Refolding and Purification of Recombinant Human (Pro)renin Receptor from Escherichia coli by Ion Exchange Chromatography Fei Wang, Jinjin Guo, Quan Bai, and Lili Wang Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Inst. of Modern Separation Science, Key Lab of Modern Separation Science in Shaanxi Province, Northwest University, Xi’an 710069, China DOI 10.1002/btpr.1916 Published online April 22, 2014 in Wiley Online Library (wileyonlinelibrary.com)
Purification of the recombinant human renin receptor (rhRnR) is a major aspect of its biological or biophysical analysis, as well as structural research. A simple and efficient method for the refolding and purification of rhRnR expressed in Escherichia coli with weak anionexchange chromatography (WAX) was presented in this work. The solution containing denatured rhRnR in 8.0 mol/L urea extracted from the inclusion bodies was directly injected into the WAX column. The aggregation was prevented and the soluble form of renatured rhRnR in aqueous solution was obtained after desorption from the column. Effects of the extracting solutions, the pH values and urea concentrations in the mobile phase, as well as the sample size on the refolding and purification of rhRnR were investigated, indicating that the above mentioned factors had remarkable influences on the efficiency of refolding, purification and mass recovery of rhRnR. Under the optimal conditions, rhRnR was successfully refolded and purified simultaneously by WAX in one step within only 30 min. The result was satisfactory with mass recovery of 71.8% and purity of 94.8%, which was further tested by western blotting. The specific binding of the purified rhRnR to recombinant human renin was also determined using surface plasmon resonance (SPR). The association constant of rhRnR to recombinant human renin was calculated to be 3.25 3 108 L/mol, which demonstrated that rhRnR was already renatured and simultaneously purified in one step using WAX. All of the above demonstrate that protein folding liquid chromatography (PFLC) should be a powerful C 2014 American Institute of Chemical tool for the purification and renaturation of rhRnR. V Engineers Biotechnol. Prog., 30:864–871, 2014 Keywords: ion exchange chromatography, protein refolding, purification, protein folding liquid chromatography, recombinant renin receptor
Introduction The renin-angiotensin system (RAS)1 plays an important role in balancing normal human activities, especially in cardiovascular and renal physiology and disease. The (Pro)renin receptor binds specifically to (pro)renin and has critical connections with diseases caused by the activation of RAS, including hypertension and diabetes mellitus,2–4 which could be a perfect target for drugs for the cure of cardiovascular and renal disease. The (Pro)renin receptor is a type of transmembrane protein that consists of 350 amino acids with a nonglycosylation and hydrophobic N-terminus.5 The highly purified (pro)renin receptor not only facilitates the further study of (pro)renin receptor blockade but also provides a target for research on intracellular signal transduction.6 However, the separation and purification of RnR has met with great difficulties due to its extremely strong hydrophobicity. The (Pro)renin receptor can be expressed in the host cell through genetic engineering methods. 7 However, recombinant human (pro)renin receptor (rhRnR) expressed in Correspondence concerning this article should be addressed to Q. Bai at [email protected]. 864
Escherichia coli (E. coli) can form inactive inclusion bodies, which are insoluble in water and can only be dissolved in solutions containing high concentrations of denaturant, such as 7.0 mol/L guanidine hydrochloride (GuHCl) or 8.0 mol/L urea. Because it is inactive in the denaturant solution, it is necessary to refold or renature the denatured (Pro)renin receptor before further separation and purification. Although scientists have developed many renaturation methods to solve this problem, such as the frequently used dilution and dialysis,8 as well as the artificial molecular chaperon,9,10 the extremely strong hydrophobicity of the