Patent Application: US-201214006587-A

Abstract:
the present technology relates to the fields of biochemistry , molecular biology and medicine . in particular , the present technology relates to methods and compositions for increased expression of recombinant proteins .

Description:
embodiments of the present invention relate to methods and compositions for high level of expression of fusion proteins . in some embodiments , a fusion protein comprising a modified protein moiety and a protein of interest has a reduced solubility in a host cell compared to the protein of interest . the reduced solubility of the fusion protein increases the yield of the fusion protein . in some embodiments , a modified protein moiety can include a modified ubiquitin moiety . ubiquitin is a small eukaryotic protein which offers a natural yield enhancement , and can be removed from a recombinant fusion protein by highly specific proteases , such as deubiquitylating enzymes . deubiquitylating enzymes do not cleave nonspecific sequences and do not leave additional amino acids at the n terminus of the protein of interest ( baker , r . t . 1996 . protein expression using ubiquitin fusion and cleavage . curr . opin . biotechnol . 7 : 541 - 546 ; hondred , d . et al ., 1999 . use of ubiquitin fusions to augment protein expression in transgenic plants . plant physiol . 119 : 713 - 724 ). this cleavage occurs precisely after a final glycine residue at the carboxyl terminal of the ubiquitin polypeptide , moreover , this cleavage is irrespective of the amino acid immediately following , with the sole exception of proline , which is cleaved inefficiently ( bachmair , a ., finley , d ., and varshaysky , a . 1986 . in vivo half - life of a protein is a function of its amino - terminal residue . science 234 : 179 - 186 ). ubiquitin fusion protein systems have been used to produce soluble proteins , for example , the phue system ( catanzariti , a .- m ., t . a . soboleva , d . a . jans , p . g . board , and r . t . baker , an efficient system for high - level expression and easy purification of authentic recombinant proteins . protein sci , 2004 . 13 ( 5 ): p . 1331 - 1339 ). nonetheless , it is often desirable to produce protein in an insoluble form . accordingly there is a need to produce high levels of recombinant protein in an insoluble form . applicant has discovered the use of particular ubiquitin variants that reduce the solubility of a fusion protein comprising the ubiquitin variants and a protein of interest . the reduced solubility of a fusion protein has several advantages . for example , applicant has discovered that the yield of particular fusion proteins with reduced solubility is increased . in addition , degradation of fusion proteins with reduced solubility is decreased . higher yields are obtained with the redesigned core variants because of reduced toxicity and proteolysis due to the “ tuned ” stability of the ubiquitin fusion ( fig6 ). for example , some proteins , such as chemokines are typically toxic to cells , and sometimes are proteolytically degraded when produced as soluble proteins . in the context of the phue system , some chemokines partition into both the soluble and insoluble fraction , making purification more difficult and resulting in lower yields . by redesigning the core amino acids of ubiquitin and altering its stability , fusions can direct chemokines into inclusion bodies . in some embodiments , a specific design can enable proteins to be refolded and efficiently cleaved from the fusion by ubiquitinase , yielding the desired sequence with the precisely desired n - terminus at significantly higher yield than the phue system . insoluble fusion proteins and fusion proteins with reduced solubility produced by the methods provided herein can be ( i ) refolded and / or ( ii ) cleaved with a protease , for example , ubiquitinase , to obtain precisely defined n - termini . precisely defined n - termini are important for the activity of some proteins , for example , chemokines , where the precise details of the termini defines affinity and activity , namely , whether the chemokines function as antagonists or agonists . in some embodiments , a protein of interest can include a chemokine chemokines are small secreted proteins with a mass about 8 - 12 kda that function by binding to and signaling through g - protein coupled receptors known as chemokine receptors . chemokines are divided into four families ( cc , cxc , c and cx3c ), depending upon the number and location of cysteine residues near the n - terminus . chemokines are sometimes described as ‘ double - edged swords ’ in the immune system , as they are necessary for correct function , but are also implicated in many inflammatory diseases , including multiple sclerosis , rheumatoid arthritis and cancer . in addition , two of the chemokine receptors ( cxcr4 and ccr5 ) are necessary for gp120 - mediated entry of hiv into cells . therefore , chemokines and their receptors are attractive targets for pharmaceutical agents . since their discovery over 20 years ago , chemokines have been the subject of a large body of research , and multiple structures have been published . however , progress in the field has been slowed by difficulties associated with obtaining sufficient quantities of pure , functional chemokines from heterologous expression systems . the system of choice for chemokine expression is e . coli , due to the cost , ease of use , and ability to isotopically label for nmr studies , and has been successfully used for expression and structure determination of multiple chemokines . however , e . coli expression of chemokines is complicated by the knowledge that in many cases , chemokines are sensitive to small modifications at their n - termini . one well - known example of this is ccl5 / rantes , for which the addition of methionine at the n - terminus ( which would occur after expression in e . coli ) renders ccl5 inactive as an agonist . instead , this version , known as met - rantes functions as a potent antagonist against both wildtype ccl5 and ccl3 in calcium flux and chemotaxis assays . some chemokines contain large , bulky residues at position 2 in the sequence , and in previous studies using ccl2 the proline at this position was utilized to specifically remove the initiating methionine residue without affecting the rest of the chemokine . however , this method is obviously dependent upon the chemokine sequence , and so cannot be applied to all chemokines . an e . coli based expression system may be used whereby the protein of interest is expressed as a fusion to poly - histidine tagged ubiquitin . during purification , a specific deubiquitylating enzyme is added , which removes the n - terminal his - tagged ubiquitin , but leaves the intact chemokine with a native n - terminus . here this method as a rapid and efficient way to produce milligram quantities of functional chemokines is used . this method has broad applicability to multiple classes of chemokines , and can be used regardless of whether the chemokine is expressed solubly , less solubly , or insolubly in inclusion bodies . finally , using a destabilizing variant of ubiquitin , it is possible to substantially increase the yield of insoluble protein or protein with reduced solubility , which can subsequently be refolded and purified . chemokines and their receptors are important immunomodulatory proteins , and as such are involved in both normal and pathophysiological functions of the immune system . however , chemokines are challenging to study in vitro , as they are often difficult to obtain in milligram quantities in a functional form . this is due to multiple reasons , including low expression levels , difficulty in purification and the importance of a native n - terminus for proper function . this last issue is especially problematic for e . coli expression due to the retention of the n - terminal methionine , which is removed in vivo during secretion . the present disclosure includes the use of a previously reported ubiquitin expression system to express mg / l quantities of multiple chemokines in e . coli , including ccl2 / mcp - 1 , ccl3 / mip - 1α , ccl7 / mcp - 3 , ccl13 / mcp - 4 , ccl14 / hcc - 1 , ccl27 / ctack , ccl28 / mec , cxcl8 / il - 8 , cxcl9 / mig , cxcl10 / ip - 10 and cxcl11 / itac . using some embodiments of the methods described herein , all of the chemokines expressed at levels & gt ; 1 mg / l . however , a number of the chemokines showed both insoluble and soluble expression , so the use of core - repacked ubiquitin mutants , with lower solubility was explored , to try to increase the final yield . using the highly soluble ccl7 as a test case , it was shown that these ubiquitin variants caused this chemokine to express at significantly higher levels , as almost exclusively insoluble protein . one particular ubiquitin variant containing i3l , v17l , i23v mutations ( 3d3 ), was recognized and efficiently cleaved by a deubiquitylating enzyme after refolding , in a similar manner to wildtype ubiquitin . the ability to use wildtype ubiquitin or ub - 3d3 as a fusion system provides a facile method for expressing functional chemokines in quantities appropriate for biophysical and biochemical characterization . moreover , the ability to rapidly and inexpensively produce labeled chemokines opens the way for their use in many applications , including non - traditional chemokine - receptor interaction studies , both on intact cells and with purified receptor reconstituted in artificial membranes in vitro . furthermore , the ability to immobilize chemokines to obtain ligand affinity columns aids in efforts to purify chemokine receptors for structural and biophysical studies , by facilitating the separation of functional proteins from their non - functional counterparts . chemokines function as extracellular proteins and as such , contain signal sequences that are cleaved during export in vivo . proper processing of chemokines to obtain a native n - terminus is necessary for function and is therefore an important factor in designing expression systems . when expressed as the native sequence in e . coli , the initiator methionine is often retained resulting in antagonists ( e . g . met - rantes ( robinson , s . c ., et al ., a chemokine receptor antagonist inhibits experimental breast tumor growth . cancer res , 2003 . 