BACKGROUND
escherichia coli have been widely used as a host to produce valuable commercial, industrial, and therapeutic proteins.
there are several disadvantages with this system, however, especially for the expression of eukaryotic proteins.
for example, when randomly selected  <dig>  full-length genes of caenorhabditis elegans were expressed in e. coli cytoplasm, only 11% of genes yielded significant amounts of soluble material  <cit> .
several different approaches have been taken to resolve the solubility problem in the past and include  truncation of long multi-domain proteins into short and separate domains  <cit>  ;  co-expression of molecular chaperones or foldases  <cit> ;  enabled secretion to the periplasm where disulfide bonds can be properly formed with the help of an oxidative environment and dsb protein families  <cit> ;  co-expression of aminoacyl trna cognates to amino acids encoded by rare codons  <cit> ; and more recently  use of a fusion expression partner  <cit> .
the most efficient method for enhancing solubility and folding efficiencies of recombinant proteins appears to be the latter  which includes maltose binding protein   <cit> , thioredoxin   <cit> , human ferritin heavy chains   <cit> , and glutathione-s-transferase   <cit> .
although these fusion partners have shown good performance in enhancing the solubility and folding of some recombinant proteins  <cit> , they likely cannot serve as universal helpers to solve all protein solubility and folding issues.
thus, novel fusion partners will continue to be developed through systematic investigations including proteome mining.
in the present study, we found that the level of rpos significantly increased during the stress caused by guanidine hydrochloride through proteome-wide mining involving the stress response of e. coli bl <dig>  as an n-terminus fusion expression partner, rpos dramatically increased the solubility of the following heterologous proteins , human epidermal growth factor , human prepro-ghrelin , human interleukin- <dig> , human activation induced cytidine deaminase , human glutamate decarboxylase , pseudomonas putida cutinase , human ferritin light chain , human granulocyte colony-stimulating factor , and cold autoinflammatory syndrome <dig>  nacht domain ).
we also have demonstrated an increased yield of a biologically active fusion mutant of heterologous bacterial cutinase that is expected to be of significant biotechnical and commercial interest.
RESULTS
guanidine hydrochloride-induced proteome response of e. coli and finding of the aggregation-resistant protein, rpos
exogenous stresses including heat shock and gdnhcl  <cit>  often results in the misfolding and aggregation of proteins within the cell.
therefore, it would seem reasonable to presume that intracellular proteins which exist in their native soluble forms even under stress conditions have an intrinsic ability to remain efficiently folded compared to proteins that unfold or aggregate under the same stresses.
gdnhcl stress facilitates the release of periplasmic proteins through increased membrane permeability  <cit>  and hence could be regarded as an osmolyte.
we investigated changes of the proteome profile of e. coli when the stress reagent, guanidine hydrochloride, was added to growing bacterial cultures, by applying 2-dimensional gel electrophoresis .
compared to non-stress condition, the total number of intracellular soluble proteins was reduced to  <dig> from  <dig>  which indicates that many proteins aggregated under the stress condition induced by gdnhcl.
what was surprising is that the rpos expression level  increased 6-fold compared to the non-stress situation.
in addition to rpos, we found several other proteins , the expression level of which more than  <dig> -fold increased in response to the stressor gdnhcl, including hsp <dig> chaperonin , chaperone htpg , transcription elongation protein nusa , dna gyrase subunit a , formate acetyltransferase  <dig> , succinate dehydrogenase flavoprotein subunit , etc.
since the synthesis yield of target protein can be significantly reduced, the molecular mass of proteins to be used as fusion expression partner should not be too high.
that is, although the large amount of fusion protein is synthesized, the actual amount of the target fusion-free protein could be very low if the fusion partner is too big.
rpos  is a relatively small protein among the stress-responsive proteins we found and was highly effective in enhancing the solubility of target proteins.
rpos, stress responsive rna polymerase sigma factor is a well-known universal stress regulator controlling many proteins under various stress conditions, e.g.
the onset of the stationary phase  <cit> , and carbon starvation  <cit> .
rpos was previously reported to be induced by osmotic stress  <cit>  and heat shock  <cit> .
we also observed that the rpos expression level increased 3- to 5-fold in response to heat shock .
muffler et al.