inclusion body of recombinant protein makes renaturation by dilution a difficult process because the aggregation and precipitation of the inclusion body become much serious when the concentration of the denaturation agents decreases, resulting in low mass recovery and renaturation efficiency. Although adding detergents into the extracting buffer is an effective way to address hydrophobic membrane proteins,11 the separation of detergents and target protein tends to be annoying and tedious, and the result is always not very satisfactory.12 Application of liquid chromatography (LC) to protein folding is one of the most interesting and exciting methods that have been developed in recent years.13–15 When it is used in protein folding, the bioactivity recovery increases, C 2014 American Institute of Chemical Engineers V
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the folded protein can be easily separated from misfolded forms, protein concentration after refolding is relatively high, and it is easy to scale up and automate; therefore, it is regarded as an efficient, and close to ideal refolding method referred to as protein folding liquid chromatography (PFLC).16 The advantages of PFLC are that it not only prevents the unfolded protein molecules from aggregating with each other, but it also simultaneously purifies or partially purifies the protein during the chromatographic process. Currently, it has become a very popular technique for protein folding, especially been applied to a large-scale industrial chromatographic technique, such as expanded bed adsorption chromatography15,17 and simulated moving bed chromatography.15,18 PFLC has been applied successfully for the renaturation and simultaneous purification of some recombinant proteins produced by E. coli, such as recombinant human interferon-c (rhINF-c),19,20 recombinant human proinsulin,21 recombinant human granulocyte colony-stimulating factor (rhG-CSF),22 recombinant human granulocyte macrophage colony stimulation factor (rhGM-CSF),23 recombinant human stem cell factor (rhSCF),24 and human bone morphogenetic protein-2,25 and the bioactivity recovery of these renatured recombinant proteins was two to three times that of other typical methods. In addition, PFLC was also used in the purification and refolding of bacterial influenza vaccine candidates.26 Ion-exchange chromatography (IEC) is a widely used chromatographic method for protein separation,27and has now become one of the most frequently used methods in PFLC. The successful purification with simultaneous renaturation of recombinant proteins by IEC have all achieved high mass and bioactivity recovery,23,25,28,29 proving that recombinant proteins that possess strong hydrophobicity really can be purified and renatured under IEC mode, which indeed has the potential to solve the purification problem of the recombinant human renin receptor (rhRnR) due to its hydrophobicity. The (Pro)renin receptor is an acid protein whose pI is 5.3 and molecular weight is 39 kDa without any disulfide bonds. In this article, the recombinant human renin receptor (rhRnR) was expressed in E. coli and then renatured and purified simultaneously with weak anion exchange chromatography (WAX). The influences of the inclusion body extraction buffer, pH, urea concentrations in the mobile phase and sample size on the purification with WAX were investigated in detail. The results indicated that rhRnR could be renatured and purified under optimal conditions only in one step with WAX.
Materials and Methods Instruments LC-20A high-performance liquid chromatographic instrument (Shimadzu, Japan), consisting of two LC-20AT vp pumps, one SPD-20A vp UV-vis detector, one SCL-20A vp system controller, and one Rheodyne 7725 valve was used. All chromatographic data were collected and evaluated using the class-VP data system. DEAE Sepharose Fast Flow was purchased from General Electric Company (America) and packed into a simple plastic column (50 3 10 mm I.D.) from Shaanxi Xida Kelin Gene-Pharmacy Co., Ltd. A Mini Protean II (Bio-Rad, Hercules, CA) apparatus was used for electrophoresis. An SORVALL RC28S centrifuge (KENDRO) was used for centrifugation. Enhanced
