Skip to content


  • Research article
  • Open Access

Solubility enhancement of aggregation-prone heterologous proteins by fusion expression using stress-responsive Escherichia coliprotein, RpoS

  • 1,
  • 1,
  • 1,
  • 1,
  • 1,
  • 1,
  • 2,
  • 1 and
  • 1Email author
Contributed equally
BMC Biotechnology20088:15

  • Received: 29 August 2007
  • Accepted: 19 February 2008
  • Published:



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 (RNA polymerase sigma factor) was significantly stress responsive. While under the stress condition the total number of soluble proteins decreased by about 7 %, 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.


  • Heterologous Protein
  • Fusion Partner
  • Guanidine Hydrochloride
  • Activation Induce Cytidine Deaminase
  • Fusion Mutant


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 2,078 full-length genes of Caenorhabditis elegans were expressed in E. coli cytoplasm, only 11% of genes yielded significant amounts of soluble material [1]. Several different approaches have been taken to resolve the solubility problem in the past and include (1) truncation of long multi-domain proteins into short and separate domains [2] ; (2) co-expression of molecular chaperones or foldases [3]; (3) enabled secretion to the periplasm where disulfide bonds can be properly formed with the help of an oxidative environment and Dsb protein families [4]; (4) co-expression of aminoacyl tRNA cognates to amino acids encoded by rare codons [5]; and more recently (5) use of a fusion expression partner [6, 7]. The most efficient method for enhancing solubility and folding efficiencies of recombinant proteins appears to be the latter (fusion expression partners) which includes maltose binding protein (MBP) [8], thioredoxin (Trx) [9], human ferritin heavy chains (hFTN-H) [10], and glutathione-S-transferase (GST) [11]. Although these fusion partners have shown good performance in enhancing the solubility and folding of some recombinant proteins [12], 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 BL21(DE3). As an N-terminus fusion expression partner, RpoS dramatically increased the solubility of the following heterologous proteins (human minipro-insulin (mp-INS), human epidermal growth factor (EGF), human prepro-ghrelin (ppGRN), human interleukin-2 (hIL-2), human activation induced cytidine deaminase (AID), human glutamate decarboxylase (GAD448–585), Pseudomonas putida cutinase (CUT), human ferritin light chain (hFTN-L), human granulocyte colony-stimulating factor (G-CSF), and cold autoinflammatory syndrome1 (NALP3) NACHT domain (NACHT)). 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 and discussion

Guanidine hydrochloride-induced proteome response of E. coliand finding of the aggregation-resistant protein, RpoS

Exogenous stresses including heat shock and GdnHCl [1315] 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 [1618] 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 (2-DE). Compared to non-stress condition, the total number of intracellular soluble proteins was reduced to 731 from 788, which indicates that many proteins aggregated under the stress condition induced by GdnHCl. What was surprising is that the RpoS expression level (i.e. protein spot intensity estimated through analysis of 2-DE gel images, Fig. 1. and Table 1) increased 6-fold compared to the non-stress situation. In addition to RpoS, we found several other proteins (Table 1), the expression level of which more than 1.9-fold increased in response to the stressor GdnHCl, including HSP60 chaperonin (57 kD), chaperone HtpG (71 kD), transcription elongation protein NusA (55 kD), DNA gyrase subunit A (97 kD), formate acetyltransferase 1 (85 kD), succinate dehydrogenase flavoprotein subunit (64 kD), 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 (38 kD) 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 [19], and carbon starvation [20]. RpoS was previously reported to be induced by osmotic stress [16] and heat shock [21]. We also observed that the RpoS expression level increased 3- to 5-fold in response to heat shock (data not shown). Muffler et al. [21] 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 [21]. 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.
Figure 1
Figure 1

E. coli proteome profiles under non-stress and GdnHCl-stress condition. (A) 2-DE gel image of E. coli proteome under the non-stress condition. (Arrow indicates RpoS spot under non-stress condition.) (B) 2-DE gel image of E. coli proteome under GdnHCl-stress condition (Arrow indicates RpoS spot under GdnHCl-stress condition). (Figure in a box presents relative spot intensities of RpoS, analyzed under non-stress and GdnHCl-stress conditions.)