63 ( 23 ): p . 8360 - 8365 ) and met - mcp - 1 hemmerich , s ., c . et al , identification of residues in the monocyte chemotactic protein - 1 that contact the mcp - 1 receptor , ccr2 . biochemistry , 1999 . 38 ( 40 ): p . 13013 - 13025 ), similarly chemical modification of n - terminus results in the same effect ( e . g . aop - rantes simmons , g ., et al ., potent inhibition of hiv - 1 infectivity in macrophages and lymphocytes by a novel ccr5antagonist . science , 1997 . 276 ( 5310 ): p . 276 - 279 ). alternatively , chemokines can be over - processed such that the n - terminal met along with additional amino acids are removed , again resulting in nonfunctional protein ( paavola , c . d ., et al ., monomeric monocyte chemoattractant protein - 1 ( mcp - 1 ) binds and activates the mcp - 1 receptor ccr2b . j biol chem , 1998 . 273 ( 50 ): p . 33157 - 65 ). in addition , chemokines contain disulfide bonds , which generally cannot be faithfully reproduced in the reducing environment of bacterial cells . for this reason , chemokines are typically expressed as inclusion bodies and then refolded , but there can be difficulties with chemokines prone to high order oligomerization . because of these problems , many different bacterial systems have been developed for chemokine expression , but for the most part have required optimization on a case by case basis , making production of every chemokine a major project . chemokines have been produced using a number of different methods , including chemical synthesis , purification directly from blood , and by expression in mammalian , yeast , insect , and e . coli cells ( paavola , c . d ., et al ., monomeric monocyte chemoattractant protein - 1 ( mcp - 1 ) binds and activates the mcp - 1 receptor ccr2b . j biol chem , 1998 . 273 : 33157 - 65 ; gong , j . h . et al ., antagonists of monocyte chemoattractant protein 1 identified by modification of functionally critical nh2 - terminal residues . j exp med , 1995 . 181 : 631 - 40 ; proost , p ., et al ., chemical synthesis , purification and folding of the human monocyte chemotactic proteins mcp - 2 and mcp - 3 into biologically active chemokines cytokine , 1995 . 7 : 97 - 104 ; harrison , j . k ., et al ., mutational analysis of the fractalkine chemokine domain . basic amino acid residues differentially contribute to cx3cr1 binding , signaling , and cell adhesion . j biol chem , 2001 . 276 : 21632 - 41 ; koopmann , w . et al ., identification of a glycosaminoglycan - binding site in chemokine macrophage inflammatory protein - 1alpha . j biol chem , 1997 . 272 : 10103 - 9 ; masure , s ., l . et al ., expression of a human mutant monocyte chemotactic protein 3 in pichia pastoris and characterization as an mcp - 3 receptor antagonist . j interferon cytokine res , 1995 . 15 ( 11 ): p . 955 - 63 ; chakravarty , l ., et al ., lysine 58 and histidine 66 at the c - terminal alpha - helix of monocyte chemoattractant protein - 1 are essential for glycosaminoglycan binding . j biol chem , 1998 . 273 ( 45 ): p . 29641 - 7 ; polo , s ., et al ., enhancement of the hiv - 1 inhibitory activity of rantes by modification of the n - terminal region : dissociation from ccr5 activation . eur j immunol , 2000 . 30 ( 11 ): p . 3190 - 8 ; mayer , m . r . et al ., identification of receptor binding and activation determinants in the n - terminal and n - loop regions of the cc chemokine eotaxin . j biol chem , 2001 . 276 ( 17 ): p . 13911 - 6 ; myers , j . a ., et al ., expression and purification of active recombinant platelet factor 4 from a cleavable fusion protein . protein expr purif , 1991 . 2 ( 2 - 3 ): p . 136 - 43 ; van coillie , e ., et al ., functional comparison of two human monocyte chemotactic protein - 2 isoforms , role of the amino - terminal pyroglutamic acid and processing by cd26 / dipeptidyl peptidase iv . biochemistry , 1998 . 37 ( 36 ): p . 12672 - 80 ; wain , j . h ., et al ., rapid site - directed mutagenesis of chemokines and their purification from a bacterial expression system . j immunol methods , 2003 . 279 ( 1 - 2 ): p . 233 - 49 ; ye , j ., et al ., characterization of binding between the chemokine eotaxin and peptides derived from the chemokine receptor ccr3 . j biol chem , 2000 . 275 ( 35 ): p . 27250 - 7 ). some of these systems are impractical based on the cost of production ( chemical synthesis ) or the final protein yield ( purification from blood ). of the remaining systems , the most attractive is e . coli expression due to its ease , low cost , high protein yields , and ability to produce chemically ( isotopic or selenomethionine ) labeled protein . the fact that chemokines are secreted proteins provides two problems that must be overcome . like most secreted proteins , chemokines contain intramolecular disulfide bonds . the reducing environment of the cytosol is not conducive to the production of disulfides . this can be overcome by secreting the protein , either into the media , in mammalian / yeast / insect cell expression , or into to the periplasm , in e . coli . these methods tend to produce lower protein yields , and so cytosolic expression is preferred . a number of chemokines have been expressed and purified from e . coli supernatants ( mayer , m . r . et al ., identification of receptor binding and activation determinants in the n - terminal and n - loop regions of the cc chemokine eotaxin . j biol chem , 2001 . 276 ( 17 ): p . 13911 - 6 ; ye , j ., et al ., characterization of binding between the chemokine eotaxin and peptides derived from the chemokine receptor ccr3 . j biol chem , 2000 . 275 ( 35 ): p . 27250 - 7 ), while other chemokines ( e . g . like ccl2 ), can be made to express solubly using thioredoxin deficient cell lines like tap - 302 ( lau , e . k ., et al ., identification of the glycosaminoglycan binding site of the cc chemokine , mcp - 1 : implications for structure and function in vivo . j . biol . chem ., 2004 . 279 ( 21 ): p . 22294 - 22305 ) and / or by decreasing the temperature at which the protein is expressed . however , many chemokines express partially or exclusively insolubly in e . coli , although their small size and presence of disulfides makes them relatively easy to refold . as a result a number of chemokines have been purified from inclusion bodies and have been shown to be properly refolded and active ( wain , j . h ., et al ., rapid site - directed mutagenesis of chemokines and their purification from a bacterial expression system . j immunol methods , 2003 . 279 ( 1 - 2 ): p . 233 - 49 ; proudfoot , a . e ., et al ., extension of recombinant human rantes by the retention of the initiating methionine produces a potent antagonist . j biol chem , 1996 . 271 ( 5 ): p . 2599 - 603 ). the second major problem with chemokine expression comes from the fact that in vivo they contain a signal peptide that gets cleaved off during their secretion , thereby removing the initiator methionine . conversely , non - secreted expression of chemokines results in the presence of the initiator methionine at the n - terminus . research has shown that the presence of an n - terminal methionine often affects the action of chemokines and turns them into powerful antagonists ( proudfoot , a . e ., et al ., extension of recombinant human rantes by the retention of the initiating methionine produces a potent antagonist . j biol chem , 1996 . 271 ( 5 ): p . 2599 - 603 ). this is not surprising since the n - terminus of chemokines has been shown to be critical for binding and signaling ( paavola , c . d ., et al ., monomeric monocyte chemoattractant protein - 1 ( mcp - 1 ) binds and activates the mcp - 1 receptor ccr2b . j biol chem , 1998 . 273 ( 50 ): p . 33157 - 65 ). it is therefore necessary to remove this residue to produce active protein . n - terminal tags that are commonly used to simplify purification also have to be removed . aminopeptidase can be used to remove residues from the n - terminus , but it will nonspecifically remove residues until it encounters a proline . this method has been used to produce active ccl2 , which fortuitously has a proline at position 2 , but cannot be used for all chemokines ( lau , e . k ., et al ., identification of the glycosaminoglycan binding site of the cc chemokine , mcp - 1 : implications for structure and function in vivo . j . biol . chem ., 2004 . 279 ( 21 ): p . 22294 - 22305 ). for those chemokines that do not have a proline at the second position , protease cleavage can be used to produce the native n - terminus . thrombin , enterokinase , and factor - x protease have all been used to produce properly cleaved products ( mayer , m . r . et al ., identification of receptor binding and activation determinants in the n - terminal and n - loop regions of the cc chemokine eotaxin . j biol chem , 2001 . 276 ( 17 ): p . 13911 - 6 ; ye , j ., et al ., characterization of binding between the chemokine eotaxin and peptides derived from the chemokine receptor ccr3 . j biol chem , 2000 . 275 ( 35 ): p . 27250 - 7 ). the problem with using these proteases is that they are not always specific , and so in many cases these proteases will also cleave at additional sites within the protein producing unwanted truncations . fusion proteins are routinely used to increase the yield of proteins , increase solubility or create specific cleavage sites for proteins during over - expression . one example of a fusion system is the baker ubiquitin / ubiquitinase system ( the phue system ) ( catanzariti , a .- m ., et al ., an efficient system for high - level expression and easy purification of authentic recombinant proteins . protein sci , 2004 . 13 : 1331 - 1339 ). described herein are embodiments which include systems for producing a wide range of fusion proteins , including chemokine fusion proteins , with high levels of expression . structural and biochemical studies of the chemokine binding proteins and their interactions with chemokines require a cheap and reliable source of chemokines . at present , most chemokines can be commercially purchased , but the cost of obtaining sufficient quantities makes their use in structural studies prohibitive . it is therefore necessary to develop an expression system to produce the chemokines in - house at a lower cost . the phue expression / cleavage system can be used to produce a very specific cleavage site described in catanzariti , a .