<cit>  reported that the duration of stability of rpos in response to heat shock stress is maintained by the direct- or indirect-binding of dnak.
bound dnak appears to assist the effective folding of rpos and also protect rpos against the action of clpp, a protease that degrades rpos  <cit> .
from this point of view, rpos may serve as a solubility enhancer in e. coli cytoplasm, when used as a fusion expression partner upon the expression of aggregation-prone heterologous proteins.
a gene name, accession number, and protein name were obtained from expasy proteomics server  <cit> .
b theoretical values of pi and mw were calculated using "compute pi/mw tool"  <cit> .
c experimental values of pi and mw were estimated through 2-de gel image analysis in the present study.
d sequence coverage and score values were calculated using "aldente : peptide mass fingerprinting tool"  <cit> .
expression of aggregation-prone heterologous proteins using rpos as fusion partner
we used rpos as an n-terminus fusion expression partner for the synthesis of numerous heterologous proteins .
stenström et al.
<cit>  reported that the codon following an aug start triplet  significantly affects gene expression in e. coli, and the second codon starting with a is most advantageous to achieve an enhanced expression level.
the second codon of rpos is agt  that seems to be favorable second codon based on the report of stenström et al.
<cit> .
as shown in figure 3b and table  <dig>  all heterologous proteins expressed directly without rpos fusion formed insoluble inclusion bodies resulting in nearly negligible solubility.
compared to these results, it seems surprising that when the same heterologous proteins were expressed with the fusion of rpos, the solubility of these foreign proteins dramatically increased , thereby indicating that e. coli rpos is a highly effective solubility enhancer for aggregation-prone heterologous proteins.
table  <dig> compares the effect of rpos- and gst fusion on the solubility enhancement for heterologous proteins.
in the fusion expression of cut, gad448– <dig>  and mpins, the effect of rpos fusion was much higher, whereas gst fusion was significantly more effective in the expression of aid, nacht, and hftn-l. this result indicates that there are no universal helpers to solve all protein solubility issues.
table  <dig> also shows that the results of direct- and fusion expression of heterologous proteins are highly reproducible.
* the solubility was defined as the fraction of the soluble recombinant protein compared to the synthesized total  recombinant protein.
average and standard deviation values were calculated based on the results of repeated triplicate experiments.
moreover, the plasmid vector for the expression of polyhistidine-tagged fusion mutant of g-csf  was constructed  to purify fusion-free g-csf.
after 6::rpos::::g-csf was bound onto the probond resin  column, the enterokinase proteolysis was carried out in a batch mode, and subsequently the digested product was collected and centrifuged.
sds-page and western blot analyses of the supernatant show that the recombinant g-csf was easily released from the rpos-fusion protein and was present in the form of soluble protein in the supernatant .
bioactivity of the recombinant fusion mutant of cutinase, rpos::cut
cutinase has been used as a lipolytic enzyme in the composition of laundry and dishwashing detergents to more efficiently remove immobilized fats  <cit> .
in addition, the oleochemistry industries  <cit> , and pollutant degradation  <cit>  represent other potential uses of cutinase.
in recent years, the esterification and transesterification properties of cutinase have been intensively exploited and could be applied usefully in other chemical synthesis processes  <cit> .
because of these extensive potential applications, we were particularly interested in the production of a bioactive recombinant cutinase.
thus, we cloned the cutinase gene from the genome of pseudomonas putida and expressed it in e. coli using the fusion of rpos.
cutinase is known for its hydrolytic activity for a variety of esters ranging from soluble p-nitrophenyl esters to insoluble long-chain triglycerides.
the hydrolytic activity of cutinase, especially on p-nitrophenyl esters of fatty acids, is extremely sensitive to fatty acid chain length.
previously, lin et al.
