incubated for 1 hour at 30 C with sets of 2-fold dilutions of the enzymes. As shown in FIG. 4A, there were significant differences in the abilities of hSENP2 and yUlp1 to cleave fusions with different SUMO proteins. Specifically, hSENP2 was able to cleave hSUMO-3-eGFP at least 4-fold better than an identical amount of yUlp1. In addition, hSENP2 was able to cleave hSUMO-1 and ySmt3 fusions somewhat more effectively (about 2-fold) than yUlp1. On the other hand, yUlp1 had a distinct advantage in the cleavage of T-SUMO-eGFP and a slight advantage in the cleavage of AtSUMO2-eGFP. Both hSENP2 and yUlp1 cleaved the AtSUMO1 fusion similarly.
In addition to dilution series, a time-course experiments were performed with hSENP2, yUlp1, and the SUMO fusions in order to assess the relative activities of fixed amounts of the proteases against the different SUMO tags. In these experiments, fixed amounts of the proteases were incubated for varying amounts of time (15 min. to 4 hrs.) with the six SUMO-eGFP fusions tested. As shown in FIG. 4B, hSENP2 was again a considerably more active protease than yUlp1 against the hSUMO-3-eGFP protein, providing nearly complete cleavage within 2 hrs., while yUlp1 was able to cleave apparently less than half of the substrate in 4 hrs., consistent with the approximate 4-fold advantage of hSENP2 in the dilution series in FIG. 4A. With the other SUMO fusions, in general yUlp1 performed somewhat better in the time course experiments than in the dilution series, cleaving not only T-SUMO but also AtSUMO1 and AtSUMO2 significantly better than did hSENP2. In addition, the proteases has similar activity to each other against hSUMO-1 and ySmt3, with both enzymes cleaving hSUMO-1-eGFP poorly and ySmt3-eGFP well.
Overall, the experiments in FIG. 4 demonstrate that human SENP2 and yeast Ulp1 have different levels of activity and robustness on SUMO tags from a variety of species, and these non-obvious specificities have been revealed in these studies. A major finding of these two sets of experiments is that hSENP2 is a much stronger protease than yUlp1 against a human SUMO-3 fusion, while yUlp1 had some advantages in the cleavage of certain plant SUMO's. The fact that hSENP2 is able to effectively cleave hSUMO-3, which was shown to be a superior expression tag (FIG. 3), gives rise to the inventive subject matter of using hSUMO-3 and hSENP2 as an effective SUMO/protease pair for an expression system offering improvement over previously described ySmt3/yUlp1 systems. In addition, hSENP2 also cleaved ySmt3 fusion as well as (FIG. 4B) or better than (FIG. 4A) did yUlp1 itself, leading to the concept that hSENP2 could alternatively be employed in an expression system using yeast Smt3—i.e. a system featuring the pairing of tag and protease from different species.
Example 9
Additional Diverse SUMO Proteases have Different Levels of Activity and Preferences for Certain SUMO Proteins In Vitro
The design and construction of all the pET vectors expressing four additional SUMO protease catalytic domains has been described above. The DNA sequences and translation frames of hSENP1, hSENP6, AtULP1c, and GST-XopD are shown in FIGS. 12-13. These proteases and each of the six SUMO-eGFP fusions described above were expressed in E. coli and purified on nickel columns by way of the 6xHis tags on the C-termini (proteases) or N-termini (SUMO-eGFP's) of the proteins. In vitro reactions were performed in which each purified protease was used in reactions with each of the purified SUMO-eGFP proteins, and the cleavage results were analyzed by Coomassie-stained SDS-PAGE gels (FIG. 5). Since the hSENP1, hSENP6, AtULP1c, and XopD protease catalytic domains displayed considerably less overall activity in vitro than those of hSENP2 and yUlp1, these 4 enzymes were tested at single concentrations with fixed incubation times, and were therefore not analyzed in as great detail as the proteases in FIG. 4, where enzyme amounts and incubation times were varied.
As shown in FIG. 5, different enzymes had different specificities against the various SUMO-eGFP fusion proteins. The most active of the proteases in FIG. 5 was hSENP1, which cleaved ySmt3 the best, followed by hSUMO-3. It also cleaved the other 4 fusions, but to a much lesser extent. Less active but seemingly more specific was hSENP6, which demonstrated partial cleavage of hSUMO-3-eGFP but did not digest the others at the incubation time and enzyme concentration used; interestingly, this protease had previously been claimed as an hSUMO-1-specific protease (Kim, K. I., S. H. Baek, Y. J. Jeon, S. Nishimori, T. Suzuki, S. Uchida, N. Shimbara, H. Saitoh, K. Tanaka, and C. H. Chung, 2000, A new SUMO-1-specific protease, SUSP1, that is highly expressed in reproductive organs, J Biol Chem 275:14102-6) based on its ability to cleave β-galactosidase fusions of hSUMO-1 but not ySmt3, but those experiments did not test hSUMO-3. Another enzyme with particular SUMO specificities is XopD, derived from a prokaryotic plant pathogen, Xanthomonas. It carried out partial cleavage of the three plant SUMO fusions (AtSUMO1, AtSUMO2, and T-SUMO), but did not cleave the fusions with yeast or human SUMO's. This is possibly consistent with the role of the pathogen in plant systems and demonstrates that despite the high level of sequence conservation among SUMO proteins and among SUMO proteases, there is evidence for certain SUMO tag/protease pairs having specificity for each other within and between species. Finally, AtULP1c did not show activity against any of the SUMO fusions. Although one possibility is that inactive enzyme was produced, it should be noted that at least 13 putative SUMO proteases and at least 8 putative SUMO proteins have been identified in Arabidopsis, so another possibility is that AtSUMO1 and AtSUMO2 are the natural substrates for a different AtULP, and perhaps AtULP1c might be active against one of the other AtSUMO proteins, which have not yet been cloned and expressed.