Table 1

Result of proteome-wide finding of some aggregation-resistant E. coli proteins

Gene namea

Access No.a

Protein namea


Sequence coveraged


Fold change







RNA polymerase sigma factor rpoS








HSP60 chaperonin








Chaperone protein htpG








Transcription elongationprotein nusA








DNA gyrase subunit A








Formate acetyltransferase 1








Succinate dehydrogenase flavoprotein subunit






a Gene name, accession number, and protein name were obtained from ExPASy Proteomics Server [35].

b Theoretical values of pI and Mw were calculated using "Compute pI/Mw tool" [36].

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" [37].

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 [mp-INS, EGF, ppGRN, hIL-2, AID, GAD448–585, CUT, hFTN-L, G-CSF, and NACHT (Fig. 2 and Table 2 for expression system construction)]. Stenström et al. [22] reported that the codon following an AUG start triplet (+2 codon) 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 (encoding Ser) that seems to be favorable second codon based on the report of Stenström et al. [22]. As shown in Figure 3B and Table 3, 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 (Fig. 3A and Table 3), thereby indicating that E. coli RpoS is a highly effective solubility enhancer for aggregation-prone heterologous proteins. Table 3 compares the effect of RpoS- and GST fusion on the solubility enhancement for heterologous proteins. In the fusion expression of CUT, GAD448–585, 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 3 also shows that the results of direct- and fusion expression of heterologous proteins are highly reproducible.
Figure 2
Figure 2

The plasmid vector constructions for direct and fusion expression of heterologous proteins in E. coli. (A) Direct expression vector, (B) RpoS- and GST-fusion expression vector, (C) Hybrid vector for metal (Ni+2) affinity purification of RpoS-CUT-(His)6, (D) Hybrid vector for metal (Ni+2) affinity purification of (His)6-RpoS-D4K-G-CSF, followed by enterokinase cleavage.

Table 2

Primers used for the cloning of genes encoding various heterologous proteins

Heterologous proteins

Primer sequences


Direct expression

Fusion expression



cat atg ttt gtc aac caa cat

ctc gag ttt gtc aac caa cat



aag ctt tta gtt aca gta gtt c

aag ctt tta gtt aca gta gtt c



cat atg aac tct gac tcc gaa tgc

ctc gag aac tct gac tcc gaa tgc



aag ctt tta acg cag ttc cca cca

aag ctt tta acg cag ttc cca cca



cat atg ggc tcc agc ttc ctg

ctc gag ggc tcc agc ttc ctg



aag ctt tca ctt gtc ggc t

aag ctt tca ctt gtc ggc t



cat atg gca cct act tca agt

ctc gag gca cct act tca agt



aag ctt tta tca agt cag tgt

aag ctt tta tca agt cag tgt



cat atg gac agc ctc ttg atg aac

ctc gag gac agc ctc ttg atg aac



aag ctt tca taa caa aag tcc ca

aag ctt tca taa caa aag tcc ca



cat atg cgc cac gtt gat gt

ctc gag cgc cac gtt gat gt



atc gat tta taa atc ttg tcc

atc gat tta taa atc ttg tcc



cat atg gct ccc ctg ccg gat ac

ctc gag gct ccc ctg ccg gat ac



aag ctt tta aag ccc gcg gcg ct

aag ctt tta aag ccc gcg gcg ct


aag ctt atg atg gtg gtg atg atg tta aag ccc gcg gcg ct (for metal affinity purification)



cat atg agc tcc cag att cgt

ctc gag agc tcc cag att cgt



aag ctt tta gtc gtg ctt gag agt

aag ctt tta gtc gtg ctt gag agt



cat atg act cca ctc gga cct g

ctc gag acc ccc ctg ggc cct gcc


ctc gag gac gat gac gat aaa acc ccc ctg ggc cct gcc (for enterokinase digestion)



aag ctt tca tgg ctg tgc aag

aag ctt tca tgg ctg tgc aag



cat atg act gtg gtg ttc cag

ctc gag act gtg gtg ttc cag



aag ctt tca cag cag gta gta c

aag ctt tca cag cag gta gta c

Table 3

Solubility of the expressed recombinant proteins

Heterologous proteins

Solubility* of the expressed recombinant proteins (%)