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chemiluminescence system (Clinx Science Instruments, Shanghai, China) was used to develop the blots in western blotting. In surface plasmon resonance (SPR) assay, specific binding was detected using Reichert SR7500DC Dual Channel SPR System (Reichert Technology). Chemicals Potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate, sodium chloride, and urea are of analytical grade. Acrylamide and bis-acrylamide were of electrophoresis grade and were obtained from Sigma-Aldrich (St Louis, MO). Tris(hydroxymethyl)aminomethane, glycine, sodium dodecyl sulfate (SDS), EDTA(ethylene diamine tetraacetic acid), Triton X-100, sodium lauroyl sareosine(SLS), and Tween 20 were obtained from Amersco. Bovine serum albumin (BSA) and molecular mass marker were purchased from Sigma-Aldrich (St Louis, MO). Anti-FLAG M2 antibody (Sigma-Aldrich, St Louis, MO) and HRP-conjugated secondary anti-mouse IgG antibody (Millipore, Billerica, MA) were used in western blotting. Purified recombinant human renin (Sino Biological Inc., China) was used in SPR assay; 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ethanolamine were obtained from Sigma-Aldrich (St Louis, MO). All other chemicals were of analytical grade and purchased from Xi’an Chemicals Co. Expression of rhRnR in E. coli The expression vector PET22B 1 RenR was transformed into Rosetta cells to obtain the E. coli system used to produce rhRnR. The bacteria were cultured in LB medium for 12 h under 37 C in a shake flask, then in M9 medium for 4 h followed by the addition of isopropyl b-D-1-Thiogalactopyranoside (IPTG, 1.0 mmol/L) which induced the production of rhRnR. The culture was harvested at an OD600 of 0.8 and then resuspended in a 0.05 mol/L NaH2PO4/NaOH, pH 7.4 by centrifugation for 10 min at 10,000 rpm, 4 C and treated as follows. Recovery of rhRnR inclusion bodies The harvested cells were washed three times at room temperature with cleaning buffer I (20 mmol/L Tris 1 1 mmol/L EDTA, pH 8.3) and were disrupted by ultrasonication in an ice-water bath, followed by centrifugation at 9,000 rpm for 15 min, 4 C. The isolated inclusion bodies were washed with cleaning buffer II (20 mmol/L Tris 1 1 mmol/L EDTA 1 2.0 mol/L urea 1 1.0 mol/L NaCl, pH 7.4), III (20 mmol/L Tris 1 1 mmol/L EDTA 1 1.0 mol/L NaCl, pH 7.4) and IV (20 mmol/L Tris 1 1 mmol/L EDTA, pH 7.4) for three times, respectively. After each washing step, the suspension was centrifuged at 14,000 rpm and 4 C for 15 min, the supernatant was discarded. Then, the pellet fraction containing rhRnR inclusion bodies were obtained and stored at 220 C. Extraction and solubilization of rhRnR inclusion bodies The clean rhRnR inclusion bodies23 were dissolved in the following extracting solutions: solution I (8.0 mol/L urea 1 0.1 mol/L Tris 1 0.01 mol/L EDTA, pH 5 8.0), solution II (5.0 mol/L urea10.1 mol/L Tris 1 0.01 mol/L EDTA 1 1% SLS, pH 5 8.0) and solution III (8.0 mol/L urea 1 0.1 mol/L Tris 1 0.01 mol/L EDTA 1 0.1% Triton X-100,
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pH 5 8.0). After incubation at 4 C for 12 h with full shaking, the supernatant containing rhRnR was obtained by centrifuging at 12,000 rpm. Chromatographic conditions The WAX column (50 3 10 mm I.D.) packed with DEAE Sepharose Fast Flow media was initially equilibrated with buffer solution A for at least 15 min. The extract solution of rhRnR (containing 10.9 mg/mL total protein) was directly injected into the column by cumulative sampling, and then a 30 min linear gradient elution of 0 to 100% buffer solution B was performed at a flow rate of 1.0 mL/min. The detection wavelength was 280 nm. The eluted fractions of the target protein were collected for the measurements of the mass recovery and purity of rhRnR. Mobile phase A: 20 mmol/L Tris 1 0 to 4.0 mol/L urea, pH 8.0; mobile phase B: 20 mmol/L Tris 1 0 to 4.0 mol/L urea 1 1.0 mol/L NaCl, pH 8.0.
Figure 1.
Analysis of SDS-PAGE of rhRnR inclusion bodies extracted with three types of solutions. Lane: 1: 8.0 mol/L urea; 2: 5.0 mol/L urea 1 1% SLS; 3: 8.0 mol/L urea 1 1% Triton X-100.