- m ., t et al ., an efficient system for high - level expression and easy purification of authentic recombinant proteins . protein sci , 2004 . 13 ( 5 ): p . 1331 - 1339 , hereby incorporated by reference for its discussion on material and methods of the phue system . briefly , the system involves fusing his6 - ubiquitin to the n - terminus of a protein . the tagged ubiquitin can be removed from the protein through the use of ubiquitinase , which results in a very specific cleavage after the second glycine at the c - terminus of ubiquitin ( fig1 ). however , using the phue system , most chemokines are expressed with high variability . moreover , particular chemokines , for example , ccl3 , shows no expression using the phue system . expression and purification of large quantities of active chemokines for biochemical experiments has proved to be difficult thus far . the requirement for properly formed disulfides and an exact n - terminus has limited the range of chemokines that can be expressed at high levels . the phue system has increased the range of chemokines that can be expressed and purified from e . coli . however , there still remain chemokines that either express at low levels or are rapidly degraded . to overcome this , the phue system was modified by altering the ubiquitin codon sequence . three different ubiquitin sequences were used : one that was merely codon optimized and two where the amino acid sequences were both computationally designed and codon optimized . computationally designed ubiquitin sequences are core - redesigns produced using the repacking of cores ( roc ) program ( lazar , g . a ., et al ., de novo design of the hydrophobic core of ubiquitin . protein sci , 1997 . 6 ( 6 ): p . 1167 - 1178 ). codon optimization of ubiquitin had little effect on the expression levels of the fusion proteins . there was some increase in expression of some insoluble chemokine fusions , but it was only minimal . in the case of solubly expressing fusions , there was either a small increase in the solubly expressed protein , cxcl11 and ccl3 , or an increase in the insoluble fraction , ccl7 . this result is not too surprising , as some of the non - codon optimized fusions ( e . g . ccl7 ) express extremely well , and suggests that the rate of expression of ubiquitin is not the limiting factor , but rather that it is the solubility of the fusion protein coupled to the chemokine itself that is the limiting factor . ccl3 expressed at higher levels , but was still subject to degradation which resulted in no net increase of protein . when ccl7 was expressed with wildtype ubiquitin , there was a small amount of protein that was found to be in inclusion bodies which increased over time . the codon optimized ccl7 construct resulted in faster inclusion body production rather than any increase in soluble expression . in an attempt to change the solubility of the ubiquitin - chemokine fusion proteins , two core redesigned ubiquitin amino acid sequences were used . the two sequences used , 2d6 and r6 , were less stable , δδg =− 3 . 2 and − 4 . 5 kcal / mol respectively , than wildtype and express insolubly . these two sequences were chosen because they were they most unstable mutants that could still be refolded . expression tests showed that all chemokines fused to either 2d6 or r6 expressed insolubly , with no trace of soluble expression . the expression levels of all five chemokines tested higher than those seen with the codon optimized or wildtype constructs . additionally , no degradation was observed in the inclusion bodies , suggesting that these designed constructs will be useful for expressing other chemokines susceptible to degradation . although refolding studies of these fusions have not yet been undertaken , this should be relatively simple , as 2d6 , r6 , and all of the phue ubiquitin chemokine - fusions tested to date have been refolded successfully . the use of the core - redesigned ubiquitin sequences produce the largest increase in protein yield of a number of different chemokines . in addition , the expression is universally insoluble which will protect the protein from possible degradation . purifying the fusion under denaturing conditions will avoid the issue seen recently in the lab where the ubiquitin - ccl2 fusion precipitated on the column during purification . the chemokines used in this study display a wide range of expression profiles in the phue system , and they all could be successfully expressed in high levels using the core - redesigned ( 2d6 and r6 ) ubiquitin systems . this indicates that these new expression vectors are generally applicable for chemokine expression and can be used to boost the expression of difficult to produce chemokines . to increase expression yields , as well as to make the ubiquitin fusion system more generally applicable to chemokines , the phue system was modified . most of the dna sequences of chemokines for e . coli expression were codon - optimized to increase their expression yields . codon - optimization of ubiquitin - chemokine fusions were performed to increase levels of expression of said ubiquitin - chemokine fusions . insoluble ubiquitin core - redesigns that can be refolded include those shown in fig2 ( lazar , g . a ., et al ., de novo design of the hydrophobic core of ubiquitin . protein sci , 1997 . 6 ( 6 ): p . 1167 - 1178 ). the following examples include descriptions of the stability and expression yields of chemokines expressed as insoluble fusion proteins or fusion proteins with reduced solubility . however , it will be appreciated that the methods and compositions described herein may be utilized with proteins of interest other than chemokines , including proteins of interest which are toxic or which provide lower yields using alternative expression systems . in particular , expression vectors and their expression profiles are described , where the replacement of wildtype ubiquitin with a core redesigned ubiquitin fusion partner had lower stability , shifting expression to inclusion bodies . such less stable fusions were expressed at higher levels than wildtype or codon optimized versions of ubiquitin . some embodiments of the methods and compositions provided herein relate to methods for producing a recombinant protein . in some embodiments , a recombinant protein can be produced by expressing a fusion protein in a host cell . in some embodiments , a fusion protein comprises a modified protein moiety and a protein of interest . the fusion protein can be expressed from a single polynucleotide sequence in a host cell . examples of host cells include eukaryotic cells , such as mammalian cells , insect cells , plant cells , yeast , and prokaryotic cells , such as e . coli . in particular embodiments , an expressed fusion protein comprising a protein of interest has a reduced solubility in a host cell compared to the solubility of the protein of interest expressed in the host cell . in some such embodiments , the expressed fusion protein can be enriched in inclusion bodies of the host cell . polynucleotides encoding a fusion protein can be constructed by methods well known in the art . a polynucleotide encoding a fusion protein can include nucleotide sequences for increasing the expression of the fusion protein in a host cell . such nucleotide sequences include promoters , enhancers , and terminal sequences . generally , a polynucleotide encoding a fusion protein can include a sequence encoding a 5 ′ modified protein moiety , a 3 ′ protein of interest , and a protease recognition cleavage site therebetween . a protease recognition cleavage site can be used to cleave a protein of interest from an expressed fusion protein . examples of proteases that can be used with the methods and compositions provided herein include ubiquitinases , such as usp2 - cc . in some embodiments , a polynucleotide encoding a fusion protein can include sequences to facilitate purification of a fusion protein . an example includes a sequence encoding a plurality of histidine residues , useful to purify a fusion protein using a nickel column . more embodiments include providing conditions for said protein of interest to refold into an active form . in some such embodiments the protein of interest is subsequently cleaved from the modified protein moiety . conditions for said protein of interest to refold into an active form can be determined by methods well known in the art . some embodiments of the methods and compositions provided herein relate to modified protein moieties . in some embodiments , a modified protein moiety includes a polypeptide sequence that reduces the solubility of an expressed fusion protein comprising a protein of interest in a cell compared to the solubility of the protein of interest in the cell . such modified protein polypeptides can be designed de novo by methods well known in the art ( see e . g ., degrado w . f ., et al ., ( 1999 ) annu rev . biochem . 68 : 779 - 819 ). examples of modified protein moieties include modified ubiquitin polypeptides ( lazar g . a . et al ., ( 1997 ) protein sci . 