RpoS-fusion expression

GST-fusion expression

Direct expression


87.4 ± 2.1

91.5 ± 1.5

4.6 ± 0.5


44.9 ± 2.7

26.2 ± 1.1

3.1 ± 0.2


18.1 ± 3.5

41.7 ± 1.5

8.7 ± 1.6


47.8 ± 2.1

87.6 ± 2.8

1.7 ± 0.4


36.4 ± 4.3

74.8 ± 2.2

8.4 ± 1.3


59.0 ± 1.8

7.8 ± 2.7

1.9 ± 0.5


60.1 ± 2.4

85.7 ± 1.7

1.3 ± 0.3


80.4 ± 0.9

92.9 ± 1.2

3.0 ± 0.9


89.2 ± 3.5

90.1 ± 2.3

7.6 ± 0.6


75.9 ± 2.8

13.3 ± 1.0

1.3 ± 0.4

* The solubility was defined as the fraction of the soluble recombinant protein compared to the synthesized total (soluble + insoluble) recombinant protein. Average and standard deviation values were calculated based on the results of repeated triplicate experiments.

Figure 3
Figure 3

Results of direct and fusion expression of heterologous proteins. SDS-PAGE analyses of the RpoS-fusion expressed proteins (A), directly (non-fusion) expressed proteins (B), and GST-fusion expressed proteins (C).

Moreover, the plasmid vector for the expression of polyhistidine-tagged fusion mutant of G-CSF [(His)6::RpoS::(D4K)::G-CSF] was constructed (Fig. 2) to purify fusion-free G-CSF. After (His)6::RpoS::(D4K)::G-CSF was bound onto the ProBond resin (Ni+2) 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 (Fig. 4).
Figure 4
Figure 4

Results of SDS-PAGE and Western blot analysis of RpoS-fusion and fusion-free G-CSF. -SDS-PAGE analysis (lane M-3): lane M, molecular markers; lane 1, supernatant of recombinant E. coli cell lysates containing recombinant (His)6-RpoS-D4K-G-CSF, which was loaded onto ProBond resin (Ni+2) column for metal affinity purification; lane 2, soluble fraction of enterokinase(EK)-digested product of (His)6-RpoS-D4K-G-CSF, containing RpoS and fusion-free G-CSF (indicated by an arrow); lane 3, purified soluble fusion-free G-SCF. -Western blot analysis (lane 4): result of immunoblotting analysis of purified soluble fusion-free G-SCF (loaded onto lane 3).

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 [23, 24]. In addition, the oleochemistry industries [25], and pollutant degradation [26, 27] 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 [28]. 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. [29] 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 (PNP).

We assayed the enzymatic activity of our fusion mutant of cutinase (RpoS::CUT) and demonstrated the same selective bioactivity as native cutinase to degrade p-nitrophenyl butyrate (PNB) but not to degrade PNP (Fig. 5A and 5B). We also purified RpoS::CUT-His6 (Fig. 6A) through Ni2+ affinity chromatography and analyzed the purified RpoS::CUT-His6 using reversed phase HPLC (Fig. 6B). As shown in Figure 6B, RpoS::CUT-His6 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.
Figure 5
Figure 5

Bioactivity of recombinant fusion mutant, RpoS::CUT. Assay results using cell-free supernatants from (A) E. coli BL21 (DE3) host and (B) recombinant E. coli BL21 (DE3) [pT7-RpoS-CUT] producing RpoS::CUT. Both PNB () and PNP () were used as substrates for the cutinase activity assay. (Concentrations: PNB = 6.6 mM; PNP = 6.6 mM).

Figure 6
Figure 6

Results of purified RpoS::CUT-6xHis analysis. (A) Results of SDS-PAGE analysis of RpoS::CUT-6xHis in soluble fraction of E. coli cell lysates (lane 1) and of purified RpoS::CUT-6xHis (lanes 2, 3). (M: molecular marker) (Arrow indicates recombinant RpoS::CUT-6xHis). (B) Result of reversed-phase HPLC analysis of the affinity-purified RpoS::CUT-6xHis. (Peaks in red rectangle and dotted circle correspond to the purified RpoS::CUT-6xHis and PBS buffer, respectively.)