The fact that different SUMO proteases from diverse species have varying levels of robustness and different and sometimes unexpected specificities for particular SUMO proteins points to the idea that multiple SUMO proteins and proteases must be investigated from a variety of species in order to maximize the effectiveness of overexpression proteins of interest and efficient generation of desired N-termini for them. Although in this inventive subject matter Applicants have already identified in hSUMO-3 and hSENP2 a SUMO tag/protease pair that gives substantial expression-system improvement over the previous state-of-the-art system, ySmt3/yUlp1, Applicants are also exploring the SUMO tags and proteases of multiple species by cloning and expressing them and testing them against one another. Examples of SUMO proteins and proteases Applicants are pursuing are provided in FIG. 1 and Table 3.
Example 10
Examples of Various Classes of Proteins Whose Fusion with SUMO Proteins Will Improve Quantity and Quality of Proteins
The design and construction of all the pET vectors expressing eGFP has been described above. As with eGFP, any DNA sequence can be cloned as a fusion with 6xHis-SUMO, such as 6xHis-hSUMO-3. Furthermore, the 6xHis-SUMO-protein of interest fusion protein can be recloned into any yeast, such as Yep, baculoviral, such as pFastBac, mammalian, such as pcDNA3, or other host vectors by, for example, a simple cut-and-paste procedure identical or similar to the one described above. Cell propagation, protein expression, harvesting, lysis, and purification of fusion are performed described above or according to one of the established procedures described elsewhere. Cleavage of fusions with an appropriate SUMO protease (such as hSENP2) can be performed as described above. The system described in this application has significant advantages as compared to other systems currently in use. One or more of these advantages may create a precedent for preferred use of the system described here over other systems. Industrial needs for protein manufacturing may suggest that the following classes or groups of proteins may be exemplified as the ones most likely to benefit from use of the current inventive subject matter. Two of the factors that may be considered when an expression system is selected are 1) the success of expression of the protein in various systems and 2) requirement for specific N-terminus. Each of these factors can be successfully addressed by the system described herein: 1) SUMO proteins such as hSUMO-3 enhance the production of proteins, and 2) robust cleavage by SUMO proteases such as hSENP2 allows generation of almost any amino-terminus. Table 3 provides representative molecules and GenBank Accession numbers for the expression of the proteins produced using the methods of the inventive subject matter.
Example 11
Comparison of Enzyme Kinetics of hSENP2 and yUlp1
To further compare the catalytic activities of hSENP2 and yUlp1, Applicants analyzed their kinetics. The kinetic parameters (Km and Kcat) for both SUMO proteases were determined using yeast and human SUMO-GFP fusions. Briefly, the SUMO proteases were assayed by mixing 6 μM enzyme with different amounts of the substrate (either the ySmt3-eGFP or the hSUMO-3-eGFP, at 12, 6, 3, 1.5, 0.75, and 0.375 mM) in PBS (pH 7.5) containing 1.0 mM DTT. Our initial experiments for time course concluded that 1 hour was well within the initial velocity rates for both SUMO proteases, in that the rates of the reactions were within in the linear region. The reactions were incubated at 30 C for 1 hour and stopped by addition of SDS-PAGE loading buffer. Samples were then heated at 95 C for 3 min and loaded onto a 15% SDS-polyacrylamide gel for analysis. The SDS-polyacrylamide gels were stained with Coomassie Blue and scanned such that each band could be quantified using the software, Scion Image version Beta 4.0.2 (Scion Corporation). Densitometry analyses were performed to determine the amount of product generated per unit time, as compared with a loaded albumin (standard as known amount). The initial velocity values used for curve fitting were the mean of three independent experiments. These initial velocity measurements were plotted against the substrate concentrations and fit to the Michaelis-Menten equation using KaleidaGraph 3.5 (Synergy Corporation). The Kcat values were calculated by assuming 100% activity for the enzyme.
Representative kinetic parameters for hSENP2 and yUlp1 are presented in Table 1, utilizing methionine at the interface of the fusion protein. Interestingly, although ySmt3 is the natural substrate for yUlp1, hSENP2 has a somewhat higher affinity and faster catalytic rate (about 2-fold difference in Kcat) for cleavage of ySmt3-eGFP than does yUlp1. But strikingly, hSENP2 has a much higher affinity and faster catalytic rate for hSUMO-3-eGFP than does yUlp1 (about 12-fold difference in Kcat). These data illustrate in a quantitative manner that hSENP2 is a much more active SUMO protease enzyme than yUlp1 on the effective expression tag hSUMO-3.