6 : 1167 - 1178 ). in some embodiments , the modified ubiquitin moiety comprises an increased frequency or number of hydrophobic amino acid residues compared to a wild type ubiquitin moiety . in some embodiments , the modified ubiquitin moiety comprises a decreased frequency or number of hydrophobic amino acid residues compared to a wild type ubiquitin moiety . examples of modified ubiquitin moieties include ubiquitin sequences with one or more mutations at positions selected from the group consisting of i3 , v5 , i13 , l15 , v17 , i23 , v26 , i30 , l43 , l50 , l56 , and l69 , relative to a wild type ubiquitin sequence such as seq id no : 09 ( fig2 ). in some embodiments , a modified ubiquitin moiety comprises a ubiquitin sequence with one or more mutations selected from the group consisting of i3l , i13v , v5i , l15i , v17l , i23v , v26l , i30l , l43i , l50i , l56i , and l69i , relative to a wild type ubiquitin sequence such as seq id no : 09 . in particular embodiments , a modified ubiquitin moiety comprises a ubiquitin sequence with one or more mutations selected from the group consisting of i3l , v17l , and i23v , relative to a wild type ubiquitin sequence such as seq id no : 09 . in particular embodiments , a modified ubiquitin moiety comprises an ubiquitin sequence with one or more mutations selected from the group consisting of i3l , v5i , i13v , v26l , i30l , and l69i , relative to a wild type ubiquitin sequence such as seq id no : 09 . in particular embodiments a modified ubiquitin moiety comprises an ubiquitin sequence with one or more mutations selected from the group consisting of l15i , v17i , i30l , l43i , l50i , and l56i , relative to a wild type ubiquitin sequence such as seq id no : 09 . in some embodiments , a modified ubiquitin moiety comprises a sequence selected from the groups consisting of seq id no : 10 , seq id no : 11 , and seq id no : 26 . in certain embodiments , a modified ubiquitin moiety comprises seq id no : 26 . some embodiments of the methods and compositions provided herein relate to proteins of interest . generally , a protein of interest can include any polypeptide . in certain embodiments , a protein of interest can be soluble , insoluble or partially soluble in a cell in which it is expressed . in preferred embodiments , the protein of interest is soluble . the methods and compositions provided herein include expression of a fusion protein comprising a modified protein moiety and a protein of interest with reduced solubility in a cell compared to the solubility of the protein of interest in the cell . in some embodiments , such methods and compositions are advantageously useful for the production of proteins of interest that would be toxic to a cell in which it is expressed in a soluble form . in some embodiments , such methods and compositions are also advantageously useful for the production of proteins of interest that would be degraded in a cell in which it is expressed in a soluble form . therefore , in some embodiments , a protein of interest includes a protein toxic to a cell in which it is expressed , in which the protein of interest is in soluble form . in addition , in some embodiments , a protein of interest includes a protein that is degraded in a cell in which it is expressed , in which the protein of interest is in soluble form . in certain embodiments , a protein of interest can include a chemokine or a cytokine examples of such proteins of interest include : ccl2 ( seq id no : 27 ), ccl3 ( seq id no : 28 ), ccl7 ( seq id no : 29 ), ccl13 ( seq id no : 30 ), ccl14 ( seq id no : 31 ), ccl27 ( seq id no : 32 ), ccl28 ( seq id no : 33 ), cxcl8 ( seq id no : 34 ), cxcl9 ( seq id no : 35 ), cxcl10 ( seq id no : 36 ), cxcl11 ( seq id no : 37 ), ccl14 ( seq id no : 38 ), cxcl1 ( seq id no : 39 ), cxcl2 ( seq id no : 40 ), cxcl3 ( seq id no : 41 ), cxcl4 ( seq id no : 42 ), cxcl5 ( seq id no : 43 ), cxcl6 ( seq id no : 44 ), cxcl7 ( seq id no : 45 ), cxcl12 ( seq id no : 46 ), cxcl13 ( seq id no : 47 ), cxcl14 ( seq id no : 48 ), cxcl16 ( seq id no : 49 ), xcl1 ( seq id no : 50 ), xcl2 ( seq id no : 51 ), cx3cl1 ( seq id no : 52 ), ccl1 ( seq id no : 53 ), ccl4 ( seq id no : 54 ), ccl5 ( seq id no : 55 ), ccl8 ( seq id no : 56 ), ccl11 ( seq id no : 57 ), ccl15 ( seq id no : 58 ), ccl16 ( seq id no : 59 ), ccl17 ( seq id no : 60 ), ccl18 ( seq id no : 61 ), ccl19 ( seq id no : 62 ), ccl20 ( seq id no : 63 ), ccl21 ( seq id no : 64 ), ccl22 ( seq id no : 65 ), ccl23 ( seq id no : 66 ), ccl24 ( seq id no : 67 ), ccl25 ( seq id no : 68 ), and ccl26 ( seq id no : 69 ). in particular embodiments , a nucleic acid encoding a protein of interest can be codon - optimized for expression in a particular cell . methods for codon optimization to achieve optimal expression in a particular organism , such as e . coli and yeast , are well known in the art . expression tests of twenty ubiquitin - chemokine fusion constructs ( five different chemokines with four versions of ubiquitin ) were undertaken . table 1 shows expression of phue / pcev ubiquitin chemokine fusions . cxcl11 , ccl7 and ccl28 showed at least small amounts of soluble expression in the phue system , while ccl13 expressed insolubly . in contrast , ccl3 showed a reasonable level of insoluble expression , but very little full - length soluble protein . however , a number of smaller molecular weight bands , presumably truncation products , were observed ( fig3 a - 3f ). to increase the yield of ubiquitin - fused chemokines , the codon sequence for ubiquitin was optimized based on e . coli codon usage . the expression patterns of the codon - optimized ubiquitin fusion constructs were similar to those seen with phue . optimization of ubiquitin did not change the solubility of any of the fusions , but did affect the overall expression levels in a number of cases . increased soluble expression was only seen in the lowest level expressers , cxcl11 and ccl3 . soluble cxcl11 expression increased slightly , but the protein was rapidly degraded , while in the case of ccl3 , there was a noticeable increase in expression of both full length protein and truncation products . conversely , codon - optimization of ubiquitin resulted in a slight decrease in soluble expression of ccl28 . ccl7 and ccl13 both show increased insoluble expression and no change in soluble expression ( fig4 ). when ccl7 was expressed in phue , a small amount of insoluble expression was seen which increased over time . this suggests that over time the protein is becoming too concentrated and therefore aggregates to form inclusion bodies . as a result , the increased expression levels of the codon optimized sequence lead to faster inclusion body formation without any increase in the soluble protein . to deal with heterogonous expression and degradation problems , the amino acid sequence of ubiquitin was altered . computational designs where the ubiquitin core was repacked led to the generation of a number of altered ubiquitin sequences which were less stable than wildtype ( paavola , c . d ., et al ., monomeric monocyte chemoattractant protein - 1 ( mcp - 1 ) binds and activates the mcp - 1 receptor ccr2b . j biol chem , 1998 . 273 ( 50 ): p . 33157 - 65 ). two of those sequences , 2d6 and r6 , were fused to chemokines via the phue system . all chemokine fusions expressed using pcev 2d6 and pcev r6 resulted in insoluble expression . in all cases the expression levels were higher than those seen for the wildtype ( phue ) or codon optimized ( pcev opt ) versions of ubiquitin ( fig5 ). codon - optimization sequences for human ccl3 , ccl13 , ccl28 , and cxcl11 for expression in e . coli were generated using computer program which replaces the natural codons of a given protein with codons observed at high frequency in bacterial cells . repetitive sequences , high gc content , and mrna structure are then eliminated with cycles of monte carlo optimization . six pcev vectors were designed to be compatible with phue chemokine vectors . the inserts were designed to have met - gly - ser - ser ( seq id no : 12 ) sequence followed by either a ( his ) 6 ( seq id no : 13 ) or a ( his ) 8 ( seq id no : 14 ) at the n - terminus of ubiquitin and use a similar c - terminal sequence that contains a sacii sequence ( fig1 ). using sacii at the end of ubiquitin allows the rapid production of expression constructs via sub - cloning from the previous phue - based chemokine expression vectors . codon optimized ( ub opt ) and r6 ( ub r6 ) ubiquitin were produced using the following primers ( mwg biotech , high point , n . c .) which resulted in a nucleic acid encoding a ( his ) 6 at the n - terminus : the 2d6 variant of ubiquitin ( ub 2d6 ) was made using the following primers which resulted in a nucleic acid encoding ( his ) 8 at the n - terminus : all modified ubiquitin fragments were first digested with ndei and ecori , after which they were ligated into pet27b and psv212 using the same restriction sites . psv212 is a pet - 21 derived expression vector that contains the same promoter / rbs region as pet - 27b , but contains ampicillin resistance instead of a kanamycin resistance . ccl3 ( mip - 1a ), ccl7 ( mcp - 3 ), ccl13 ( mcp - 4 ), ccl28 ( mec ) and cxcl11 ( 1 - tac ) were subcloned from respective phue based - vectors into pcevopt - 27 , pcev2d6 - 27 and pcevr6 - 27 using sacii and hindiii . table 2 lists the names and genes of constructs made . all constructs were transformed into bl21 ( de3 ) plyss cells and plated on lb plates containing chloramphenicol and either carbenicillin ( phue ) or kanamycin ( pcev - 27 based vectors ). once single colonies were visible , a few were picked and transferred to 50 ml of lb + antibiotic and grown overnight at 37 ° c . 100 ml samples of lb + antibiotics in baffled flasks were inoculated to an od 600nm = 0 . 05 the following morning . cultures were grown at 37 ° c . with shaking ( 200 rpm ). od 600nm time points were taken every 30 - 60 min until the od 600nm was between 0 . 5 and 0 . 6 , at which time a 1 ml sample was taken and the rest of the culture induced with 1 mm iptg . 1 ml samples were taken at time points of 1 , 2 and 4 hrs post induction . all 1 ml samples were treated as follows : samples were spun down at 16 , 000 × g in a tabletop microfuge ( eppendorf 5415 c ) for 1 minute . excess media was removed and the cell pellet was resuspended in 50 mm tris 8 . 0 , 300 mm nacl , 5 mm mgso 4 , 0 . 1 % tween - 20 at a volume of 100 μl of buffer / 1 odu 600nm . 1 μl of 1 mg / ml dnase and 1 μl of 1 mg / ml lysozyme were added to each sample prior to freezing at − 20 ° c . to assay the levels of soluble and insoluble expression the samples were run on 15 % sds - page gels . protein bands were visualized using coomassie stain . table 3 shows a comparison of the estimated yields ( mg / l ) of chemokine from the phue system , compared to yields with redesigned ubiquitin . the redesigned ubiquitin can be used to shift protein from the soluble to the insoluble fraction ( fig7 ). dna sequences encoding human ccl2 , ccl3 ccl7 , ccl13 , ccl14 , ccl27 , ccl28 , cxcl8 , cxcl9 , cxcl10 and cxcl11 were codon - optimized for e . coli using an in - house program and constructed using overlapping primer synthesis . table 4 provides examples of vectors with chemokine sequences inserted therein . to generate chemokines with native n - termini after usp2 - cc digestion , codon - optimized sequences were cloned into the phue vector using the sacii and hindiii restriction sites . sequences for the codon - optimized and destabilizing ubiquitin variants were cloned using the nde i and sacii restriction sites . fusion constructs were expressed using bl21 ( de3 ) plyss cells or tap302 cells in luria broth at 30 ° c . or 37 ° c . cultures were grown to an od600 nm = 0 . 