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 BL21(DE3). The results of these findings may, therefore, be useful in the production of other biologically active industrial enzymes, as successfully demonstrated by cutinase.


Bacterial strain and plasmids

E. coli strain BL21(DE3) (F-ompT hsdSB(rB- mB-)) was selected under both non-stress and GdnHCl-stress conditions for 2-dimensional gel electrophoresis analysis. Through PCR amplification using appropriate primers (Table 2), the genes encoding mp-INS, EGF, ppGRN, hIL-2 [30], AID, GAD448–585 [31], CUT, hFTN-L [32], G-CSF, and NACHT were cloned using previously cloned heterologous genes, except for CUT gene that was cloned from chromosomal DNA of Pseudomonas putida (ATCC 53552). The gene clones of mp-INS, ppGRN, AID, G-CSF, and NACHT were kindly donated by other researchers, as acknowledged (see Acknowledgements). The EGF gene was cloned using pCMV6-XL vector (OriGene Technologies, USA). Each of the recombinant genes above and various fusion/hybrid genes [rpoS(or GST gene)::(each heterologous gene)] were inserted into the NdeI-HindIII site of the same plasmid pT7-7 to construct the expression vectors. All the heterologous genes above were fused directly to the RpoS gene (cloned from the chromosomal DNA of E. coli BL21(DE3)) or GST gene [cloned from the GST-fusion vector pET42a(+) (Novagen, USA)] without any linker sequence. Therefore, each expression vector has no enzymatic cleavage site between rpoS (or GST gene) and heterologous gene, except for the case of G-CSF. For the purification of fusion-free recombinant G-CSF, the D4K sequence for enterokinase digestion was inserted between RpoS and G-CSF genes to synthesize the polyhistidine-tagged fusion mutant of G-CSF, i.e. (His)6::RpoS::(D4K)::G-CSF. For the purification of RpoS::CUT, hexahistidine (His6) was added to the C-terminus of CUT by PCR (Fig. 2). For this, the sequence coding for N-RpoS::CUT-His6-C was inserted into the NdeI-HindIII site of plasmid pT7-7.

After complete DNA sequencing of all gel-purified hybrid plasmids, the E. coli strain BL21(DE3) (F- ompT hsdSB(rB- mB-)) was transformed with the hybrid plasmids, and ampicillin-resistant transformants were subsequently selected using LB-agar plates supplemented with ampicillin (100 mg/l).

Recombinant E. coliculture, gene expression, and recombinant protein purification

For shake flask experiments, 250 ml Erlenmeyer flasks containing 50 ml LB media and ampicillin at 100 mg/l of culture (37°C and 150 rpm) was used. When the culture turbidity (OD600) reached 0.5, gene expression was induced with the addition of IPTG (1 mM), and after a further 4 h of cultivation the all recombinant cells (80 mg wet cell mass) were harvested by centrifugation (13,000 rpm (MICRO17TR, Hanil Science Industrial, Korea) × 5 min) and cell pellets resuspended in 5 ml lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM EDTA). Cell disruption was achieved using a Branson Sonifier (Branson Ultrasonics Corp., Danbury, CT). The cell-free supernatant and insoluble protein aggregates were separated at 13,000 rpm (MICRO17TR, Hanil Science Industrial, Korea) for 10 min. The isolated inclusion bodies, if any, were washed twice with 1 % Triton X-100. Cell-free supernatants and the washed inclusion bodies were subjected to polyacrylamide (14%) gel electrophoresis (PAGE) analysis. Coomassie-stained protein bands were ultimately scanned, and the intensity of each recombinant protein band was estimated using densitometry (Duoscan T1200, Bio-Rad, Hercules, CA). The solubility of recombinant proteins was determined by analyzing the fraction of the soluble recombinant protein compared to the synthesized total (soluble + insoluble) recombinant protein. Average and standard deviation values of the solubility were calculated based on the results of repeated triplicate experiments.