Determination of protein concentration The protein concentration was estimated according to the Bradford method.30 SDS-PAGE and western blotting analysis The purity of rhRnR was determined by SDS-PAGE followed by western blotting. SDS-PAGE was performed using the Mini-protean II system (Bio-Rad, Hercules, CA). The purified protein was visualized on the SDS-PAGE gel by staining with Coomassie brilliant blue (CBB) R-250. After SDS-PAGE, protein was electroblotted onto polyvinylidene difluoride (PVDF) membrane. After blocking in phosphate buffered saline containing 0.1% Tween 20 and 10% nonfat dry milk, the membrane was incubated in 1:1,000 diluted anti-FLAG M2 antibody solution overnight at 4 C. After washing, the membrane was incubated for 1 h at 37 C in 1:3,000 diluted HRP-conjugated secondary anti-mouse IgG antibody. Blots were developed using the enhanced chemiluminescence system. Binding assay of rhRnR using SPR analysis SPR was applied to detect the specific binding of the purified rhRnR to recombinant human renin, which was immobilized onto a 10% COOH-PEG self-assembled monolayer sensor chip. The desalting process of the IEC fraction of rhRnR was necessary before the injection of the sample. After pumping an adequate amount of running buffer (PBST), a mixture of 0.20 mol/L EDC and 0.05 mol/L NHS was injected to activate the carboxyl group on the surface of the sensor chip. Then, rhRnR dissolved in PBST was injected until the desired amount was immobilized. Unreacted carboxymethyl groups on the sensor chip were blocked with ethanolamine to prevent non-specific binding. Then, the solution with different concentrations of purified rhRnR was injected into the sensor chip, respectively. Data analysis was performed with SPRAutolink post-processing system and Clamp98 software.
Results and Discussion Extraction and solubilization of rhRnR inclusion bodies Because of the extremely strong hydrophobicity, rhRnR expressed in E. coli formed inactive inclusion bodies, which
Figure 2. Chromatograms of rhRnR inclusion bodies extracted with three different solutions separated by WAX. The rhRnR inclusion bodies were dissolved in a: 8.0 mol/L urea; b: 5.0 mol/L urea containing 1% SLS; c: 8.0 mol/L urea containing 1% TritonX-100, respectively. Chromatographic conditions: stationary phase: DEAE Sepharose FF; mobile phase A: 20 mmol/L Tris 1 4.0 mol/L urea, pH 7.5; mobile phase B: mobile phase A containing 1.0 mol/L NaCl, pH 7.5; linear gradient elution for a and b: 0 to 5 min, 0%B; 5 to 35 min, 0%B to 100%B; linear gradient elution for c: 0 to 10 min, 0%B; 10 to 40 min, 0%B to 100%B; Flow rate: 1.0 mL/min; Detection wavelength: 280 nm. (*): Target protein.