6 , in the presence of 200 μg / ml carbenicillin , and were induced using 1 mm iptg for 3 - 4 hr . cells were harvested via centrifugation at 5000 × g for 15 min at 4 ° c . for test expressions , cell pellet weights were normalized by pelleting cells and resuspending them in lysis buffer ( 50 mm tris 8 . 0 , 300 mm nacl , 5 mm mgso4 , 0 . 1 % tween - 20 ) based on their relative od600 &# 39 ; s . cells were lysed through the addition of 10 μl of 1 mg / ml lysozyme followed by 2 rounds of freeze - thawing , and then spun down at 18 , 000 × g for 10 min at room temperature . 5 × sds - loading buffer was added to the supernatant , while the pellets were resuspended in an identical volume of 1 × loading buffer . 10 μl of pellet and supernatant from each sample fractions was run on 15 % sds - polyacrylamide gels and stained with coomassie blue . for large - scale purification , cell pellets were resuspended in 50 mm tris 8 . 0 , 300 mm nacl , 10 mm imidazole . dnasei , lysozyme ( 40 μg / ml ) and edta - free protease inhibitor tablets ( roche , indianapolis , ind .) were added . cells were lysed via sonication for 4 × 30 sec at 60 % power , and the supernatant was clarified by centrifugation at 18 , 000 × g for 15 min at 4 ° c . for soluble proteins , the supernatant was loaded onto a ni - sepharose column ( ge healthcare , piscataway , n . j .) or a ni - nta column ( qiagen , valencia , calif . ), using an akta fplc system ( ge healthcare ). peak fractions containing fusion proteins were eluted with 50 mm tris 8 . 0 , 300 mm nacl , 500 mm imidazole , and dialyzed against 50 mm tris 8 . 0 , 300 mm nacl prior to usp2 - cc cleavage and further purification . cells were lysed and centrifuged as described herein . pellets obtained after centrifugation were resuspended and washed twice with 20 mm tris 8 . 0 , 0 . 25 % ( w / v ) deoxycholate . inclusion bodies were pelleted at 18 , 000 × g for 15 min at 4 ° c . and then resuspended in 20 mm tris 8 . 0 , 6 m guhcl using a dounce homogenizer . the proteins were loaded onto a ni - nta column and eluted with 20 mm tris 8 . 0 , 6 m guhcl 500 mm imidazole . all proteins were refolded prior to usp2 - cc cleavage , and refolding conditions were determined using the fold - it screen ( hampton research , aliso viejo , calif .). briefly , each fusion protein was rapidly diluted into each of sixteen refolding conditions . the refolded proteins were then dialyzed against 50 mm tris 8 . 0 , 300 mm nacl , and conditions that produced no visible precipitation were used for larger scale purifications . the final refolding buffer conditions for each of the six chemokines tested in this study ( ccl13 , ccl27 , ccl28 , cxcl9 - 11 ). after refolding , chemokines were purified using ni - affinity chromatography and dialyzed into cleavage buffer , as described herein . refolding conditions are summarized in table 5 usp2 - cc was expressed and purified using a modified version of a previously published protocol . briefly , pusp2 - cc was transformed into bl21 ( de3 ) plyss cells and grown in 2 × yeast tryptone ( 2xyt ) broth at 37 ° c . to an od600 nm = 0 . 6 . cultures were then induced with 1 mm iptg and grown for a further 4 hrs . cells were harvested via centrifugation at 5000 × g for 15 min at 4 ° c . pellets were resuspended in 50 mm tris 8 . 0 , 500 mm nacl , 20 mm imidazole , 5 % glycerol ( v / v ), 5 mm f3 - mercaptoethanol , and sonicated for 4 × 30 sec @ 60 % power . cell debris was removed by centrifugation at 18 , 000 × g for 15 min at 4 ° c . supernatant was further clarified by filtering through a 0 . 22 μm filter ( millipore ) before loading onto a ni - sepharose hp column ( ge healthcare ) using an akta fplc ( ge healthcare ). usp2 - cc was eluted using 500 mm imidazole , and concentrated using 10 kda - cutoff amicon concentrators ( millipore , billerica , mass .). for final purification , usp2 - cc was loaded onto a sephadex s75 size exclusion chromatography column ( ge healthcare ), equilibrated with 50 mm tris , ph8 . 0 , 300 mm nacl , 1 mm dtt , 5 % glycerol . peak fractions containing usp2 - cc were concentrated as above , and glycerol was added to a final concentration of 25 % ( v / v ). purified enzyme was stored at − 20 ° c . usp2 - cc was incubated with dialyzed fusion protein at a molar ratio of 1 : 100 usp2 - cc : ubiquitin - chemokine reactions were mixed and allowed to proceed at room temperature for typically 1 - 4 hrs . most reactions went to completion within 2 hrs . to facilitate removal of his - tagged ubiquitin , usp2 - cc and any remaining fusion protein , an optional ni - nta column was added . flow - through from this column containing chemokine was loaded onto a c18 - reverse phase hplc column ( grace , deerfield , ill . ), pre - equilibrated with 0 . 1 % trifluoroacetic acid , 25 % acetonitrile ( acn ). chemokines were eluted using a 25 - 90 % gradient of acn , lyophilized and stored at − 80 ° c . hek293 cells expressing ccr1 were utilized . calcium flux assays were performed using a flipr calcium 4 assay kit ( molecular devices , sunnyvale , calif . ), using 2 × 104 cells / well in a 384 - well assay format . ccl7 - dependent increases in cytosolic ca 2 + were measured using a flipr instrument ( molecular devices ). expression of ubiquitin - chemokine fusions : eleven ubiquitin - chemokine fusions were transformed and tested for expression in bl21 ( de3 ) plyss cells . all of the chemokine fusions successfully expressed , although the total yield and the proportion of soluble protein varied , depending upon the chemokine table 6 lists chemokines tested in this study , and their relative expression levels , as determined by gel analysis . five of the eleven fusions ( ccl2 , ccl7 , ccl14 , cxcl8 and cxcl10 ) showed soluble expression , although the estimated yield varied from ˜ 1 mg / l to & gt ; 10 mg / l . the remaining fusions ( ccl3 , ccl13 , ccl27 , ccl28 , cxcl9 and cxcl11 ) showed very little or no soluble expression , but they all showed good levels (& gt ; 1 mg / l ) of insoluble expression . this indicates that the presence of ubiquitin was unable to increase the solubility of otherwise insoluble chemokines , when expressed in this cell line . fig8 shows a representative selection of the expression gels in which the amount of protein loaded in each lane was normalized to the cell density , and highlights differences in solubility and overall expression levels of the different chemokines tested . as chemokines contain one or more disulfide bonds , it is not surprising that a number of them expressed insolubly in bl21 ( de3 ) plyss cells . to address this , the effect of using the disulfide - permissive tap302 cells , and decreasing the expression temperature from 37 ° c . to 30 ° c ., upon the yield of soluble protein was tested . the use of tap302 cells to increase the yield of soluble his - tagged ccl2 has been reported . however , the combination of tap302 cells and lower temperature did not enhance the solubility of the ubiquitin fusions ( data not shown ), so all further expressions were undertaken using bl21 ( de3 ) plyss cells . purification of chemokines : after successfully demonstrating that all of the chemokines that were tested as ubiquitin fusions , expressed at high levels . the fusions proteins were then tested to determine whether they could be successfully purified , and ubiquitin removed to generate active chemokine . for this study nine representative chemokines were selected ( table 8 ), three of which ( ccl2 , ccl7 and ccl14 ) were chosen to purify from soluble expression , while the other six ( ccl13 , ccl27 , ccl28 , cxcl9 , cxcl10 and cxcl11 ) were predominantly insoluble , so were refolded from inclusion bodies before purification . table 7 provides final yields and characterization of chemokines tested in this study . in order to purify these insoluble proteins , they were first solubilized in guanidine hydrochloride , which yielded up to 50 mg / l of fusion protein , and used a commercially available screen to help identify optimal refolding conditions for each protein . in all cases ni - affinity chromatography as a first pass purification step was used , and this served to significantly enrich for the protein of interest . a representative gel from ni - affinity chromatography of ccl7 is shown in fig9 a . after partial purification , ubiquitin was removed from the fusions , using the catalytic core of the de - ubiquitylating enzyme usp2 - cc . fig9 b shows a typical result from the cleavage of the ubiquitin - ccl7 fusion . at a 100 : 1 molar ratio of fusion protein : usp2 - cc , the fusion was completely cleaved within 1 hr at room temperature . cleavage rates and efficiencies were similar for the other chemokine fusions , although in some cases , a small amount of fusion protein was left uncleaved . this was most likely due to a small fraction of misfolded protein that was not recognized by the ubiquitinase . after further purification to remove usp2 - cc enzyme , uncleaved fusion protein and ubiquitin , all samples were tested by mass spectrometry to confirm that cleavage by usp2 - cc occurred at the correct site in the sequence . for each chemokine , only one species was observed by mass spectrometry , and this corresponded to the mass of the chemokine with the native n - terminus ( table 7 ), indicating that usp2 - cc cleaves specifically at the desired site . the final yields of purified chemokine are shown in table 7 . in general , yields after purification and usp2 - cc cleavage tracked well with starting expression levels . for example , ccl7 , ccl27 and cxcl10 all expressed very well , and could be purified to levels 10 mg / l , with ccl27 providing the highest final yield , at 30 mg / l . in contrast , ccl14 showed modest expression , and was purified with a final yield of 0 . 