<cit>  reported that microbial cutinase lacked a large hydrophobic surface around the active site, in contrast to other lipases and esterases.
this structural characteristic of cutinase may be strongly related to high substrate-specificity, i.e.
the extremely low activity on p-nitrophenyl ester of a long chain fatty acid such as p-nitrophenyl palmitate .
we assayed the enzymatic activity of our fusion mutant of cutinase  and demonstrated the same selective bioactivity as native cutinase to degrade p-nitrophenyl butyrate  but not to degrade pnp .
we also purified rpos::cut-his <dig>  through ni2+ affinity chromatography and analyzed the purified rpos::cut-his <dig> using reversed phase hplc .
as shown in figure 6b, rpos::cut-his <dig> was analyzed as a single peak, which seems to indicate that the crafted mutant molecules of cutinase have uniform and correctly folded conformation due probably to the help of fusion partner, e. coli rpos.
CONCLUSIONS
using 2-dimensional gel electrophoresis, we found that e. coli rpos, rna polymerase sigma factor was gdnhcl stress-responsive and highly effective as a strong solubility enhancer when used as fusion partner for the expression of aggregation-prone heterologous proteins in e. coli bl <dig>  the results of these findings may, therefore, be useful in the production of other biologically active industrial enzymes, as successfully demonstrated by cutinase.

 escherichia coli have been widely used as a host to produce valuable commercial, industrial, and therapeutic proteins. there are several disadvantages with this system, however, especially for the expression of eukaryotic proteins. for example, when randomly selected full-length genes of caenorhabditis elegans were expressed in e. coli cytoplasm, only 11% of genes yielded significant amounts of soluble material. several different approaches have been taken to resolve the solubility problem in the past and include truncation of long multi-domain proteins into short and separate domains; co-expression of molecular chaperones or foldases; enabled secretion to the periplasm where disulfide bonds can be properly formed with the help of an oxidative environment and dsb protein families; co-expression of aminoacyl trna cognates to amino acids encoded by rare codons; and more recently use of a fusion expression partner. the most efficient method for enhancing solubility and folding efficiencies of recombinant proteins appears to be the latter which includes maltose binding protein, thioredoxin, human ferritin heavy chains, and glutathione-s-transferase. although these fusion partners have shown good performance in enhancing the solubility and folding of some recombinant proteins, they likely cannot serve as universal helpers to solve all protein solubility and folding issues. thus, novel fusion partners will continue to be developed through systematic investigations including proteome mining. in the present study, we found that the level of rpos significantly increased during the stress caused by guanidine hydrochloride through proteome-wide mining involving the stress response of e. coli bl as an n-terminus fusion expression partner, rpos dramatically increased the solubility of the following heterologous proteins, human epidermal growth factor, human prepro-ghrelin, human interleukin-, human activation induced cytidine deaminase, human glutamate decarboxylase, pseudomonas putida cutinase, human ferritin light chain, human granulocyte colony-stimulating factor, and cold autoinflammatory syndrome nacht domain ). we also have demonstrated an increased yield of a biologically active fusion mutant of heterologous bacterial cutinase that is expected to be of significant biotechnical and commercial interest.  guanidine hydrochloride-induced proteome response of e. coli and finding of the aggregation-resistant protein, rpos exogenous stresses including heat shock and gdnhcl often results in the misfolding and aggregation of proteins within the cell. therefore, it would seem reasonable to presume that intracellular proteins which exist in their native soluble forms even under stress conditions have an intrinsic ability to remain efficiently folded compared to proteins that unfold or aggregate under the same stresses. gdnhcl stress facilitates the release of periplasmic proteins through increased membrane permeability and hence could be regarded as an osmolyte. we investigated changes of the proteome profile of e. coli when the stress reagent, guanidine hydrochloride, was added to growing bacterial cultures, by applying 2-dimensional gel electrophoresis. compared to non-stress condition, the total number of intracellular soluble proteins was reduced to from which indicates that many proteins aggregated under the stress condition induced by gdnhcl. what was surprising is that the rpos expression level increased 6-fold compared to the non-stress situation. in addition to rpos, we found several other proteins, the expression level of which more than -fold increased in response to the stressor gdnhcl, including hsp