were insoluble in water and could only be dissolved with solutions containing high concentrations of denaturant, such as 7.0 mol/L guanidine hydrochloride (GuHCl) or 8.0 mol/L urea. To purify rhRnR from the inclusion bodies formed in E. coli, we first determined the conditions for dissolving the inclusion bodies. Firstly, the inclusion bodies of rhRnR were dissolved in 8.0 mol/L urea. In general, the detergents are always used to solubilize the membrane proteins.12 To enhance the solubility of the inclusion bodies and to inhibit the aggregation of rhRnR, two types of detergents were added to the urea solution as the additives for dissolving the inclusion bodies: Triton X100 and sodium lauroyl sareosine (SLS). The SDS-PAGE analysis of the three extractions of rhRnR inclusion bodies is shown in Figure 1. In addition, the three types of extractions of rhRnR inclusion bodies were injected directly into the column and separated with WAX, respectively. The chromatograms are shown in Figure 2. The comparisons of the purity,
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Table 1. Comparison of the Protein Content, Purity, and Mass Recovery of rhRnR Extracted with Three Different Extractions Between Before and After Purification with WAX
Solutions Aim protein concentration (mg/mL) Purity of rhRnR (%) Purity of rhRnR after purification (%) Mass recovery (%)
8.0 mol/L Urea
5.0 mol/L Urea 1 1% SLS
8.0 mol/L Urea 1 1% Triton X-100
10.9
13.3
13.5
60.2 94.7
79.3 97.0
56.2 68.4
43.1
31.3
21.1
the content and mass recovery of the aim protein before and after purification with WAX are also listed in Table 1. From Figure 1 and Table 1, to compare the three extraction methods for dissolving the inclusion bodies, the result indicated that the least impurities of the extraction with 5.0 mol/L urea containing 1% SLS was observed, and the purity of rhRnR was 79.3% (Lane 2). On the contrary, when 1% Triton X-100 was added to 8 mol/L urea as the additive to dissolve the rhRnR inclusion bodies, much more impurities were extracted together with aim protein and the purity of rhRnR was only 56.2% (Lane 3). From Figure 2, it could be observed that when the detergents were added in the extraction solutions as the additives, the peak shapes of the target protein (marked with the asterisk) became distorted and multiple peaks appeared in Figures 2b,c. Only when 8.0 mol/L urea was used to dissolve the inclusion bodies did the peak shape become more symmetric and the highest mass recovery (43.1%) and purity (94.7%) of the target protein were obtained after purification with WAX. As a result, 8.0 mol/L urea was used to extract the rhRnR inclusion bodies.
Application of IEC to simultaneous refolding and purification of rhRnR Many types of recombinant proteins have been produced in E. coli resulting in the formation of inclusion bodies. The challenge is how to convert the insoluble inclusion body proteins into soluble and bioactive proteins. However, activity recovery in general is only 5% to 20% when refolding of denatured proteins using traditional dilution and dialysis methods. In addition, the impure proteins were difficult to separate. Therefore, protein refolding or renaturation has become the bottleneck during the production of recombinant proteins by biotechnology. As mentioned above, LC should be a powerful tool for protein refolding with high yield and is becoming one of the most interesting and exciting protein refolding methods developed in recent years; it is referred to as protein folding liquid chromatography (PFLC).14 It was reported that ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), affinity chromatography (AFC) and size exclusion chromatography (SEC) have been employed for PFLC at laboratory or industrial scales.15 The target proteins may be purified simultaneously during the protein refolding by PFLC. Now IEC has become one of the most frequently used methods in PFLC. In this work, refolding rhRnR with simultaneous purification with relatively high yield was set as the aim. Due to its isoelectric point at approximately 5.3, anion exchange chromatography (AEC) should be used to obtain an effective retention. In the
presented work, weak anion exchange chromatography (WAX) with a stationary phase DEAE Sepharose FF was used to refold rhRnR expressed in E. coli with simultaneous purification. When the sample solution containing rhRnR in 8.0 mol/L urea was loaded onto the IEC column, the denatured rhRnR was captured by the stationary phase. The nonretarded or very weakly retained proteins were eluted out the column directly. With the mobile phase flowing through the column, the urea in the denatured protein solution flowed out of the column without any retention. Thus the urea concentration surrounding the rhRnR molecules reduced, initiating the collapse of rhRnR molecules. The electrostatic interaction between the denatured rhRnR molecules and the IEC stationary phase suppressed the non-specific association of the unfolded molecules or incompletely folded molecules, preventing rhRnR molecules from aggregating with each other. Actually, the operation of both PFLC and normal LC are essentially the same. A sample solution containing the recombinant protein is directly injected into a suitable chromatographic column, and then the fractions containing the renatured target protein are collected. Because both refolding and purification are accomplished by PFLC, the