PEDS Advance Access originally published online on October 30, 2007
Protein Engineering Design and Selection 2007 20(11):543-549; doi:10.1093/protein/gzm055
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Solubilization of aggregation-prone heterologous proteins by covalent fusion of stress-responsive Escherichia coli protein, SlyD
Department of Chemical and Biological Engineering, Korea University, Anam-Dong 5-1, Sungbuk-Ku, Seoul 136-713, South Korea
2 To whom correspondence should be addressed. E-mail: leejw{at}korea.ac.kr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionFundingAcknowledgementsReferences |
|---|
The proteome profile of Escherichia coli BL21(DE3) generated in response to heat shock stress was analyzed by two-dimensional electrophoresis (2-DE), wherein we identified a FKBP-type peptidyl–prolyl cis–trans isomerse (PPIases), SlyD, as a stress-responsive (i.e. aggregation-resistant) protein. Even under an imposed severe stress condition where 29 out of 858 soluble proteins were totally eliminated and the synthesis levels of 171 proteins decreased over 5-fold, a 3.37-fold increase induced by heat shock treatment was observed in the synthesis level of SlyD compared with a non-stress condition. As a fusion partner, as well as solubility enhancer, SlyD facilitated folding and significantly increased the solubility of many aggregation-prone heterologous proteins in E. coli cytoplasm. SlyD was very effective in sequestering interactive surfaces of heterologous proteins associated with non-specific protein–protein interactions and the formation of inclusion bodies, most likely as a result of intrinsic folding efficiencies and/or chaperone-like activities. SlyD was also shown to be suitable for the production of a biologically active fusion mutant of Pseudomonas putida cutinase that is of considerable biotechnological and commercial interest.
Keywords: Escherichia coli BL21(DE3)/proteome/SlyD/solubility enhancer/stress response
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingAcknowledgementsReferences |
|---|
The enteric bacterium Escherichia coli is generally the first choice as a production host for the commercial cellular manufacture of proteins to be used for industrial and medical purposes (De Bernardez Clark, 1998
; Eiteman and Altman, 2006). Owing to a high production yield, low manufacturing cost and a well established expression system, a wide variety of recombinant proteins have been produced using E. coli expression system (Baneyx and Mujacic, 2004; Davis et al., 1999; Eiteman and Altman, 2006). To be biologically active, however, recombinant proteins need fold into their native three-dimensional conformation (Kaiser et al., 2006). In a crowded E. coli cytosol, recombinant proteins, even those of bacterial origin, tend to fail in rapidly attaining a precise three-dimensional conformation and interact with non-specific hydrophobic patches leading to protein aggregation (De Bernardez Clark, 1998; Lilie et al., 1998; Baneyx and Mujacic, 2004).
To eliminate this production obstacle and enhance soluble recombinant protein expression, various strategies have been suggested and include identifying suitable alternative hosts, N-terminus and/or C-terminus truncated mutant formations, chaperone co-expression and fusion protein technologies (Sorensen and Mortensen, 2005). Currently, Shistosoma japonicum glutathione S-transferase (GST), E. coli maltose-binding protein (MBP), E. coli N utilization substance A (NusA) and E. coli thioredoxin are the most extensively examined fusion partners for overcoming inclusion body formation and simultaneously increasing expression levels (Davis et al., 1999; LaVallie et al., 1993; Smith and Johnson, 1988; Nallamsetty and Waugh, 2006).