TABLE 1







Kinetic parameters (Km and Kcat) for hSENP2 and yUlp1










hSENP2
yUlp1





hSUMO-3-eGFP
Km = 2.41 mM
Km = 15.8 mM



Kcat = 0.33 mM−1S−1
Kcat = 0.027 mM−1S−1


ySmt3-eGFP
Km = 2.72 mM
Km = 4.5 mM



Kcat = 0.75 mM−1S−1
Kcat = 0.40 mM−1S−1









Example 12
Comparison of hSENP2 and yUlp1 on Cleavage of hSUMO-3-eGFP Fusions with Different Amino Acid Termini
To determine if hSENP2 can cleave hSUMO-3-eGFP fusions with each of the 20 amino acids at the N-terminus of eGFP and to determine if it is more powerful than yUlp1, we have designed and made a set of hSUMO-3-X-eGFP fusions in which the methionine (M) that follows the C-terminal Gly-Gly motif of the hSUMO-3 has been replaced with one of the other 19 amino acids (X).
The purified hSUMO-3-X-eGFP fusions were incubated with either hSENP2 or yUlp1 in PBS (pH 7.4) containing 2 mM DTT at 30 C for 1 hour. Cleavage of the fusions was determined by quantitative comparison of the released proteins (eGFP and hSUMO-3) with the fusions observed on 15% SDS-polyacrylamide gels. Briefly, after the proteins were separated on the gels and stained with Coomassie Blue, the gels were scanned and saved as TIFF images. The protein bands were quantitated using the Scion Image (version Beta 4.0.2) (Scion Corporation, Bethesda, Md.) by densitometric analyses. The values of pixels (in arbitrary units) for each protein band (hSUMO-3-eGFP fusion, released eGFP and hSUMO-3) were recorded. Percentages of cleavage were determined by the ratio of the densities of the released eGFP and hSUMO-3 divided by the total densities (hSUMO-3-eGFP plus released proteins).
The results for cleavage of all versions of hSUMO-3-X-eGFP (with different amino acids at the eGFP N-terminus) by hSENP2 and yUlp1 are shown in Table 2 and FIG. 4C. Both hSENP2 and yUlp1 can partially or totally cleave hSUMO-3-X-eGFP for 19 of the amino acids, but the enzymes cannot effectively cleave the fusion where proline is at the N-terminus of eGFP. Compared to yUlp1, hSENP2 has much higher efficiency of cleavage (at least 5-10-fold higher in most cases) for all the 19 amino acid junctions. When a low amount of SENP2 was used (1:1000 ratio of enzyme to substrate), 14 amino acid junctions were >90% cleaved while five others (D, I, L, Q, and T) were at least >50% cleaved. In contrast, with the same amount of yUlp1 used, the enzyme gave only 10-27% cleavage for most of the amino acid junctions, and for the six other fusions (D, I, K, L, Q and V) only 0-4% cleavage was achieved. At a 1:100 ratio of enzyme to substrate SENP2 gave >95% cleavage for all the amino acid junctions except the proline fusion, but yUlp1 still poorly cleaves the I, K, L, and V fusions in that only  ̃2-22% cleavage was observed. Overall, the data demonstrate that hSENP2, unlike Ulp1, is an effective enzyme for cleavage of any amino acid (except proline) fused to hSUMO-3.