chromatographic conditions must be carefully optimized. Therefore, several factors that influence the refolding yield of rhRnR, including urea concentration in the mobile phase, pH and sample size, were investigated in detail in subsequent experiments. Effect of pH The pH of the mobile phase is the significant factor in the IEC system because the pH value of the system strongly affects the retention of proteins by altering the net charge on both the protein and the stationary phase.31 rhRnR can be separated with the WAX column because it is an acid protein with an isoelectric point (pI) of 5.3. As is well-known, electrostatic interaction is the main interaction in ion exchange chromatography, proteins can be separated in the solution with a certain pH based on the difference of net charges. Therefore, the pH value of the mobile phase solution has a great effect on the retention behaviors of proteins in a chromatographic process. As a result, the refolding and purification of rhRnR depends on the pH of the mobile phase solution. The rhRnR extract was separated with different pHs of the mobile phase on the WAX column, and the chromatograms were shown in Figure 3. All of the eluted fractions were collected and analyzed by SDS-PAGE, in which the target rhRnR protein was found as the peak indicated with the asterisk in Figure 3. Furthermore, the mass recovery and the purity of rhRnR at different pH are shown in Figure 4. The two figures illustrated that a higher chromatographic peak was obtained and a better result was achieved when a pH of 7.5 was used. The purity of rhRnR rose to 94.8% from that of 60.2% in rhRnR inclusion body extract, and mass recovery was obtained to be 43.2%. Effect of urea concentration in mobile phase Although PFLC has so many advantages, several problems still exist. If proteins have strong hydrophobicity, some aggregates may form when loading the denatured protein solutions onto an LC column, resulting in increased back pressure of the employed column, which may even block it.
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Figure 3.
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The effects of pH on the separation of rhRnR with WAX. Chromatographic conditions: mobile phase A:20 mmol/L Tris14.0 mol/L urea; mobile phase B: 20 mmol/L Tris 1 4.0 mol/L urea 1 1.0 mol/L NaCl; (1) pH 6.5; (2) pH 7.0; (3) pH 7.5; (4) pH 8.0; detection wavelength: 280 nm; linear gradient: 100% A to 100% B; sample size: 100 lL; flow rate: 1.0 mL/ min; (*): target protein.
Figure 5. Comparisons of the chromatograms of rhRnR separated with WAX under the different concentrations of urea in the mobile phase. Chromatographic conditions: mobile phase A: 20 mmol/L Tris 1 1 to 5.0 mol/L urea, pH 5 7.5; mobile phase B: 20 mmol/L Tris 1 1 to 5.0 mol/L urea 1 1.0 mol/L NaCl, pH 5 7.5; urea concentration: 1, 1.0 mol/L; 2, 2.0 mol/L; 3, 3.0 mol/L; 4, 4.0 mol/L; 5, 5.0 mol/L; linear gradient: 100% A to 100% B, 30 min; sample size: 180 lL; flow rate: 1.0 mL/min; detection wavelength: 280 nm. (*): Target protein.
Figure 4. Effects of the different pH of the mobile phase on the mass recovery and purity of rhRnR separated with WAX. 䊏: mass recovery; ~: purity.
Figure 6. Purity and mass recovery of rhRnR separated by WAX with different urea concentrations in mobile phase. 䊏: mass recovery; ~: purity.
Additionally, mass and bioactivity recoveries of the target protein would decrease. rhRnR was refolded and purified in 30 min by one step on the WAX column, but its mass recovery was lower. This decrease might be caused by the aggregation of denatured rhRnR on the column. It is well known that urea is a strong denaturant for proteins as well as an effective blocking agent against the aggregation of protein molecules. It was reported that the refolding efficiency of recombinant protein could be dramatically increased by a gradually changing concentration of urea in the mobile phase,32 because the addition of urea is able to effectively prevent the serious aggregation of protein caused by the sudden separation from the denaturant. The effects of various concentrations of urea in the mobile phase on the refolding with simultaneous purification of rhRnR were also investigated in detail, and the result is shown in Figure 5. The mass recovery and the purity of rhRnR with the different urea concentration in the range from 1.0 to 5.0 mol/L are presented in Figure 6. The results indicated that rhRnR could not be eluted or partially eluted with lower urea
concentrations in the mobile phase leading to the lower mass recovery. With the increase in urea concentration in the mobile phase from 1.0 to 5.0 mol/L, the chromatographic peak of rhRnR became higher and the mass recovery increased. With the presence of 4.0 mol/L of urea in the mobile phase, the peak of rhRnR was the highest, and the mass recovery reached 41.0%. However, when the urea concentration was increased to 5.0 mol/L, a lower purity of 87.1% of rhRnR was obtained because of the co-eluting of impurities. As a result, 4.0 mol/L urea was the best choice. Effect of sample size The effects of sample sizes on the purification and refolding of rhRnR were also investigated. The different sample sizes, such as 100 lL, 200 lL, 300 lL, 400 lL and 500 lL of rhRnR extracts were loaded directly onto the column, and a linear gradient elution was performed. The chromatograms
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Figure 7.