chaperonin, chaperone htpg, transcription elongation protein nusa, dna gyrase subunit a, formate acetyltransferase, succinate dehydrogenase flavoprotein subunit, etc. since the synthesis yield of target protein can be significantly reduced, the molecular mass of proteins to be used as fusion expression partner should not be too high. that is, although the large amount of fusion protein is synthesized, the actual amount of the target fusion-free protein could be very low if the fusion partner is too big. rpos is a relatively small protein among the stress-responsive proteins we found and was highly effective in enhancing the solubility of target proteins. rpos, stress responsive rna polymerase sigma factor is a well-known universal stress regulator controlling many proteins under various stress conditions, e.g. the onset of the stationary phase, and carbon starvation. rpos was previously reported to be induced by osmotic stress and heat shock. we also observed that the rpos expression level increased 3- to 5-fold in response to heat shock. muffler et al. reported that the duration of stability of rpos in response to heat shock stress is maintained by the direct- or indirect-binding of dnak. bound dnak appears to assist the effective folding of rpos and also protect rpos against the action of clpp, a protease that degrades rpos. from this point of view, rpos may serve as a solubility enhancer in e. coli cytoplasm, when used as a fusion expression partner upon the expression of aggregation-prone heterologous proteins. a gene name, accession number, and protein name were obtained from expasy proteomics server. b theoretical values of pi and mw were calculated using "compute pi/mw tool". c experimental values of pi and mw were estimated through 2-de gel image analysis in the present study. d sequence coverage and score values were calculated using "aldente: peptide mass fingerprinting tool". expression of aggregation-prone heterologous proteins using rpos as fusion partner we used rpos as an n-terminus fusion expression partner for the synthesis of numerous heterologous proteins. stenström et al. reported that the codon following an aug start triplet significantly affects gene expression in e. coli, and the second codon starting with a is most advantageous to achieve an enhanced expression level. the second codon of rpos is agt that seems to be favorable second codon based on the report of stenström et al.. as shown in figure 3b and table all heterologous proteins expressed directly without rpos fusion formed insoluble inclusion bodies resulting in nearly negligible solubility. compared to these results, it seems surprising that when the same heterologous proteins were expressed with the fusion of rpos, the solubility of these foreign proteins dramatically increased, thereby indicating that e. coli rpos is a highly effective solubility enhancer for aggregation-prone heterologous proteins. table compares the effect of rpos- and gst fusion on the solubility enhancement for heterologous proteins. in the fusion expression of cut, gad448– and mpins, the effect of rpos fusion was much higher, whereas gst fusion was significantly more effective in the expression of aid, nacht, and hftn-l. this result indicates that there are no universal helpers to solve all protein solubility issues. table also shows that the results of direct- and fusion expression of heterologous proteins are highly reproducible. * the solubility was defined as the fraction of the soluble recombinant protein compared to the synthesized total recombinant protein. average and standard deviation values were calculated based on the results of repeated triplicate experiments. moreover, the plasmid vector for the expression of polyhistidine-tagged fusion mutant of g-csf was constructed to purify fusion-free g-csf. after 6::rpos::::g-csf was bound onto the probond resin column, the enterokinase proteolysis was carried out in a batch mode, and subsequently the digested product was collected and centrifuged. sds-page and western blot analyses of the supernatant show that the recombinant g-csf was easily released from the rpos-fusion protein and was present in the form of soluble protein in the supernatant. bioactivity of the recombinant fusion mutant of cutinase, rpos::cut cutinase has been used as a lipolytic enzyme in the composition of laundry and dishwashing detergents to more efficiently remove immobilized fats. in addition, the oleochemistry industries, and pollutant degradation represent other potential uses of cutinase. in recent years, the esterification and transesterification properties of cutinase have been intensively exploited and could be applied usefully in other chemical synthesis processes. because of these extensive potential applications, we were particularly interested in the production of a bioactive recombinant cutinase. thus, we cloned the cutinase gene from the genome of pseudomonas putida and expressed it in e. coli using the fusion of rpos. cutinase is known for its hydrolytic activity for a variety of esters