TABLE 2





Percent cleavage of hSUMO-3-eGFP fusions by hSENP2 and yUlp1



























Control
A1
C
D
E
F
G
H
I
K
L





hSENP22
0
92.5
98.5
69.8
92.2
98.9
98.9
98.4
56.2
92.8
75.2


(1:1000)


hSENP2
0
99.6
100
99.5
100
100
100
100
98.4
100
98.6


(1:100)


yUlp1
0
27.1
25.3
3.3
11.3
27.0
27.0
25.5
0
0
0


(1:1000)


yUlp1
0
96.3
98.4
92.3
80.1
95.4
96.5
90.8
1.9
22.4
4.4


(1:100)























M
N
P
Q
R
S
T
V
W
Y







hSENP22
95.4
98.3
0
66.7
98.7
99.1
82.0
99.8
99.4
98.7



(1:1000)



hSENP2
99.4
96.2
0
95.9
99.7
99.6
99.0
100
100
100



(1:100)



yUlp1
18.4
16.9
0
3.7
17.2
16.7
14.3
4.0
16.8
16.9



(1:1000)



yUlp1
98.3
97.9
0
65.5
96.6
98.2
95.8
20.2
89.7
96.9



(1:100)







Control is hSUMO-3-M-eGFP treated with the same conditions, but with no enzyme added.



1The letters are the one-letter code indicating the amino acid that is present at the N-terminus of eGFP, fused with hSUMO-3.



2Ratio of enzyme to substrate is indicated in parentheses













TABLE 3







Summary of representative SUMO proteases











Reference or accession


SUMO protease
Species
number





yUlp1
Saccharomyces cerevisiae
Li 1999, Malakhov


yUlp2
Saccharomyces cerevisiae
Li 2000


hSENP1
Homo sapiens
Gong, Xu


hSENP2
Homo sapiens
Nishida 2001, Zhang,




Reverter


hSENP3
Homo sapiens
Nishida 2000


hSENP5
Homo sapiens
Q96HI0


hSENP6
Homo sapiens
Kim


hSENP7
Homo sapiens
Q9BQF6


AtULP1a, b, c, d
Arabidopsis thalania
Kurepa


AtULP2a, b, c, d,
Arabidopsis thalania
Kurepa


e, f, g, h




AtESD4
Arabidopsis thalania
Murtas


XopD
Xanthomonas campestris
Hotson


XSENP1a, b
Xenopus laevis
Yukita


DmUlp1
Drosophila melanogaster
Smith


CeULP-1
Caenorhabditis elegans
AAK21468


CeULP-2
Caenorhabditis elegans
AAA98019


SpUlp1
Schizosaccharomyces pombe
Taylor


SpUlp2
Schizosaccharomyces pombe
O13769


KlUlp1
Kluyveromyces lactis
CAG98289


PfUlp
Plasmodium falciparum
AAN36413


AnUlp
Aspergillus nidulans
EAA63091





Indicated are references for studies in which a given SUMO protease was identified and/or characterized; protein database accession numbers are given for putative SUMO proteases lacking published data.













TABLE 4







Summary of activity levels of SUMO protease


catalytic domains on various SUMO fusions














hSUMO1-
hSUMO3-
AtSUMO1-
AtSUMO2-
T-SUMO-
ySmt3-



eGFP
eGFP
eGFP
eGFP
eGFP
eGFP

















hSENP2
++
+++
+
+
+
+++++


yULP1
+
+
++
++
++
++++


hSENP1
+/−
+
+/−
+/−
+/−
++


hSENP6
−
+/−
−
−
−
−


AtULP1c
−
−
−
−
−
−


XopD
−
−
−
+/−
+/−
+/−
















TABLE 5







Examples of Proteins and Protein Classes for use with the Current Inventive Subject Matter












Success rate with

Requirement for



Protein class
conventional systems
Protein
specific N-termini
ACC #





Cytokine
High/inclusion
IL-15
Yes
P40933



bodies
IFN□

P01579






P01375


Chemokine
High/inclusion
Myb-1□
Yes
P13236



bodies





Growth factors
low
TGF□
Yes
P01135


Enzymes
Low
DNA
Yes
1RDR




polymerase





Very low
DNase II alpha
Yes
AAC77366



Very low
Cathepsin
Unknown
P07858




(peptidase)





Very low
BMP-1
Unknown
NP_001190




protease




Peptides including
Low/proteolysis
Calcitonin
Yes
CAA26189


therapeutic






Nuclear receptors
high
LXR
No
Q13133


Cytokine/chemokine/
Low/inclusion
IFNAR,
No
P15260


growth factor receptors
bodies
VEGFR,

P35968




CCR9

NP_034043






P00533


Ser-Thr kinases
Low
MAPK
No
P49137


Tyr kinases
Extremely low/
Zap 70
No
P43403



inactive





Transcription factors
low
CREB
Unknown
NP_005185


Initiation factors
low
eIF2
Unknown
NP_004085


Viral/parasite/bacterial
Low
Spike protein,
Varies
AAP33697


proteins (vaccines)