The purification of recombinant G-SCF was accomplished using metal affinity chromatography. That is, polyhistidine-tagged fusion mutant of G-CSF [(His)6::RpoS::(D4K)::G-CSF] (Fig. 4) were loaded onto ProBond resin (Ni+2) column. Prior to sample loading, the resin was washed twice with 10 column volumes of binding buffer (50 mM potassium phosphate, 300 mM KCl, 20 mM imidazole, pH 7.0). Binding buffer contains 20 mM imidazole to minimize non-specific binding of untagged protein contaminants, and binding was carried out in a batch mode at 4°C. Afterwards the resin was washed twice with 5–8 ml Tris-HCl (10 mM Tris, pH 8.0) prior to enterokinase digestion step. The enterokinase digestion was carried out in a batch mode at 4°C for 10 h using 5-unit enterokinase (Invitrogen, CA, USA). Then, the proteolytic product was collected and centrifuged [3,000 rpm (MICRO17TR, Hanil Science Industrial, Korea) for 10 min], and the supernatant fraction was subjected to being analyzed by SDS-PAGE and western blotting.

Purification and HPLC analysis of cutinase fusion mutant

For the purification of RpoS::CUT-His6, the soluble fraction was separated after cell disruption by centrifugation (13,000 rpm (MICRO17TR, Hanil Science Industrial, Korea)) for 30 min. The cell-free supernatant containing RpoS::CUT-His6 was loaded onto the ProBond resin (Ni2+) column (Invitrogen) for affinity purification. Before the sample loading, the resin was washed twice with ten column volumes of binding buffer (pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole). Binding was carried out in a batch mode at 4°C. Afterwards, the resin was washed twice with 8 ml washing buffer (pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 50 mM imidazole) and eluted with elution buffer (pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole). The elution buffer in the eluted solution containing the purified RpoS::CUT-His6 was changed with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) using Amicon Ultra-4 centrifugal filter (Millipore, Ireland). Analysis of the purified RpoS::CUT-His6 was performed with high-performance liquid chromatography (HPLC) (LC-20A Prominence, Shimadzu Co. Ltd., Japan). A Shim-pack CLC-NH2 column (60 × 150 mm, Shimadzu Co. Ltd., Japan) was equilibrated with 10 % acetonitrile containing 0.1 % trifluoroacetic acid at a flow rate of 1 ml/min. The sample was loaded onto the column and eluted with 75 % acetonitrile. The elution profile was monitored at 215 nm.

Sample preparation for proteome analysis and 2-dimensioanl gel electrophoresis

Flask culture conditions were identical to those for recombinant gene expressions. Cells were grown at 37°C and then 100 mM GdnHCl was added for the GdnHCl-induced proteome response when culture turbidity (OD600) reached 0.5 in the LB media. After further 3 h cultivation, the cells were harvested by centrifugation at 6,000 rpm (MEGA17R, Hanil Science Industrial, Korea)for 15 min (4°C) and then washed twice with 40 mM Tris buffer (pH 8.0). Cell pellets were resuspended in 500 μl of lysis buffer (8 M urea, 4% (w/v) CHAPS, 40 mM Tris, protease inhibitor cocktail; Roche Diagnostics GmbH, Mannheim, Germany) and disrupted by sonication. After sonication (Branson Sonifier 450, USA), the cell debris and the aggregated proteins were removed by centrifugation at 12,000 rpm (MICRO17TR, Hanil Science Industrial, Korea) for 60 min (4°C), and only soluble proteins were obtained. 2-dimensional gel electrophoresis was performed as described previously Kim et al. [33]. Stained gels were scanned using a UMAX powerlook 1100 scanner and Image Master software v 4.01 (Amersham Biosciences, Uppsala, Sweden) was used for gel image analysis, including quantification of spot intensities performed on volume bases (i.e. values calculated from the integration of spot optical intensity over spot area).