In the present study, we have identified an aggregation-resistant protein, SlyD, through a proteome-wide analysis of stress responsive proteomes of E. coli. Wondrous histidine-rich protein (WHP), SlyD was originally discovered as a contaminant during immobilized metal affinity chromatography (IMAC) used to purify recombinant proteins (Wülfing et al., 1994; Mitterauer et al., 1999; Scholz et al., 2006). In addition to the peptidyl–proline cis–trans isomerase activity involved in accelerating a rate-limiting intermediate formation (Mitterauer et al., 1999), a truncated form of SlyD also exhibited chaperone-like properties (Scholz et al., 2006) and the fusion mutants with SlyD tended to demonstrate high solubility and immunoactivity (Scholz et al., 2005). The efficacy of SlyD as a fusion partner was demonstrated during the synthesis of several aggregation-prone proteins in E. coli cytoplasm. SlyD facilitated folding and dramatically increased solubility of the various heterologous proteins. SlyD was also well suited for the production of biologically active fusion mutants of a heterologous bacterial cutinase that is of significant biotechnology and commercial interest.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingAcknowledgementsReferences |
|---|
Bacterial strain and plasmids
Escherichia coli strain BL21(DE3) [F– ompT hsdSB(rB–mB–)] was selected under both non-stress and heat shock stress conditions for the two-dimensional electrophoresis (2-DE) analysis. After PCR amplification using appropriate primers, each of the recombinant genes and various fusion mutants were inserted into the NdeI–HindIII site of plasmid pT7-7 to construct the fusion expression vector (see Fig. 1 and Table I). After complete DNA sequencing of all gel-purified plasmid vectors, the E. coli strain BL21(DE3) was transformed with the plasmid expression vectors, and ampicillin-resistant transformants were subsequently selected using LB-agar plates supplemented with ampicillin (100 mg/l).
|
|
Recombinant E. coli culture, gene expression and recombinant protein purification
For shake flask experiments (250 ml Erlenmeyer flasks), LB media containing ampicillin at 100 mg/l of culture (37°C) was used. When the culture turbidity (OD600nm) reached 0.5, gene expression was induced with the addition of IPTG (1 mM). After a further 3–4 h of cultivation, the recombinant cells were harvested by centrifugation (17 000 g x 5 min), and the cell pellets were resuspended in 5 ml distilled water. Cell disruption was achieved using a Branson Sonifier (Branson Ultrasonics Corp., Danbury, CT, USA). The cell-free supernatant and insoluble protein aggregates were separated at 17 000 g 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 analysis. Coomassie-stained protein bands were ultimately scanned and analyzed by densitometer (Duoscan T1200, Bio-Rad, Hercules, CA, USA).
The purification of recombinant granulocyte colony-stimulating factor (G-CSF) was accomplished using metal affinity chromatography. That is, polyhistidine-tagged fusion mutants of G-CSF [(His)6::SlyD::(D4K)::G-CSF] (Fig. 1) 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, centrifuged (17 000 g for 10 min), and analyzed by SDS–PAGE.
Sample preparation for proteome analysis and 2-DE
Flask culture conditions were same as those for recombinant gene expressions. Cells were grown at 37°C and then cells for the heat shock response analysis were shifted to 48°C when the culture turbidity (OD600nm) reached 0.5 (LB media was used). After a further 3 h cultivation, the cells were harvested by centrifugation at 3620 g 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 [8M Urea, 4% (w/v) CHAPS, 40 mM Tris and Protease inhibitor cocktail; Roche Diagnostics GmbH, Mannheim, Germany] and disrupted by sonication. After sonication, the cell debris and the aggregated proteins were removed by centrifugation at 14 480 g for 60 min (4°C). The protein samples were resuspended in rehydration solution [8M Urea, 0.5% (V/V) Triton X-100, 0.005% Orange G, 1% w/v DTT and 1% v/v carrier ampholyte, pH3-10; final volume, 320 µl]. Urea, CHAPS, Tris, DTT, orange G, Triton X-100 and SDS were purchased from Sigma (St Louis, MO, USA).