5 mg / l . to confirm activity , selected chemokines were tested in chemokine binding , calcium flux and / or chemotaxis assays ( fig9 c ). to compare activity of ccl7 expressed as an ubiquitin fusion to commercially available ccl7 directly , the effects of ccl7 from the two sources upon calcium mobilization in ccr1 - expressing hek293 cells was tested ( fig9 c ). both proteins showed identical activity in this assay , indicating that ccl7 expressed as an ub - fusion behaves very similarly to that obtained by other methods . optimization and redesign of ubiquitin : to explore whether it would be possible to increase chemokine yields , a number of different forms of ubiquitin was tested . initially the wildtype ubiquitin dna sequence was replaced with one that had been optimized by mutating the rare arginine codons . when fused to ccl7 , this new optimized sequence did appear to slightly increase the overall yield , though primarily through increasing the expression of the insoluble fraction ( fig1 a vs fig1 b ). similar results were also observed using optimized fusions to ccl3 and ccl13 ( data not shown ). in some cases , as with ccl28 and cxcl11 , no appreciable increase in expression could was seen . it is unclear why the benefit from codon optimization varied among the fusions . since the use of a codon optimized sequence tended to increase the insoluble expression of the fusions , a variety of destabilized ubiqutin proteins to try and drive the expression into inclusion bodies was used . a number of computationally core - repacked mutants of ubiquitin were generated and characterized . three of these mutants , 3d3 , 2d6 and * r6 were selected , to cover a range of destabilizing mutations . to test the effect of these mutants to promote inclusion body expression ccl7 was used , which normally expresses solubly . in all three cases , there was a significant increase in the overall expression level and the resulting destabilized ubiquitin - fusion expressed insolubly ( fig1 a , versus fig1 c , fig1 d , and fig1 e ). it should be noted , that some soluble expression was observed at intermediate time points , but by the final harvest point all of the fusion was seen in the inclusion body . similar results were seen for other chemokines . one concern with using the core - repacked ubiquitin mutants was whether these variants could be efficiently recognized and cleaved using usp2 - cc . to test this , refolding and cleavage studies using ccl7 as a test case were carried out ( fig1 ). 3d3 ubiquitin ( 3d3 - ccl7 ) was recognized and cleaved with efficiency similar to that observed for wildtype ubiquitin ( wt - ccl7 ). in contrast , the 2d6 fusion ( 2d6 - ccl7 ) was only partially cleavable , with large amounts of fusion remaining uncleaved after 12 hr . based on the published cd spectra for the two mutant proteins , the cleavage efficiency could be related to how close to wildtype the spectra are . expressing chemokines as ubiquitin fusion proteins , and subsequently cleaving using usp2 - cc , it is possible to routinely express and purify milligram quantities of pure , active chemokines . based on our experience with both major families of chemokines , this system is generally applicable to the production of most , if not all chemokines finally , by driving the expression of insoluble protein , destabilizing mutants of ubiquitin such as ub - 3d3 show promise in their ability to improve the yield of otherwise low expressing chemokines chemokines include a large family of signaling molecules that are produced by a variety of cells in response to signals associated with host defense and wound repair . they play a pivotal role in the immune system , controlling migration and activation of leukocytes , by binding to and signaling through seven transmembrane g - protein coupled receptors ( gpcrs ) on the surface of leukocytes [ 1 , 2 ]. however , chemokines and their receptors are often likened to double - edged swords because they are also associated with a number of pathologies and are essential cofactors during viral entry of hiv into host cells [ 3 - 5 ]. studies of chemokine : receptor binding in intact cells or membrane fragments have been traditionally conducted using 125 i - radiolabeled chemokines . however , this method has a number of drawbacks , including changes in receptor binding affinity caused by iodination of the chemokine , and the necessity of custom labeling if a particular chemokine of interest is not commercially available . also , the assays are time - consuming and require multiple wash steps to remove unbound chemokine . more recently , homogeneous assays such as the scintillation proximity assay ( spa ) ( perkinelmer ) have become popular , particularly in the pharmaceutical industry , as they can be easily miniaturized and require no wash steps . however , they still use radiolabeled chemokines that may need to be custom - labeled , and require safe handling procedures and hazardous waste disposal . it is possible to express , purify and solubilize gpcrs , including chemokine receptors at sufficient levels for structural and biophysical studies [ 6 - 11 ]. progress in this area recently culminated in the first high resolution crystal structures of a chemokine receptor ; those of cxcr4 in complex with small molecule and peptide antagonists [ 7 ]. nevertheless , there are few reports describing chemokine binding to their receptors in solution and due to the lack of alternative approaches , radiolabeled ligands ( e . g . 125 i - chemokine and 35 s - gtp - γs ) have been used for this purpose too . expression and purification of non - radiolabeled chemokines has been described previously [ 12 - 14 ], though sporadically , and for only one or two ligands . chemical synthesis of fluorescently labeled chemokines is also possible [ 15 ], but is expensive , making the use of these ligands impractical for large - scale studies . described herein are methods to systematically express and purify milligram quantities of four different functional , derivatized chemokines for use in a range of assays . to date , fluorescent and / or biotinylated versions of ccl7 , ccl14 , ccl3 and cxcl8 have been obtained ; this method is also valid for many other chemokines . the applications tested in this study include non - traditional radioligand assays , in which biotinylated chemokines are used in conjunction with 125 i - labeled streptavidin , circumventing the need for radiolabeled chemokines and problems associated with reduced binding affinity of the radiolabeled probe . in addition , described herein is the use of biotinylated chemokines for use as ligand affinity columns , and the production of fluorescently labeled chemokines for solution - based fluorescence anisotropy - based binding assays . the ability to readily produce these chemokine reagents should also aid in many other types of studies including fluorescence resonance energy transfer - based studies , drug discovery efforts , and array based screening of interacting partners such as chemokine : glycosaminoglycan interactions [ 16 ]. fluorescein - 5 - maleimide and alexa fluor 647 c2 - maleimide were obtained from invitrogen ( carlsbad , calif .). e . coli codon optimized variants of full - length ccl3 , ccl7 , and cxcl8 , along with the functional form of ccl14 containing residues 9 - 74 [ 17 ] were purchased from genscript ( piscataway , n . j .). the phue vector containing an n - terminal his tag and ubiquitin ( ub ), along with pet15b containing the catalytic core of the deubiquitylating enzyme usp2 ( usp2 - cc ) were obtained from jcsmr , the australian national university , act0200 , can berra , australia . a variant of ubiquitin in which three destabilizing mutations were introduced ( i3l , v17l , i23v ) [ 18 ] was cloned into pet27 ( emd chemicals , gibbstown , n . j .) in - house to obtain phue - 3d3 . 125 i - labeled ccl3 and streptavidin were purchased from perkin elmer ( waltham , mass . ), and anti - ccr1 antibody was obtained from thermo fisher scientific ( rockford , ill .). chemokines were cloned onto the 3 ′ end of ubiquitin in phue [ 19 ] ( ccl7 , ccl14 ) or ubiquitin - 3d3 in phue - 3d3 ( ccl3 , cxcl8 ) using sacii and hindiii restriction sites at the chemokine n and c - termini respectively . for derivatization , dna coding for a cysteine residue or an avitag sequence ( klgsglndifeaqkiewhe ; seq id no : 23 ) was also added to the c - terminus of the chemokine . after verification by sequencing , phue or phue3d3 - based constructs were transformed into bl21 ( de3 ) plyss cells ( novagen , madison , wis .). for phue constructs , cells were grown in luria broth in the presence of carbenicillin ( 200 μg / ml ) and chloramphenicol ( 34 μg / ml ) at 30 ° c . for phue3d3 constructs , cells were grown in luria broth in the presence of kanamycin ( 40 μg / ml ) and chloramphenicol ( 34 μg / ml ) at 37 ° c . in all cases , expression of the ubiquitin - chemokine fusions was induced using 0 . 5 mm iptg at od 600nm ˜ 0 . 6 . after induction , cells were grown for 4 hours before harvesting . cell pellets containing ubiquitin - chemokine fusions were resuspended in buffer a ( 50 mm hepes , ph 7 . 4 , 0 . 5 m nacl , 40 mm imidazole , protease inhibitor tablets ( no edta ) ( roche , indianapolis , ind .)). 5 mm mgcl 2 , dnase i ( roche ) and 0 . 1 % tween - 20 ( sigma , st . louis , mo .) were added and cells were disrupted using an emulsiflex ( avestin , ottawa , canada ) ( 2 runs , 12 , 000 psi ). for soluble chemokines expressed using phue , insoluble material was removed by centrifugation ( 48 , 000 g , 45 minutes , 4 ° c .) and the supernatant was loaded onto a ni - sepharose column ( ge life sciences , piscataway , n . j .). ub - chemokine fusion proteins were eluted using a linear gradient from 40 mm to 500 mm imidazole . ccl3 expressed predominantly insolubly as a phue3d3 fusion , so was purified and refolded from inclusion bodies before being loading onto a ni - affinity column as described above . inclusion body preparation and refolding of ccl3 was undertaken according to a modified version of zabel et al . [ 20 ]. briefly , cell pellets were detergent - solubilized by three rounds of homogenization and centrifugation ( 40 , 000 g , 45 mins ) in the presence of 0 . 25 % ( w / v ) sodium deoxycholate . the insoluble inclusion body pellet was solubilized in a denaturing buffer ( 6 m guanidine hcl , 0 . 1 m sodium phosphate , 10 mm tris , ph8 . 0 ) and loaded onto a nickel - nitrilotriacetic acid ( ni - nta ) column ( qiagen , valencia , calif .). ccl3 was eluted by decreasing the ph to 4 , and refolded by rapid dilution into refolding buffer ( 55 mm mes , ph 6 . 5 , 264 mm nacl , 11 mm kcl , 0 . 055 % ( w / v ) peg3350 , 1 . 1 mm edta , 550 mm l - arginine , 0 . 