MALDI-TOF-MS analysis and protein identification

Samples for MALDI-TOF MS analysis were prepared through the extraction from silver-stained protein spots according to the previous protocol [33]. Enzymatic digestion was performed with 10 mg/ml of sequencing grade modified trypsin (Promega, WI, USA) in 25 nM ammonium bicarbonate (pH 8.0) for overnight at 37°C in a stationary incubator. The trypsin-digested protein spots was analyzed using a MALDI-TOF-MS system (Voyager DE-STR, PE Biosystem, Framingham, MA, U.S.A.) by the Korea Basic Science Institute (Seoul, Republic of Korea), and peptide mass fingerprinting for protein identification was performed using MS-Fit [34]. Spectra were calibrated using a matrix and tryptic autodigestion ion peaks as internal standards. Peptide mass fingerprints were analyzed using the MS-Fit [34]. The identification of a protein with respect to theoretical parameters (pI, molecular mass, etc.) was accepted if the peptide mass matched within a mass tolerance of 10 ppm.

Bioactivity assay

A hydrolytic enzyme activity of the recombinant cutinase fusion mutant was assessed as described below. The hydrolysis reactions occurred in 96-well microplates at 37°C for 15 min where each well contained 200 μl enzyme/substrate solution comprised of 106.7 μl phosphate buffer (0.1 M, pH 8.0), 13.3 μl Triton X-100 solution (4 g/l), cutinase solution. The reaction was initiated by adding 66.7 μl of substrate reagent solution to each well in the 96-well microplate. Absorbance changes (ΔA415nm per min) were measured using a Bio-Rad microplate reader (Tecan, Austria), and solution A with no enzyme solution was used as blank. From the absorbance changes measured at each column, an average absorbance for a specific reaction condition could then be calculated.




2-dimensional gel electrophoresis


human activation induced cytidine deaminase


Pseudomonas putida cutinase


human epidermal growth factor


human granulocyte colony-stimulating factor


Guanidine hydrochloride




human ferritin heavy chain


human ferritin light chain


human interleukin-2


maltose binding protein




human cold autoinflammatory syndrome 1 protein (NALP3) NACHT domain


p-nitrophenyl butyrate


p-nitrophenyl phamitate


human prepro-ghrelin





We thank Professor Hang Chul Shin at Soongsil University for kindly providing the gene clones of mpINS and G-CSF. We also appreciate Professors Won Tae Lee and Hyun Soo Cho at Yonsei University for the kind donation of ppGRN, AID, and NACHT clones, respectively. This study was supported by the National Research Laboratory Project of the Ministry of Science and Technology (grant no. ROA-2007-000-20084-0) of the Republic of Korea. This work was also supported by the Korea Health 21 R&D Project of the Ministry of Health & Welfare (grant no. A050750), by grant 031-061-029 of the Ecotechnopia 21 project of the Ministry of Environment, and by the Second Brain Korea 21 Project. Further supports from the Korea Science and Engineering Foundation (grant no. R01-2005-000-10355-0) and the Korea Research Foundation (grant no. KRF-2004-041-D00180) are also appreciated.

Authors’ Affiliations

Department of Chemical and Biological Engineering, Korea University, Anam-Dong 5-1, Sungbuk-Ku, Seoul, 136-713, South Korea
Department of Chemical Engineering, Sungkyunkwan University, Suwon, South Korea