The first dimension of 2-DE was performed on an IPGphor Electrophoresis System (Amersham Bioscience, Uppsala, Sweden) at 20°C. Linear IPG (immobilized pH gradient) gel strips, pH 4–7, were rehydrated for 12 h. Isoelectic focusing of rehydrated protein samples (45 µg) was performed at 500 V for 2 h, at 1000 V for 30 min, at 2000 V for 30 min, at 4000 V for 30 min and finally maintained at 8000 V until 70 000 V h was achieved. For the second dimension, the IPG gel strips were equilibrated for 15 min in equilibration solution [50 mM Tris/HCl, pH 8.8/6 M Urea, 30% (v/v) glycerol/2% (w/v) SDS and trace element Bromophenol Blue] in 1% dithiothreitol for 15 min followed by 2.5% (w/v) iodoacetamide for 15 min. The second-dimensional separation was performed using a PROTEAN II Xi cell system (Bio-Rad) in a cold chamber at 4°C on 12.5% polyacrylamide gels. SDS/PAGE was performed at 30 mA/gel for 12 h. The silver stained gels were scanned using a UMAX powerlook 1100 scanner. Image Master Software v 4.01 (Amersham Biosciences) was used for gel image analyze, including quantification of spot intensities that is performed on a volume basis (i.e. values were calculated from the integration of spot optical intensity over spot area). Under non-stress or stress condition, three independent bacterial cultures were grown. Using the three bacterial culture samples harvested at a fixed time point, the three 2-DE gels were prepared. By the Image Master software, an average gel image was constructed from the three 2-DE gels and used for comparative image analysis.
MALDI-TOF-MS analysis and protein identification
Samples for the MALDI-TOF mass spectrometry analysis were extracted from silver stained spots according to the previous protocol (Farzin et al., 1999). Enzymatic digestions were performed overnight at 37°C in stationary incubator using 10–15 µg/ml of sequencing grade modified trypsin (Proma, WI, USA) in 25 mM ammonium bicarbonate (pH 8.0). In-gel-digested peptide fragments were extracted from gel pieces using solution prepared by adding of 5% v/v trifluoroacetic acid to 50% v/v acetonitrile followed by vortexing for 1 h. After three times repeated, solute materials including peptide fragments were dried down by vacuum centrifugation. Ziptip column (Millipore, Bedford, USA) in which C18 resin is fixed at the end of the tip was used to eliminate impurities of samples. The peptide solution was prepared with an equal volume of saturated 
-cynao-4-hydroxy-cinnamic acid solution in 50% ACN/0.1% TFA on a sample plate of MALDI-TOF mass spectrometer. Protein analyzes were performed by the Korea Basic Science Institute (Seoul, Korea) using MALDI-TOF mass spectrometry system (Voyager DE-STR, PE Biosystem, Framingham, MA, USA). Spectra were calibrated using a matrix and tryptic autodigestion ion peaks as internal standards. Peptide mass fingerprints were analyzed using the MS-Fit (http://prospector.ucsf.edu/). The identification of a protein with respective theoretical parameters (pI, molecular mass) was accepted if the peptide mass matched with a mass tolerance within 10 ppm.
The circular dichroism (CD) spectrum of the purified G-CSF (74 mg/l) was taken in a JASCO J-710 spectropolarimeter (Korea Basic Science Center, Ochang, Korea) at room temperature.
The 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 phosphate buffer 106.7 µl (0.1 M, pH 8.0), Triton X-100 solution 13.3 µl (4 g/l), enzyme solution 13.3 µl and substrate (PNB or PNP 6.6 mM) reagent solution 66.7 µl. The reaction was initiated by adding 66.7 µl of substrate reagent solution to each well in the 96-well microplate. Absorbance changes (
OD415nm) were measured every 6 s using a Bio-Rad microplate reader (Tecan, Austria), and enzyme-free reagent solution above was used as blank. The eight wells in each of 12 columns of the 96-well microplate represented the same reaction condition and contained equal contents of enzyme and substrate. From the absorbance changes measured at each column, an average absorbance for a specific reaction condition was then calculated. Fungal cutinase (Novozyme) was used as standard (positive control). Cutinase activity is defined as initial maximum rate of PNB hydrolysis (PNB µmol/ml/min) that is estimated based on the pre-determined linear correlation between OD415nm and concentration of hydrolysis product (p-nitrophenol).