Apa (Rv1860),

Q50906




RAP-1

AAF15365









REFERENCE LIST
The following references are cited to provide the reader with background information relating to the inventive subject matter and the general state of the art. The fact that a document is cited herein is not an indication that Applicants believe that any document is prior art or is material to patentability. Each of the following documents is incorporated by reference in the Specification of this application as though set forth in full herein.
1. Bayer, P., A. Arndt, S. Metzger, R. Mahajan, F. Melchior, R. Jaenicke, and J. Becker, 1998, Structure determination of the small ubiquitin-related modifier SUMO-1, J Mol Biol 280:275-86.
2. Ben-Bassat, A, 1991, Methods for removing N-terminal methionine from recombinant proteins, Bioprocess Technol 12:147-59.
3. Buchberger, A., M. J. Howard, M. Proctor, and M. Bycroft, 2001, The UBX domain: a widespread ubiquitin-like module, J Mol Biol 307:17-24.
4. Butt, T. R., S. Jonnalagadda, B. P. Monia, E. J. Sternberg, J. A. Marsh, J. M. Stadel, D. J. Ecker, and S. T. Crooke, 1989, Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli, Proc Natl Acad Sci USA 86:2540-4.
5. Davis, G. D., C. Elisee, D. M. Newham, and R. G. Harrison, 1999, New fusion protein systems designed to give soluble expression in Escherichia coli, Biotechnol Bioeng 65:382-8.
6. di Guan, C., P. Li, P. D. Riggs, and H. Inouye, 1988, Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein, Gene 67:21-30.
7. Ecker, D. J., J. M. Stadel, T. R. Butt, J. A. Marsh, B. P. Monia, D. A. Powers, J. A. Gorman, P. E. Clark, F. Warren, A. Shatzman, and et al., 1989, Increasing gene expression in yeast by fusion to ubiquitin, J Biol Chem 264:7715-9.
8. Georgiou, G. and P. Valax, Isolating inclusion bodies from bacteria, 1999, Methods Enzymol 309:48-58.
9. Goettsch, S., and P. Bayer, 2002, Structural attributes in the conjugation of ubiquitin, SUMO and RUB to protein substrates, Front Biosci 7:a148-62.
10. Hanania, U., N. Furman-Matarasso, M. Ron, and A. Avni, 1999, Isolation of a novel SUMO protein from tomato that suppresses EIX-induced cell death, Plant J 19:533-41.
11. Hotson, A., R. Chosed, H. Shu, K. Orth, and M. B. Mudgett, 2003, Xanthomonas type III effector XopD targets SUMO-conjugated proteins in plants, Mol Microbiol 50:377-89.
12. Hu, M., P. Li, M. Li, W. Li, T. Yao, J. W. Wu, W. Gu, R. E. Cohen, and Y. Shi, 2002, Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde, Cell 111:1041-54.
13. Ikonomou, L., Y. J. Schneider, and S. N. Agathos, 2003, Insect cell culture for industrial production of recombinant proteins, Appl Microbiol Biotechnol 62:1-20.
14. Jentsch, S., and G. Pyrowolakis, 2000, Ubiquitin and its kin: how close are the family ties? Trends Cell Biol 10:335-42.
15. Johnson, E. S., 2004, Protein modification by SUMO, Annu Rev Biochem 73:355-82.
16. Johnson, E. S., I. Schwienhorst, R. J. Dohmen, and G. Blobel, 1997, The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer, Embo J 16:5509-19.
17. Jonasson, P., S. Liljeqvist, P. A. Nygren, and S. Stahl, 2002, Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli, Biotechnol Appl Biochem 35:91-105.
18. Kapust, R. B., and D. S. Waugh, 1999, Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused, Protein Sci 8:1668-74.
19. Kim, K. I., S. H. Baek, and C. H. Chung, 2002, Versatile protein tag, SUMO: its enzymology and biological function, J Cell Physiol 191:257-68.
20. Kim, K. I., S. H. Baek, Y. J. Jeon, S. Nishimori, T. Suzuki, S. Uchida, N. Shimbara, H. Saitoh, K. Tanaka, and C. H. Chung, 2000, A new SUMO-1-specific protease, SUSP1, that is highly expressed in reproductive organs, J Biol Chem 275:14102-6.
21. Kretz-Remy, C., and R. M. Tanguay, 1999, SUMO/sentrin: protein modifiers regulating important cellular functions, Biochem Cell Biol 77:299-309.
22. Kurepa, J., J. M. Walker, J. Smalle, M. M. Gosink, S. J. Davis, T. L. Durham, D. Y. Sung, and R. D. Vierstra, 2003, The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis, Accumulation of SUMO1 and -2 conjugates is increased by stress. J Biol Chem 278:6862-72.
23. Larsen, C. N., and H. Wang, 2002, The ubiquitin superfamily: members, features, and phylogenies, J Proteome Res 1:411-9.
24. Layfield, R., K. Franklin, M. Landon, G. Walker, P. Wang, R. Ramage, A. Brown, S. Love, K. Urquhart, T. Muir, R. Baker, and R. J. Mayer, 1999, Chemically synthesized ubiquitin extension proteins detect distinct catalytic capacities of deubiquitinating enzymes, Anal Biochem 274:40-9.
25. Li, S. J., and M. Hochstrasser, 1999, A new protease required for cell-cycle progression in yeast, Nature 398:246-51.
26. Li, S. and M. Hochstrasser, 2000, The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein, Mol Cell Biol 20:2367-77.
27. Link, A. J., K. Robison, and G. M. Church, 1997, Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12, Electrophoresis 18:1259-313.
28. Lo Conte, L., B. Ailey, T. J. Hubbard, S. E. Brenner, A. G. Murzin, and C. Chothia, 2000, SCOP: a structural classification of proteins database, Nucleic Acids Res 28:257-9.
29. Lois, L. M., C. D. Lima, and N. H. Chua, 2003, Small ubiquitin-like modifier modulates abscisic acid signaling in Arabidopsis, Plant Cell 15:1347-59.