ranging from soluble p-nitrophenyl esters to insoluble long-chain triglycerides. the hydrolytic activity of cutinase, especially on p-nitrophenyl esters of fatty acids, is extremely sensitive to fatty acid chain length. previously, lin et al. reported that microbial cutinase lacked a large hydrophobic surface around the active site, in contrast to other lipases and esterases. this structural characteristic of cutinase may be strongly related to high substrate-specificity, i.e. the extremely low activity on p-nitrophenyl ester of a long chain fatty acid such as p-nitrophenyl palmitate. we assayed the enzymatic activity of our fusion mutant of cutinase and demonstrated the same selective bioactivity as native cutinase to degrade p-nitrophenyl butyrate but not to degrade pnp. we also purified rpos::cut-his through ni2+ affinity chromatography and analyzed the purified rpos::cut-his using reversed phase hplc. as shown in figure 6b, rpos::cut-his was analyzed as a single peak, which seems to indicate that the crafted mutant molecules of cutinase have uniform and correctly folded conformation due probably to the help of fusion partner, e. coli rpos.  using 2-dimensional gel electrophoresis, we found that e. coli rpos, rna polymerase sigma factor was gdnhcl stress-responsive and highly effective as a strong solubility enhancer when used as fusion partner for the expression of aggregation-prone heterologous proteins in e. coli bl the results of these findings may, therefore, be useful in the production of other biologically active industrial enzymes, as successfully demonstrated by cutinase. 
rpos is highly effective as a strong solubility enhancer for aggregation-prone heterologous proteins when it is used as a fusion expression partner in an e. coli expression system. while under the stress condition the total number of soluble proteins decreased by about %, but a 6-fold increase in the level of rpos was observed, indicating that rpos is a stress-induced protein. the results of these findings may, therefore, be useful in the production of other biologically active industrial enzymes, as successfully demonstrated by cutinase. the most efficient method for enhancing solubility of recombinant proteins appears to use the fusion expression partners. as an n-terminus fusion expression partner, rpos increased significantly the solubility of many aggregation-prone heterologous proteins in e. coli cytoplasm, indicating that rpos is a very effective solubility enhancer for the synthesis of many recombinant proteins. although commercial fusion partners including maltose binding protein and glutathione-s-transferase have shown good performance in enhancing the solubility, they cannot be used for the proprietory production of commercially value-added proteins and likely cannot serve as universal helpers to solve all protein solubility and folding issues. we analyzed the escherichia coli proteome response to the exogenous stress of guanidine hydrochloride using 2-dimensional gel electrophoresis and found that rpos was significantly stress responsive. thus, novel fusion partners will continue to be developed through systematic investigations including proteome mining presented in this study. rpos was also well suited for the production of a biologically active fusion mutant of pseudomonas putida cutinase.
 the most efficient method for enhancing solubility of recombinant proteins appears to use the fusion expression partners. although commercial fusion partners including maltose binding protein and glutathione-s-transferase have shown good performance in enhancing the solubility, they cannot be used for the proprietory production of commercially value-added proteins and likely cannot serve as universal helpers to solve all protein solubility and folding issues. thus, novel fusion partners will continue to be developed through systematic investigations including proteome mining presented in this study.  we analyzed the escherichia coli proteome response to the exogenous stress of guanidine hydrochloride using 2-dimensional gel electrophoresis and found that rpos was significantly stress responsive. while under the stress condition the total number of soluble proteins decreased by about %, but a 6-fold increase in the level of rpos was observed, indicating that rpos is a stress-induced protein. as an n-terminus fusion expression partner, rpos increased significantly the solubility of many aggregation-prone heterologous proteins in e. coli cytoplasm, indicating that rpos is a very effective solubility enhancer for the synthesis of many recombinant proteins. rpos was also well suited for the production of a biologically active fusion mutant of pseudomonas putida cutinase.  rpos is highly effective as a strong solubility enhancer for aggregation-prone heterologous proteins when it is used as a fusion expression partner in an e. coli expression system. the results of these findings may, therefore, be useful in the production of other biologically active industrial enzymes, as successfully demonstrated by cutinase. 