  1. Finley JB, Qui SH, Luan CH, Luo M: Structural genomics for Caenorhabditis elegans : high throughput protein expression analysis. Protein Expr Purif. 2004, 34: 49-55. 10.1016/j.pep.2003.11.026.View ArticleGoogle Scholar
  2. Himanen JP, Rajashankar KR, Lackmann M, Cowan CA, Henkemeyer M, Nikolov DB: Crystal structure of an Eph receptor-ephrin complex. Nature. 2001, 414: 933-938. 10.1038/414933a.View ArticleGoogle Scholar
  3. de Marco A, Deuerling E, Mogk A, Tomoyasu T, Bukau B: Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli. BMC Biotechnol. 2007, 7: 32-10.1186/1472-6750-7-32.View ArticleGoogle Scholar
  4. Qiu J, Swartz JR, Georgiou G: Expression of active human tissue-type plasminogen activator in Escherichia coli. Appl Environ Microbiol. 1998, 64: 4891-4896.Google Scholar
  5. Tan WS, Dyson MR, Murray K: Hepatitis B virus core antigen: enhancement of its production in Escherichia coli, and interaction of the core particles with the viral surface antigen. Biol Chem. 2003, 384: 363-371. 10.1515/BC.2003.042.View ArticleGoogle Scholar
  6. Braun P, Hu Y, Shen B, Halleck A, Koundinya M, Harlow E, LaBaer J: Proteome-scale purification of human proteins from bacteria. Proc Natl Acad Sci USA. 2002, 99: 2654-2659. 10.1073/pnas.042684199.View ArticleGoogle Scholar
  7. Hammarstrom M, Hellgren N, van Den Berg S, Berglund H, Hard T: Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci. 2002, 11: 313-321. 10.1110/ps.22102.View ArticleGoogle Scholar
  8. Bedouelle H, Duplay P: Production in Escherichia coli and one-step purification of bifunctional hybrid proteins which bind maltose. Export of the Klenow polymerase into the periplasmic space. Eur J Biochem. 1998, 171: 541-549. 10.1111/j.1432-1033.1988.tb13823.x.View ArticleGoogle Scholar
  9. LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM: A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (N. Y.). 1993, 11: 187-193. 10.1038/nbt0293-187.View ArticleGoogle Scholar
  10. Ahn JY, Choi H, Kim YH, Han KY, Park JS, Han SS, Lee J: Heterologous gene expression using self-assembled supra-molecules with high affinity for HSP70 chaperone. Nucleic Acids Res. 2005, 33: 3751-3762. 10.1093/nar/gki692.View ArticleGoogle Scholar
  11. Smith DB, Johnson KS: Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase. Gene. 1988, 67: 31-40. 10.1016/0378-1119(88)90005-4.View ArticleGoogle Scholar
  12. Hammarstrom M, Woestenenk EA, Hellgren N, Hard T, Berglund H: Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein. J Struct Funct Genomics. 2006, 7: 1-14. 10.1007/s10969-005-9003-7.View ArticleGoogle Scholar
  13. Sakane I, Hongo K, Motojima F, Murayama S, Mizobata T, Kawata Y: Structural stability of covalently linked GroES heptamer: Advantages in the formation of oligomeric structure. J Mol Biol. 2007, 367: 1171-1185. 10.1016/j.jmb.2007.01.037.View ArticleGoogle Scholar
  14. Eaglestone SS, Ruddock LW, Cox BS, Tuite MF: Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI(+)] of Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 2000, 97: 240-244. 10.1073/pnas.97.1.240.View ArticleGoogle Scholar
  15. Park JS, Ahn JY, Lee SH, Lee H, Han KY, Seo HS, Ahn KY, Min BH, Sim SJ, Choi IS, Kim YH, Lee J: Enhanced stability of heterologous proteins by supramolecular self-assembly. Appl Microbiol Biotechnol. 2007, 75: 347-355. 10.1007/s00253-006-0826-3.View ArticleGoogle Scholar
  16. Weber A, Kögl SA, Jung K: Time-dependent proteome alterations under osmotic stress during aerobic and anaerobic growth in Escherichia col. J Bacteriol. 2006, 188: 7165-7175. 10.1128/JB.00508-06.View ArticleGoogle Scholar
  17. Chen YC, Chen LA, Chen SJ, Chang MC, Chen TL: A modified osmotic shock for periplasmic release of a recombinant creatinase from Escherichia coli. Biochem Eng J. 2004, 19: 211-215. 10.1016/j.bej.2004.03.001.View ArticleGoogle Scholar