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingAcknowledgementsReferences |
|---|
Proteome responses to heat shock stressand stress-responsive protein mining
The misfolding and aggregation of proteins are major damaging consequences of heat shock stress. Our laboratory was interested in identifying any E. coli protein(s) that could exist in their soluble and native form under a stressful environmental condition. It seemed reasonable to presume that such an aggregation-resistant protein(s) would have the intrinsic capability of folding more efficiently thereby attaining a native structure when compared with proteins that aggregate under the same stress conditions. We investigated changes in the E. coli proteome profile based on 2-DE analysis after heat shock stress to a growing bacterial culture. An averaged gel image was constructed by three 2-DE gels showing high degree of reproducibility that were obtained from independent experiments. The average gel image was used for the comparative image analysis. As shown in Fig. 2, the cells under the condition of heat shock continued to grow up to the final culture OD600nm of 3.19 after temperature shift (when OD600nm of the bacterial culture reached 0.5), whereas the culture OD600nm of non-stressed cells reached 5.08. Compared with a non-stress control environment, 29 proteins out of the 858 soluble proteins that were present in the non-stress proteome were totally aggregated or eliminated, and the synthesis levels of 171 host proteins significantly (over 5-fold) decreased under the heat shock condition. Although the synthesis level of many E. coli proteins was reduced, we identified several proteins, the expression level of which significantly increased even under the stress condition of heat shock. A nearly 3.4-fold increase in expression level (i.e. the protein spot intensity estimated through the comparative analysis of 2-DE gel image) of SlyD was detected despite the imposed heat shock stress (Fig. 3, Table II).
|
|
|
PPIases that accelerate cis–trans-isomerization of polypeptide chains are classified into three families: parvulins; FK506-binding proteins (FKBP) which include the putative subfamily of trigger factors and cyclophilins (Hottenrott et al., 1997
). Members of all three PPIase families have been found in E. coli, and SlyD is a putative folding helper protein and a member of the FKBP type PPIase (Hottenrott et al., 1997). SlyD consists of an N-terminal peptidyl–prolyl isomerase domain of the FKBP type and an additional C-terminal domain with histidine-rich metal-binding residues (Hottenrott et al., 1997; Zhang et al., 2005). Scholz et al. (2005) recently reported on an in vitro study wherein SlyD, which behaves as a chaperone in stabilizing a hydrophobic viral protein, could also aid the refolding of inclusion bodies and solubilize retroviral envelope proteins that even MBP fusion protein showed a tendency to aggregate. As presented in Fig. 3, the synthesis level of SlyD increased significantly in response to heat shock stress, which suggests that SlyD is a stress-responsive or aggregation-resistant protein possibly as a result of an intrinsic high folding efficiency. On the basis of predictive evaluations of potential solubility of overexpressed fusion partner candidates, the cold shock protein NusA was used as a fusion expression partner in an attempt to increase cytoplasmic solubility of aggregation-prone human interleukin-3 (predicted solubility of overexpressed NusA at 95%, corresponded to the highest predicted value) (Davis et al. 1999). The same recombinant protein solubility prediction (http://www.biotech.ou.edu) showed 98% solubility of SlyD, which exceeds the solubility of any fusion partner candidates previously reported (Davis et al., 1999). Hence, it is strongly suggested that SlyD may serve as a highly effective solubility enhancer in the bacterial cytoplasm when used as a fusion partner upon overexpression of aggregation-prone heterologous proteins. Expression of aggregation-prone heterologous proteins using the SlyD protein as fusion expression partner
We used SlyD as an N-terminus fusion expression partner and cis-acting folding enhancer upon the synthesis of the following heterologous proteins: [minipro-insulin(mp-INS), human epidermal growth factor (EGF), human prepro-ghrelin (ppGRN), human interleukin-2 (hIL-2), human activation induced cytidine deaminase (AID), deletion mutant of human glutamate decarboxylase (GAD448–585), human ferritin light chain (hFTN-L), human G-CSF, human cold autoinflammatory syndrome 1 protein (NALP3) NACHT domain (NACHT) and Pseudomonas putida cutinase (CUT)]. We initially attempted in vivo synthesis of the hybrid protein, NH2-[SlyD]-[heterologous protein]-COOH in E. coli cytoplasm. All heterologous proteins mentioned above aggregated and formed inclusion bodies when expressed directly without an N-terminus fusion tag, and as a result, solubility’s were almost negligible (Fig. 4).