30. Malakhov, M. P., O. A. Malakhova, K. I. Kim, K. J. Ritchie, and D. E. Zhang, 2002, UBP43 (USP18) Specifically Removes ISG15 from Conjugated Proteins, J Biol Chem 277:9976-81.
31. Malakhov, M. P., Mattern, M. R., Malakhova, O. A., Drinker, M., Weeks, S. D., and Butt, T. R., 2004, SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins, J Structural and Functional Genomics 5:75-86.
32. Melchior, F., M. Schergaut, and A. Pichler, 2003, SUMO: ligases, isopeptidases and nuclear pores, Trends Biochem Sci 28:612-8.
33. Mossessova, E., and C. D. Lima, 2000, Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast, Mol Cell 5:865-76.
34. Muller, S., C. Hoege, G. Pyrowolakis, and S. Jentsch, 2001, SUMO, ubiquitin's mysterious cousin, Nat Rev Mol Cell Biol 2:202-10.
35. Murtas, G., P. H. Reeves, Y. F. Fu, I. Bancroft, C. Dean, and G. Coupland, 2003, A nuclear protease required for flowering-time regulation in Arabidopsis reduces the abundance of SMALL UBIQUITIN-RELATED MODIFIER conjugates, Plant Cell 15:2308-19.
36. Ryan, T. E., and S. D. Patterson, 2002, Proteomics: drug target discovery on an industrial scale, Trends Biotechnol 20:S45-51.
37. Saitoh, H., and J. Hinchey, 2000, Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3, J Biol Chem 275:6252-8.
38. Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
39. Smith, D. B., and K. S. Johnson, 1988, Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase, Gene 67:31-40.
40. Terpe, K., 2003, Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems, Appl Microbiol Biotechnol 60:523-33.
41. Varshavsky, A., 2003, The N-end rule and regulation of apoptosis, Nat Cell Biol 5:373-6.
42. Varshavsky, A., 1996, The N-end rule: functions, mysteries, uses, Proc Natl Acad Sci USA 93:12142-9.
43. Waldo, G. S., B. M. Standish, J. Berendzen, and T. C. Terwilliger, 1999, Rapid protein-folding assay using green fluorescent protein, Nat Biotechnol 17:691-5.
44. Weickert, M. J., D. H. Doherty, E. A. Best, and P. O. Olins, 1996, Optimization of heterologous protein production in Escherichia coli, Curr Opin Biotechnol 7:494-9.
45. Yeh, E. T., L. Gong, and T. Kamitani, 2000, Ubiquitin-like proteins: new wines in new bottles, Gene 248:1-14.
46. Zhang, G., V. Gurtu, and S. R. Kain, 1996, An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells, Biochem Biophys Res Commun 227:707-11.
47. Gong, L., Millas, S., Maul, G. G., and E. T. H. Yeh, 2000, Differential regulation of sentrinized proteins by a novel sentrin-specific protease, J. Biol. Chem. 275:3355-3359.
48. Xu, Z., and S. W. Au, 2005, Mapping residues of SUMO precursors essential in differential maturation by SUMO-specific protease, SENP1, Biochem. J., in press.
49. Nishida, T., Kaneko, F., Kitagawa, M., and H. Yasuda H, 2001, Characterization of a novel mammalian SUMO-1/Smt3-specific isopeptidase, a homologue of rat axam, which is an axin-binding protein promoting beta-catenin degradation, J. Biol. Chem. 276:39060-39066.
50. Reverter, D., and C. D. Lima CD, 2004, A basis for SUMO protease specificity provided by analysis of human Senp2 and a Senp2-SUMO complex, Structure (Camb.) 12:1519-1531.
51. Nishida, T., Tanaka, H., and H. Yasuda, 2000, A novel mammalian Smt3-specific isopeptidase 1 (SMT3IP1) localized in the nucleolus at interphase, Eur. J. Biochem. 267:6423-6427.
52. Yukita, A., Michiue, T., Fukui, A., Sakurai, K., Yamamoto, H., Ihara, M., Kikuchi, A., and M. Asashima, 2004, XSENP1, a novel sumo-specific protease in Xenopus, inhibits normal head formation by down-regulation of Wnt/beta-catenin signalling, Genes Cells 9:723-736.
53. Smith, M., Bhaskar, V., Fernandez, J., and A. J. Courey, 2004, Drosophila Ulp1, a nuclear pore-associated SUMO protease, prevents accumulation of cytoplasmic SUMO conjugates, J. Biol. Chem. 279:43805-43814.
54. Taylor, D. L., Ho, J. C., Oliver, A., and F. Z. Watts, 2002, Cell-cycle-dependent localisation of Ulp1, a Schizosaccharomyces pombe Pmt3 (SUMO)-specific protease, J. Cell Sci. 115:1113-1122.
55. Marblestone, J. Edavettal, S., Lim, Y., Lim, P., Zuo, X., Butt, T. R. (2006) Comparison of SUMO Fusion Technology with Traditional Gene Fusion System: Effects of Recombinant Protein Expression and Purification. Protein Science 15(1):182-9.
56. Butt, T. R., Edavettal S., Hall, J. and Mattern M. R (2005) SUMO Fusion Technology for Difficult to Express Proteins. Protein Expr. Purif. 43:1-9.
57. Zuo, Xun., Mattern, M. R., Tan, R., Li, S., Hall, H., Sterner, D. E., Shoo, J., Lim, P., Sarafianos, S. G., Kazi, L., Navas-Martin., Weiss, S. R. and Butt, T. R. (2005) Expression and Purification of SARS Coronavirus Proteins Using SUMO-Fusions. Protein Expr. Purif. 42:100-110.
58. Zuo, Xun, Li, Shuisen, Hall John, Mattern, Michael R., Tran, Tran, Shoo, Joshua, Tan, Robin, Weiss, Susan and Butt, T. R. (2005) Enhanced expression and purification of membrane proteins by SUMO fusion in Echerichia coli. J. Struct. Funct. Genomics 6(2-3):103-11.
The inventive subject matter being thus described, it will be obvious that the same may be modified or varied in many ways. Such modifications and variations are not to be regarded as a departure from the spirit and scope of the invention and all such modifications and variations are intended to be included within the scope of the following claims.