3 mm n - dodecyl maltoside , 1 mm reduced l - glutathione , 0 . 1 mm oxidized l - glutathione ), at a final ccl3 concentration of 0 . 1 mg / ml . after stirring overnight at 4 ° c ., refolded ccl3 was dialyzed into buffer a before loading onto a ni - affinity column . after ni - affinity chromatography , ubiquitin fusion proteins were either directly cleaved using the usp2 - cc , or further purified by loading onto a c18 reversed - phase liquid chromatography column ( grace vydac , deerfield , ill .) in 0 . 1 % trifluoroacetic acid ( tfa )/ 25 % acetonitrile and eluted using an increasing gradient of acetonitrile . in the latter case , chemokines were lyophilized and stored as fusion proteins . for deubiquitylation by usp2 - cc , samples were resuspended to approximately 1 mg / ml in a buffer containing 20 mm tris , ph 8 . 0 , 0 . 2 m nacl . usp2 - cc , prepared as described in catanzariti et al . [ 19 ], was added at a molar ratio of 50 : 1 chemokine : usp2 - cc , and samples were placed at room temperature for 2 hours before loading onto a ni - nta column ( qiagen ) in 50 mm tris , ph 7 . 5 , 40 mm imidazole , 500 mm nacl . ni - nta was used at this stage instead of ni - sepharose , due to the tendency of untagged chemokines to bind to ni - sepharose resin ( data not shown ). column flow - through samples containing chemokine were quickly pooled and derivatized by addition of fluorophore or bira enzyme as described below . for fluorescent labeling , chemokines containing a c - terminal cysteine were mixed with a 1 : 5 molar ratio of chemokine to fluorescein - 5 - maleimide or alexa fluor 647 , and incubated in the dark at 4 ° c . overnight . biotinylation of avitagged chemokines was undertaken using the bira enzyme , prepared as previously described [ 21 ]. after derivatization , labeled chemokines were further purified using a c18 reversed - phase liquid chromatography column as described above . this step efficiently removed unbound small molecules such as fluorophore and biotin , along with bira and any remaining unbiotinylated chemokine . after purification , samples were flash - frozen and lyophilized . the identity and purity of all samples was verified using electrospray mass spectrometry . hek293 cells expressing ccr1 were cultured as previously described [ 8 ]. calcium flux activity assays were performed using a flipr calcium 4 assay kit ( molecular devices ), using 1 . 3 × 105 cells per well in a 96 - well assay format . chemokine - dependent increases in cytosolic ca2 + were measured using a flexstation 3 microplate reader ( molecular devices ). for each column , 200 μl slurry of high capacity streptavidin agarose resin ( thermo scientific ) was equilibrated in 50 mm tris , ph 8 . 5 , 150 mm nacl . resin was mixed with 1 mg biotinylated ccl14 or buffer alone in a total volume of 1 ml . after 20 minute incubation at 20 ° c ., columns were washed with 40 column volumes of equilibration buffer followed by binding buffer ( 50 mm tris ph 8 . 5 , 150 mm nacl , 10 % glycerol , 0 . 1 % ( w / v ) dodecyl maltoside and 0 . 01 % ( w / v ) cholesterol hemisuccinate ). detergent - solubilized ccr1 , prepared as previously described [ 8 ], was loaded onto the ccl14 and control columns by gravity flow , and the column was washed three times with 1 ml binding buffer . ccr1 was eluted by washing twice with 1 ml binding buffer supplemented with 1 m nacl . scintillation proximity assay ( spa ) binding assays ( ge healthcare , piscataway , n . j .) were conducted essentially as previously described [ 8 , 22 , 23 ]. hek293 cells stably expressing ccr1 were used for these assays , and untransfected hek293 cells were used as a control . assays were undertaken in a 96 - well plate format and each data point was assayed in triplicate on a microbeta plate counter ( perkin elmer ) for 1 min per well . each well contained 20 , 000 cells , 0 . 2 mg wga - pvt - spa beads , increasing concentrations of unlabeled competitor chemokine , and either 50 μm 125 i - ccl3 or 100 μm 125 i - streptavidin + 400 pm ccl3 - biotin , in a total volume of 100 μl . ic 50 values were obtained by nonlinear regression curve fitting using prism software ( graphpad , la jolla , calif .). e . coli codon - optimized variants of four chemokines ( ccl3 , ccl7 , ccl14 , and cxcl8 ) were cloned into phue and phue3d3 vectors to produce a fusion protein containing an n - terminal 6 - his tag , ubiquitin and the chemokine of interest . depending upon the application , the chemokine was either native , or modified at the c - terminus by addition of an extra cysteine residue or an avitag sequence , to permit labeling ( fig1 a ). the n - terminal ubiquitin fusion system was developed , as cleavage with usp2 - cc results in the native sequence . this is important because the exact nature of the n - terminal residues of chemokines affects their activity . in contrast , chemokines are generally tolerant to modifications at their c - termini , so this region was chosen for the addition of non - native residues for labeling . two different versions of ubiquitin were used in this study : wildtype ubiquitin ( phue vector ) was used for expression of ccl7 , ccl14 and cxcl8 , as these express solubly at high levels and ubiquitin promotes soluble expression . however , a core - repacked mutant of ubiquitin ( ub3d3 in the phue - 3d3 vector ) was used for the expression of ccl3 , in order to increase the overall yield of this protein by enhancing its expression in inclusion bodies . an overview of the purification scheme is described in fig1 b , and representative gels and mass spectrometry data for ccl14 are shown in fig1 a / b as an example . for the avi - tagged chemokine constructs , 6 - his - ubiquitin - chemokine - avitag fusions were purified by ni - affinity chromatography , the chemokines were cleaved from 6 - his - ubiquitin using usp2 - cc , derivatized as required and loaded onto ni - nta resin prior to a final reverse phase chromatography and lyophilization . as ubiquitin elutes from a c18 reverse phase chromatography column at a similar acetonitrile concentration as many chemokines , the addition of a ni - nta column served to remove his - tagged ubiquitin , and increase the purity of the final product . this procedure was modified slightly for chemokines containing c - terminal cysteine residues such that an additional reversed phase hplc column was inserted into the method prior to cleavage and derivatization . this optional additional step allowed chemokines to be stored in an underivatized state with minimal aggregation , as described herein . as shown in fig1 for ccl14 , cleavage of ubiquitin - chemokine fusion is & gt ; 90 % efficient ( fig1 a , lane 1 ), and the column efficiently separates chemokine that flows through the column ( fig1 a , lanes 2 and 3 ) from his - tagged ubiquitin that efficiently binds to the resin ( fig1 a , lane 4 ). after purification , mass spectrometry confirmed the correct mass of the purified , derivatized proteins ( fig1 b ). table 8 provides a summary of masses of labeled chemokines , and final yields after derivatization fluorescently labeled chemokines are attractive as they can be used to easily determine dissociation constants for the binding of chemokines to solubilized chemokine receptors [ 8 ]. in order to obtain fluorescently labeled chemokines , the proteins were expressed with a non - native c - terminal cysteine , and maleimide chemistry was used to couple fluorescent labels at this position after purification . during preliminary purifications of ccl7 - cys , cleavage of ubiquitin early in the purification and store the protein as lyophilized underivatized protein was attempted , as was customary for wildtype ccl7 . however , after cleavage and storage the efficiency of derivatization was very low indicating that the reactive cysteine was not inaccessible . electrospray mass spectrometry confirmed that chemokine dimers had formed , as the major mass observed was 18108 . 5 ( expected mass of dimer = 18109 . 0 ). this dimer was presumably facilitated by the formation of an additional time - dependent disulfide bond between the non - native c - terminal cysteine residues . chemokines contain multiple disulfide bonds ( ccl3 , ccl7 , ccl14 and cxcl8 each contain two ) and attempts to selectively reduce the non - native disulfide bond with reasonable efficiency failed . however , in small - scale purification studies the ccl7 fusion protein was monomeric until the ubiquitin was cleaved , based on sds - page gels run under non - reducing conditions . therefore , purification of the chemokine as a fusion was carried out , and remove ubiquitin as a last step , just prior to derivatization . this method was successful for both chemokines tested , and the yields of purified ccl7 and ccl14 prior to derivatization were approximately 5 mg / l and 1 mg / l cells respectively . how ubiquitin is able to prevent dimerization of chemokines is unclear without the existence of a high - resolution structure , but is likely due to steric hindrance . the recovery of pure , labeled ccl7 and ccl14 was 2 . 5 mg / l and 0 . 5 mg / l respectively ( table 8 ). fluorescently tagged chemokines displayed activity indistinguishable from native chemokines on cell membranes [ 8 ]. fig1 c shows representative calcium flux data generated by exposing hek293s cells stably expressing ccr1 to a dilution series of ccl14 - fluorescein . the ec 50 for the dose response curve was 2 nm , similar to that ( 2 . 8 nm ) previously published for unlabeled ccl14 [ 17 ]. fluorescently labeled chemokines may be used in fluorescence polarization studies to obtain dissociation constants for chemokine binding to solubilized chemokine receptors [ 8 ]. the addition of an avitag , and biotinylation with the bira enzyme is a well characterized method for producing protein that is biotinylated at a single , specific lysine residue [ 21 ]. biotin has been used as a molecular handle in numerous published studies , but the ability to biotinylate chemokines is especially attractive because chemokine ligand affinity columns can be used to separate functional chemokine receptors from non - functional chemokine receptors during receptor purification . three chemokines ( ccl3 , ccl14 and cxcl8 ) were tested in this study , and all three could be purified and biotinylated with an efficiency of & gt ; 95 %. in all three cases , the final yield of biotinylated chemokine was at least 0 . 