  18. Ewis HE, Lu CD: Osmotic shock: A mechanosensitive channel blocker can prevent release of cytoplasmic but not periplasmic proteins. FEMS Microbial Lett. 2005, 253: 295-301. 10.1016/j.femsle.2005.09.046.View ArticleGoogle Scholar
  19. Hirsch M, Elliott T: Stationary-phase regulation of RpoS translation in Escherichia coli. J Bacteriol. 2005, 187: 7204-7213. 10.1128/JB.187.21.7204-7213.2005.View ArticleGoogle Scholar
  20. Wei B, Shin S, LaPorte D, Wolfe AJ, Romeo T: Global regulatory mutations in csr A and rpoS cause severe central carbon stress in Escherichia coli in the presence of acetate. J Bacteriol. 2000, 182: 1632-1640. 10.1128/JB.182.6.1632-1640.2000.View ArticleGoogle Scholar
  21. Muffler A, Barth M, Marschall C, Hengge-Aronis R: Heat shock regulation of sigmaS turnover: a role for DnaK and relationship between stress responses mediated by sigmaS and sigma32 in Escherichia coli. J Bacteriol. 1997, 179: 445-452.Google Scholar
  22. Stenström CM, Jin H, Major LL, Tate WP, Isaksson LA: Codon bias at the 3'-side of the initiation codon is correlated with translation initiation efficiency in Escherichia col. Gene. 2001, 263: 273-284. 10.1016/S0378-1119(00)00550-3.View ArticleGoogle Scholar
  23. Flipsen JAC, Appel ACM, Van der Hijden HTWM, Verrips CT: Mechanism of removal of immobilized triacylglycerol by lipolytic enzymes in a sequential launcry wash process. Enzyme Microb Technol. 1998, 23: 274-280. 10.1016/S0141-0229(98)00050-7.View ArticleGoogle Scholar
  24. Murphy CA, Cameron JA, Huang SJ, Vinopal RT: Fusarium polycaprolactone depolymerase is cutinase. Appl Environ Microbiol. 1996, 62: 456-460.Google Scholar
  25. Cristina MLC, Maria RAB, Joaquim MSC: Cutinase: From molecular level to bioprocess development. Biotechnol Bioeng. 1999, 66: 17-34. 10.1002/(SICI)1097-0290(1999)66:1<17::AID-BIT2>3.0.CO;2-F.View ArticleGoogle Scholar
  26. Kim YH, Lee J, Ahn JY, Gu MB, Moon SH: Enhanced degradation of an endocrine-disrupting chemical, butyl benzyl phthalate, by Fusarium oxysporum f. sp. pisi cutinase. Appl Environ Microbiol. 2002, 68: 4684-4688. 10.1128/AEM.68.9.4684-4688.2002.View ArticleGoogle Scholar
  27. Martinez C, De Geus P, Lauwereys M, Matthysens G, Cambillau C: Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent. Nature. 1992, 356: 615-618. 10.1038/356615a0.View ArticleGoogle Scholar
  28. Gerard HC, Fett WF, Osman SF, Moreau RA: Evaluation of cutinase activity of various industrial lipases. Biotechnol Appl Biochem. 1993, 17: 181-189.Google Scholar
  29. Lin TS, Kolattukudy PE: Structural studies on cutinase, a glycoprotein containing novel amino acids and glucuronic acid amide at the N terminus. Eur J Biochem. 1980, 106: 341-351. 10.1111/j.1432-1033.1980.tb04580.x.View ArticleGoogle Scholar
  30. Kim DY, Lee J, Saraswat V, Park YH: Glucagon-induced self-association of recombinant proteins in Escherichia coli and affinity purification using a fragment of glucagon receptor. Biotechnol Bioeng. 2000, 69: 418-428. 10.1002/1097-0290(20000820)69:4<418::AID-BIT8>3.0.CO;2-C.View ArticleGoogle Scholar
  31. Choi H, Ahn JY, Sim SJ, Lee J: Glutamate decarboxylase -derived IDDM autoantigens displayed on self-assembled protein nanoparticles. Biochem Biophys Res Commun. 2005, 327: 604-608. 10.1016/j.bbrc.2004.12.046.View ArticleGoogle Scholar
  32. Kim SW, Kim YH, Lee J: Thermal stability of human ferritin: concentration dependence and enhanced stability of an N-terminal fusion mutant. Biochem Biophys Res Commun. 2001, 289: 125-129. 10.1006/bbrc.2001.5931.View ArticleGoogle Scholar
  33. Kim YH, Han KY, Lee K, Heo JH, Kang HY, Lee J: Comparative proteome analysis of Hansenula polymorpha DL1 and A16. Proteomics. 2004, 4: 2005-2013. 10.1002/pmic.200300739.View ArticleGoogle Scholar
  34. MS Fit. []
  35. ExPASy Proteomics Server. []
  36. Compute pI/Mw tool. []
  37. Aldente: peptide mass fingerprinting tool. []