## Claims

1. A nucleic acid composition for enhancing expression levels of a protein of interest, comprising, in-frame:
(a) an isolated nucleic acid sequence which encodes a human SUMO-3 protein, wherein said human SUMO-3 protein is SEQ ID NO: 3;
(b) an isolated nucleic acid sequence encoding said protein of interest; and
(c) an isolated nucleic acid sequence encoding at least one purification tag,
wherein, upon expression in a suitable host cell, said nucleic acid composition produces a fusion protein in which said human SUMO-3 protein is attached to the amino terminal of said protein of interest and said purification tag is N-terminal to said human SUMO-3 protein.

2. The composition of claim 1, wherein said purification tag is selected from the group consisting of polyhistidine tags, polyarginine tags, glutathione-S-transferase, maltose binding protein, S-tag, influenza virus HA tag, thioredoxin, staphylococcal protein A tag, the FLAGTM epitope, AviTag epitope, and the c-myc epitope.

3. The composition of claim 1, wherein said host cell is selected from the group consisting of bacteria, yeast, mammalian cells, and insect cells.

4. An expression vector comprising, in-frame:
(a) an isolated nucleic acid sequence which encodes a human SUMO-3 protein, wherein said human SUMO-3 protein is SEQ ID NO: 3;
(b) an isolated nucleic acid sequence encoding a protein of interest; and
(c) an isolated nucleic acid sequence encoding at least one purification tag,
wherein, upon expression in a suitable host cell, said expression vector produces a fusion protein in which said human SUMO-3 protein is attached to the amino terminal of said protein of interest and said purification tag is N-terminal to said human SUMO-3 protein.

5. The expression vector of claim 4, wherein said purification tag is selected from the group consisting of polyhistidine tags, polyarginine tags, glutathione-S-transferase, maltose binding protein, S-tag, influenza virus HA tag, thioredoxin, staphylococcal protein A tag, the FLAGTM epitope, AviTag epitope, and the c-myc epitope.

6. The expression vector of claim 4, wherein said host cell is selected from the group consisting of bacteria, yeast, mammalian cells, and insect cells.