5 mg / l , and masses were as expected according to electrospray mass spectrometry ( table 8 ). to demonstrate the utility of this approach , ccr1 solubilized in detergent micelles [ 8 ] was loaded onto a column containing biotinylated ccl14 immobilized on streptavidin - agarose resin . fig1 shows a representative anti - ccr1 western blot of column samples from this experiment . the ccr1 sample likely contains a mixture of functional and non - functional ccr1 , as illustrated by the presence of ccr1 in both the flow - through and the elution fractions of the chemokine column . a control column without immobilized ccl14 did not bind ccr1 . another useful application of biotinylated chemokines is in non - conventional radioligand binding assays . radioligand assays are accepted as the gold - standard for determining binding affinities between chemokines and their receptors , and as such are desired for chemokine receptor studies . the drawback of this approach is that although many common chemokines can be purchased as 125 i versions , some chemokines need to be custom labeled , and it is generally accepted that iodination can affect receptor binding affinities . however , it is possible to purchase 125 i - labeled streptavidin , and to use it in conjunction with biotinylated chemokine as a surrogate for labeled chemokine in binding assays . in order to test the use of 125 i - streptavidin as a surrogate radiolabel in a chemokine spa assay , competition assays using membranes containing the chemokine receptor ccr1 were performed . ccl3 was used for these assays because 125 i - ccl3 is commercially available , allowing for a direct comparison . after immobilization of membranes to spa beads , either radiolabeled chemokine , or a combination of biotinylated chemokine and 125 i - labeled streptavidin was added . fig1 shows a representative spa assay for ccr1 , and demonstrates that unlabeled ccl14 competes with 125 i - ccl3 or a combination of ccl3 - biotin / 125 i - streptavidin for binding to hek293 membranes containing ccr1 with a similar affinity ( ic 50 = 2 . 6 nm versus 0 . 9 nm for displacement of 125 i - ccl3 or ccl3 - biotin / 125 i - streptavidin respectively ). in contrast , no binding of ccl3 or competition with ccl14 was observed when ccr1 - expressing membranes were replaced with untransfected hek293 membranes . in addition , the 125 i - streptavidin signal was ccl3 - biotin dependent and while the highest signal was observed with a 4 : 1 ratio of chemokine to streptavidin , avidity effects due to the tetrameric state of streptavidin were not observed . these data indicate that the assay is a valid surrogate for traditional radioligand assays , and opens the door to radioligand assays using any chemokine , while only purchasing a single radiolabeled species ( 125 i - streptavidin ). these assays may be further improved with appropriately engineered versions of monomeric streptavidin [ 24 ]. accordingly , it is possible to efficiently and inexpensively produce milligram quantities of labeled chemokines that can be used in many cell - based , biochemical and biophysical assays . these assays include fluorescence - based binding assays , radioligand binding assays , and the generation of chemokine affinity columns . easily separating functional chemokine receptors from non - functional counterparts , and conducting binding studies in solution enables the identification of detergents / lipids that retain receptors in a chemokine - binding competent state [ 8 ]. dna constructs comprising the 3d3 ubiquitin encoding nucleic acid fused to nucleic acids encoding various chemokines were generated and are summarized in table 9 . fig1 shows protein expression of a 3d3 - mip - 1α polypeptide ( left ) or ub - mip - 1α polypeptide ( right ). expression fractions were taken at time 0 and 4 hours post induction and divided into pellet / insoluble ( p ) and supernatant / soluble ( s ) fractions . full length fusion protein is circled in red and degradation products are indicated with arrows . each polypeptide was expressed in an insoluble form . cleavage products of ub - mip - 1α were observed in expression fractions . in contrast , no cleavage products of 3d3 - mip - 1α were observed in expression fractions . thus , the 3d3 ubiquitin moiety protects the linked fusion polypeptide from degradation . this protection increases the yield of a fusion polypeptide linked to a 3d3 ubiquitin moiety , compared to the yield of a fusion polypeptide linked to a wild type ubiquitin moiety . fusion polypeptides comprising either ( 1 ) a wildtype ubiquitin moiety and ccl14 ( ub - ccl14 ); or ( 2 ) a 3d3 ubiquitin moiety and ccl14 ( 3d3 - ccl14 ), were expressed in e . coli . fig1 shows protein expression of ub - ccl14 ( a ) and 3d3 - ccl14 ( b ). expression fractions were at 4 hours post induction and divided into pellet / insoluble ( p ) and supernatant / soluble ( s ) fractions . full length fusion protein is circled in red . the ub - ccl14 fusion polypeptide was expressed in both the soluble ( supernatant ) and insoluble ( pellet / inclusion body ) fractions . in contrast , the 3d3 - ccl14 fusion polypeptide was expressed entirely in insoluble ( pellet / inclusion body ) fractions . this demonstrates the utility of the 3d3 system and its destabilizing effect on chemokine expression to enhance protein yields . 3d3 ubiquitin moiety is a useful expression system when the quality of the soluble fusion protein is not optimal fusion polypeptides comprising either ( 1 ) a wildtype ubiquitin moiety and cxcl8 ( ub - cxcl8 ); or ( 2 ) a 3d3 ubiquitin moiety and cxcl8 ( 3d3 - cxcl8 ), were expressed in e . coli . fig1 shows protein purification of ub - cxcl8 ( a ) and 3d3 - cxcl8 ( b ). the ub - cxcl8 fusion polypeptide was expressed mostly in a soluble fraction , and was purified over a ni - nta column and eluted with increased imidazole . the 3d3 - cxcl8 fusion polypeptide was expressed mostly in insoluble inclusion bodies , and was purified over a ni - nta column and eluted with decreased ph . although the ub - cxcl8 fusion polypeptide appeared as a uniform species by sds - page ( a ), an hplc chromatogram of this material revealed that there were multiple species containing both folded and misfolded proteins ( c ). in particular , while the ub - cxcl8 fusion polypeptide was expressed in a soluble fraction , hplc analysis indicated that about 30 - 40 % of the expressed ub - cxcl8 fusion polypeptide was misfolded . the misfolded fusion polypeptide was prone to precipitation during dialysis , resulting in a further decreased protein yield . here , the use of the 3d3 ubiquitin moiety provided a good alternative expression and purification strategy . expression of the 3d3 - cxcl8 fusion polypeptide was greater than expression of the ub - cxcl8 fusion polypeptide , with & gt ; 90 % of the 3d3 - cxcl8 fusion polypeptide expressed into inclusion bodies ( data not shown ). both the ub - cxcl8 and the 3d3 - cxcl8 fusion polypeptides produced ubiquitin cleavage products ( denoted by *), however , the ub - cxcl8 fusion polypeptide exhibited a greater level of cleavage . table 11 summarizes the results of examples 11 - 13 . in 3 out of 4 cases shown in table 11 , 3d3 fusion polypeptides had at least a 2 - fold increase in yield ( mg / l ) compared to the wildtype ubiquitin fusion polypeptides . these examples demonstrate the advantages of using 3d3 - ubiquitin moieties to express fusion polypeptides where the polypeptide is : ( 1 ) highly prone to degradation ( e . g . ccl3 ); ( 2 ) partitioned into both soluble and insoluble fractions ( e . g . ccl14 ); or ( 3 ) is misfolded in the soluble fraction ( e . g . cxcl8 ). use of the 3d3 system overcomes these issues , in part , by increasing expression of the fusion polypeptide into inclusion bodies . wu b , chien e y , mol c d , fenalti g , liu w , katritch v , abagyan r , brooun a , wells p , bi f c , hamel d j , kuhn p , handel t m , cherezov v , stevens r c . structures of the cxcr4 chemokine gpcr with small - 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92 . vaidehi n , schlyer s , trabamino r j , floriano w b , abrol r , sharma s , kochanny m , koovakat s , dunning l , liang m , fox j m , de mendonca f l , pease j e , goddard w a , 3rd , horuk r . predictions of ccr1 chemokine receptor structure and bx 471 antagonist binding followed by experimental validation . j biol chem 2006 ; 281 : 27613 - 20 . wu s c , wong s l . engineering soluble monomeric streptavidin with reversible biotin binding capability . j biol chem 2005 ; 280 : 23225 - 31 . chiu m l , tsang c , grihalde n , macwilliams m p . over - expression , solubilization , and purification of g protein - coupled receptors for structural biology . comb chem high throughput screen 2008 ; 11 : 439 - 62 . hanson m a , stevens r c . discovery of new gpcr biology : one receptor structure at a time . structure 2009 ; 17 : 8 - 14 . the disclosure of u . s . provisional application no . 61 / 114 , 412 is incorporated herein by reference in its entirety . the above description discloses several methods and systems of the present invention . this invention is susceptible to modifications in the methods and materials , as well as alterations in the fabrication methods and equipment . such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein . for example , the invention has been exemplified using nucleic acids but can be applied to other polymers as well . consequently , it is not intended that this invention be limited to the specific embodiments disclosed herein , but that it cover all modifications and alternatives coming within the true scope and spirit of the invention . all references cited herein including , but not limited to , published and unpublished applications , patents , and literature references , are incorporated herein by reference in their entirety and are hereby made a part of this specification . to the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification , the specification is intended to supersede and / or take precedence over any such contradictory material . the term “ comprising ” as used herein is synonymous with “ including ,” “ containing ,” or “ characterized by ,” and is inclusive or open - ended and does not exclude additional , unrecited elements or method steps .