|
It was observed, however, that cytoplasmic solubility of these foreign proteins increased dramatically when they were expressed with the N-terminus fusion of SlyD (Fig. 4), indicating that the fusion expression partner, E. coli SlyD, was a highly effective solubility enhancer. On the other hand, for a different heat shock up-regulated protein (FlgL), the predicted solubility was only 52.4% using the software cited by Davis et al. (1999)
, and in practice failed to promote heterologous protein solubility’s (data not shown). In vivo inclusion body formation would presumably be a situation of protein overexpression in a heterologous environment, where nascent polypeptide chains are continuously synthesized and may aggregate because of non-specific protein–protein interactions among partially folded intermediates of the recombinant or various host proteins, or a combination of the two sources within the cytoplasm. We speculate that SlyD may be very effective in shielding potentially interactive surfaces of heterologous proteins associated with non-specific protein–protein interactions which lead to the formation of inclusion bodies. In the E. coli cytoplasm, both SlyD and a trigger factor have peptidyl–prolyl isomerase activities and chaperone functions (Hottenrott et al., 1997; Scholz et al., 2006). During protein synthesis, it may be that a trigger factor binds first to both a nascent polypeptide and the ribosome, and the void formed by ribosome and trigger factor might serve as a cage-like compartment to sequester hydrophobic patches of target proteins and protect nascent proteins from degradation and interaction leading to formation of inclusion bodies (Ferbitz et al., 2004; Maier et al., 2005). The function of a trigger factor in eliminating misfolding or the aggregation of nascent peptides presumably stems from sequestering exposed hydrophobic patches in the confined cage-like space (Yonath, 2006). As also, as DnaK and SlyD are in the set of chaperones that can play as general Tat signal-binding proteins, these chaperones would similarly recognize exposed hydrophobic residues of target proteins (Graubner, 2007). Therefore, we presume that the chaperone function of SlyD, the N-terminus fusion partner of heterologous proteins, would be to enhance cytoplasmic solubility in the same manner as a trigger factor and/or DnaK.
Moreover, the fusion mutant of G-CSF with polyhistidine tag [(His)6-SlyD-(D4K)::G-CSF (Fig. 1) were affinity purified, and then the enterokinase digestion was carried out in a batch mode, and subsequently the proteolytic products were collected and centrifuged (17 000 g for 10 min). SDS–PAGE analysis showed that the recombinant G-CSF released from SlyD was present in the supernatant fraction (Fig. 5A), i.e. in the form of soluble protein. The CD spectrum of the purified G-CSF was analyzed at room temperature using a spectropolarimeter (Fig. 5B). The analysis result (Fig. 5B) shows that CD spectrum of the recombinant G-CSF is identical to what was previously reported (Bae et al., 1998; Jeong and Lee, 2001) for native human G-CSF, thereby indicating that the recombinant G-CSF has the correct secondary structure. Therefore, it seems reasonable to conclude that the recombinant G-CSF was actually folded. Consequently it seems reasonable to presume that the other recombinant proteins expressed with the SlyD fusion were folded to correct conformation like G-CSF, although all the analysis data are not presented here.