7. A method for enhancing expression of a protein of interest, comprising the steps of:
(a) transforming a host cell with an expression vector comprising:
(1) a vector which is suitable for transforming a host cell, and
(2) a nucleic acid insert comprising, in-frame (i) an isolated nucleic acid sequence encoding a human SUMO-3 protein, wherein said human SUMO-3 protein is SEQ ID NO: 3, (ii) an isolated nucleic acid sequence encoding said protein of interest, and (iii) an isolated nucleic acid encoding at least one purification tag, operably linked;
(b) expressing said expression vector in a suitable host cell; and
(c) isolating the protein produced by expression of said nucleic acid insert,
wherein upon expression in said host cell, said expression vector encodes a fusion protein in which said purification tag is N-terminal to said human SUMO-3 protein, and said human SUMO-3 protein is attached to the amino terminal of the protein of interest.

8. The method of claim 7, wherein said host cell is selected from the group consisting of bacteria, yeast, mammalian cells, and insect cells.

9. The method of claim 7, optionally comprising one or more of the following additional step(s):
(d) lysing the host cell expressing the fusion protein;
(e) purifying the fusion protein by way of said at least one purification tag; and
(f) cleaving the SUMO moiety and purification tag(s) from the protein of interest with human SUMO protease SENP2 encoded by SEQ ID NO: 43, an equivalent thereof, a homologue thereof, a catalytic domain thereof, or a combination thereof.

10. The method of claim 7, wherein said purification tag is selected from the group consisting of polyhistidine tags, polyarginine tags, glutathione-S-transferase, maltose binding protein, S-tag, influenza virus HA tag, thioredoxin, staphylococcal protein A tag, the FLAGTM epitope, AviTag epitope, and the c-myc epitope.

11. The method of claim 7, wherein said protein of interest has a desired native N-terminal amino acid following cleavage with SENP2 protease, and provided that said N-terminal amino acid is not proline.

12. A kit for enhancing expression levels of a protein of interest, comprising:
(a) a vector which is suitable for transforming a host cell and which has at least one cloning site for cloning a nucleic acid encoding a protein of interest, operably linked to an nucleic acid insert comprising, in-frame, (i) an isolated nucleic acid sequence encoding a human SUMO-3 protein, wherein said human SUMO-3 protein is SEQ ID NO: 3, and (ii) an isolated nucleic acid encoding at least one purification tag,
wherein said at least one cloning site is oriented in said vector so that upon cloning in a nucleic acid sequence encoding said protein of interest, and expression of said vector in a host cell, said vector encodes a fusion protein in which said purification tag is N-terminal to said human SUMO-3 protein, and said human SUMO-3 protein is attached to the amino terminal of the protein of interest; and
(b) a cleavage enzyme for cleaving the SUMO moiety and purification tag(s) from the protein of interest, comprising human SUMO protease SENP2 encoded by SEQ ID NO: 43, an equivalent thereof, a homologue thereof, a catalytic domain thereof, or a combination thereof.

13. The kit of claim 12, wherein said purification tag is selected from the group consisting of polyhistidine tags, polyarginine tags, glutathione-S-transferase, maltose binding protein, S-tag, influenza virus HA tag, thioredoxin, staphylococcal protein A tag, the FLAGTM epitope, AviTag epitope, and the c-myc epitope.

14. The kit of claim 12, wherein said host cell is selected from the group consisting of bacteria, yeast, mammalian cells, and insect cells.

15. The kit of claim 12, further comprising:
i) a solid support resin for binding the purification tag,
ii) cell lysis buffers,
iii) wash buffers,
iv) elution buffers,
v) SENP2 cleavage buffers,
vi) an instruction manual, or
vii) a combination thereof.

16. The kit of claim 12, wherein said protein of interest has a desired native N-terminal amino acid following cleavage with SENP2 protease, and provided that said N-terminal amino acid is not proline.

17. The composition of claim 1, wherein said isolated nucleic acid sequence which encodes a human SUMO-3 protein is SEQ ID NO: 4.

18. The expression vector of claim 4, wherein said isolated nucleic acid sequence which encodes a human SUMO-3 protein is SEQ ID NO: 4.

19. The method of claim 7, wherein said isolated nucleic acid sequence which encodes a human SUMO-3 protein is SEQ ID NO: 4.

20. The kit of claim 12, wherein said isolated nucleic acid sequence which encodes a human SUMO-3 protein is SEQ ID NO: 4.

21. The method of claim 3, wherein said host cell is a yeast cell.

22. The method of claim 6, wherein said host cell is a yeast cell.

23. The method of claim 8, wherein said host cell is a yeast cell.

24. The kit of claim 14, wherein said host cell is a yeast cell.