|
Bioactivity assay of the fusion mutant of cutinase
Cutinase is a known hydrolytic enzyme that degrades cutin, a circular polymer (i.e. polyester composed of hydroxy and epoxy fatty acids, usually n-C16, n-C18) (Kim et al., 2005). Potential roles for cutinase in the biotechnological applications continue to be the subject of much research. Cutinases have been used for their lipolytic properties in dishwashing and laundry detergents to degrade immobilized fats (Flipsen et al., 1998). Other biocatalytic uses of cutinases are involved in the oleochemistry (Carvalho et al., 1999), and additional and potential applications of cutinases are being sought for environmental pollutant control, such as waste plastics and water-soluble synthetic polymers (Shimao, 2001). Cutinase, an enzyme with a high hydrolytic activity for a range of esters, from soluble p-nitrophenyl esters to insoluble long-chain triglycerides, presented an extremely low enzyme on p-nitrophenyl esters of long-chain fatty acids like p-nitrophenyl phamitate (PNP) (Kim et al., 2003). A cutinase enzyme gene from P.putida was cloned and expressed in E. coli cytoplasm using the N-terminus fusion of E. coli SlyD as a solubility enhancer. We have assayed the enzymatic activity of the fusion mutant of cutinase, SlyD-CUT using p-nitrophenyl butyrate (PNB) and PNP as substrates. As shown in Fig. 6, PNB was hydrolyzed significantly by the cutinase fusion mutant, whereas PNP, a long-chain fatty acid, was not degraded by the recombinant fusion enzyme. This result clearly shows that the fusion mutant of cutinase was correctly folded into its native conformation and as biologically active as native cutinase. As presented in Fig. 6, the hydrolytic activity of the fusion mutant of cutinase [SlyD-CUT (1 g/l)] was 227 µmol/min/ml, which is even higher than the previously reported activity (90–150 µmol/min/ml) of P.putida cutinase (Sebastian et al., 1987). Figure 6 also shows that the specific activity of fungal cutinase was much higher than that of bacterial cutinase, and the hydrolytic activity of cutinase seems to significantly depend on microbial origin. Consequently, the stress-responsive protein, SlyD, was shown to be a potent solubility enhancer for an aggregation-prone protein such as cutinase when structured as cis-acting fusion partner in an E. coli expression system. Moreover, the potential applications of SlyD as a solubility enhancer seem very promising in the commercial production of biologically active aggregation-prone heterologous enzymes.
|
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingAcknowledgementsReferences |
|---|
National Research Laboratory Project (ROA-2007-000-20084-0) from the Korea Science and Engineering Foundation (KOSEF); Korean government (MOST), the Korea Health 21 R&D Project (A050750) of the Ministry of Health and Welfare of the Republic of Korea; Second Brain Korea 21 Project; KOSEF grant (R01-2005-000-10355-0); Korea Research Foundation (KRF-2004-041-D00180); Microbial Genomics and Applications Center (Taejon, Republic of Korea).
|
Footnotes |
|---|
1 These authors contributed equally to the work.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingAcknowledgementsReferences |
|---|
The authors appreciate the support of Professor Hang Chul Shin at Soonnsil University for kindly providing the gene clones of mp-INS and G-CSF. We also thank Professors Won Tae Lee and Hyun Soo Cho at Yonsei University for kindly donating the gene clones of ppGRN, AID and NACHT.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingAcknowledgementsReferences |
|---|
Bae C.S., Yang D.S., Chang K.R., Seong B.L., Lee J. Biotechnol. Bioeng. (1998) 57:600–609.[CrossRef][Web of Science][Medline]
Baneyx F., Mujacic M. Nat. Biotechnol. (2004) 22:1399–1408.[CrossRef][Web of Science][Medline]
Carvalho C.M., Aires-Barros M.R., Cabral J.M.S. Biotechnol. Bioeng. (1999) 66:17–34.[CrossRef][Web of Science][Medline]
Davis G.D., Elisee C., Newham D.M., Harrison R.G. Biotechnol. Bioeng. (1999) 65:382–388.[CrossRef][Web of Science][Medline]
De Bernardez Clark E. Curr. Opin. Biotechnol. (1998) 9:157–163.[CrossRef][Web of Science][Medline]
Eiteman M.A., Altman E. Trends Biotechnol. (2006) 24:530–536.[Web of Science][Medline]
Farzin G., Catherine R.W., Denise A.M., Brian S.I., Sheenah M.M. Electrophoresis (1999) 20:601–605.[CrossRef][Web of Science][Medline]
Ferbitz L., Maier T., Patzelt H., Bukau B., Deuerling E., Ban N. Nature (2004) 431:590–596.[CrossRef][Medline]
Flipsen J.A.C., Appel A.C.M., Van der Hijden H.T.W.M., Verrips C.T. Enzyme Microb. Technol. (1998) 23:274–280.[CrossRef][Web of Science]
Graubner W., Schierhorn A., Bruser T. J. Biol. Chem. (2007) 282:7116–7124.
Hottenrott S., Schumann T., Plückthun A., Fischer G., Rahfeld J.U. J. Biol. Chem. (1997) 272:15697–15701.
Jeong K.J., Lee S.Y. Protein Expr. Purif. (2001) 23:311–318.[CrossRef][Web of Science][Medline]
Kaiser C.M., Chan H.C., Agashe V.R., Lakshmipathy S.K., Etchells S.A., Hayer-Hartl M., Hartl F.U., Barral J.M. Nature (2006) 444:455–460.[CrossRef][Medline]
Kim Y.H., Lee J., Moon S.H. J. Microbiol. Biotechnol. (2003) 13:57–63.
Kim Y.H., Ahn J.Y., Moon S.H., Lee J. Chemosphere (2005) 60:1349–1355.[Medline]
Lavallie E.R., Diblasio E.A., Kovacic S., Grant K.L., Schendel P.F., McCoy J.M. Nat. Biotechnol. (1993) 11:187–193.[CrossRef]
Lilie A., Schwarz E., Rudolph R. Curr. Opin. Biotechnol. (1998) 9:497–501.[CrossRef][Web of Science][Medline]
Maier T., Ferbitz L., Deuerling E., Ban N. Curr. Opin. Struct. Biol. (2005) 15:204–212.[CrossRef][Web of Science][Medline]
Mitterauer T., Nanoff C., Ahorn H., Freissmuth M., Hohenegger M. Biochem. J. (1999) 342:33–39.[CrossRef][Web of Science][Medline]
Nallamsetty S., Waugh D.S. Protein Expr. Purif. (2006) 45:175–182.[CrossRef][Web of Science][Medline]
Scholz C., Schaarschmidt P., Engel A.M., Andres H., Schmitt U., Faatz E., Balbach J., Schmid F.X. J. Mol. Biol. (2005) 345:1229–1241.[CrossRef][Web of Science][Medline]
Scholz C., Eckert B., Hagn F., Schaarshmidt P., Balbach J., Schmid F.X. Biochemistry (2006) 45:20–33.[CrossRef][Medline]
Sebastian J., Chandra A.K., Kolattukudy P.E. J. Bacteriol. (1987) 169:131–136.
Shimao M. Curr. Opin. Biotechnol. (2001) 12:242–247.[CrossRef][Web of Science][Medline]
Smith D.B., Johnson K.S. Gene (1998) 67:31–40.[CrossRef]
Sorensen H.P., Mortensen K.K. J. Biotechnol. (2005) 115:113–128.[CrossRef][Web of Science][Medline]
Wülfing C., Lombardero J., Plückthun A. J. Biol. Chem. (1994) 269:2895–2901.
Yonath A. Nature (2006) 444:435–436.[CrossRef][Medline]
Zhang J.W., Butland G., Greenblatt J.F., Emili A., Zamble D.B. J. Biol. Chem. (2005) 280:4360–4366.
Received July 23, 2007; revised September 8, 2007; accepted September 12, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||