The effects of sample sizes of rhRnR extract loaded onto the column on the separation of rhRnR with WAX. Chromatographic conditions: mobile phase A: 20 mmol/L Tris 1 4.0 mol/L urea, pH 5 7.5; mobile phase B: 20 mmol/L Tris 1 4.0 mol/L urea 1 1.0 mol/L NaCl, pH57.5; sample size: 1, 100 lL; 2, 200 lL; 3, 300 lL; 4, 400 lL; 5, 500 lL; linear gradient: 100% A to 100% B, 30 min; flow rate: 1.0 mL/min; detection wavelength: 280 nm. (*): target protein.
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Figure 9. SDS-PAGE and western blot analysis of rhRnR separated by WAX. Purified rhRnR was analyzed by SDS-PAGE (a) and western blot (b). Lane 1: molecular weight marker (from bottom to top 14,400, 20,100, 31,000, 43,000, 66,200, 97,400 Da); 2: rhRnR extract; 3: rhRnR after refolding and purification by WAX.
phase was 4.0 mol/L, the pH was 7.5 and the sample size was 400 lL. The chromatogram and the results of SDSPAGE analysis are shown in Figures 8 and 9a, respectively. Western blot analysis indicated that the purified rhRnR can bind successfully to anti-FLAG M2 antibody and the purity of renatured rhRnR was further demonstrated as no higher molecular weight multimers were observed in Figure 9b. As a result, rhRnR could be purified successfully within 30 min by one step, and the purity of 94.8% and mass recovery of 71.8% were achieved. Binding assay using SPR
Figure 8. Chromatograms of rhRnR under the optimal conditions by WAX. Chromatographic conditions: mobile phase A: 20 mmol/L Tris 1 4.0 mol/L urea, pH 5 7.5; mobile phase B: 20 mmol/L Tris 1 4.0 mol/L urea 1 1.0 mol/L NaCl; pH 5 7.5; sample size: 400 lL; (*): target protein; detection wavelength: 280 nm; liner gradient: 100% A to 100% B; flow rate: 1.0 mL/min.
of rhRnR separated by WAX with loading different sample sizes are shown in Figure 7. Although the purities of all cases above were obtained to be more than 94%, the mass recoveries of rhRnR with different sample sizes were determined to be 43.2%, 39.6%, 69.8%, 71.8% and 69.2%, respectively. The result indicated that the mass recovery of rhRnR separated with WAX increased with increasing of the sample size loaded on the column. The average mass recovery of rhRnR was 70%. Thus, the sample size was selected to be 400 lL.
Purification of rhRnR under optimal conditions rhRnR was finally purified and refolded by WAX under optimal conditions when the urea concentration in the mobile
Unfortunately, there is no suitable and standardized bioactivity assay method for rhRnR, the specific activity of rhRnR renatured with WAX cannot currently be determined. However, the specific binding assay was available for purified rhRnR using SPR. After the desalting process, rhRnR was dissolved in PBST to make an aqueous solution of purified rhRnR with different concentrations. Considering the fact that aggregation and precipitation will occur in aqueous rhRnR solution at concentrations higher than 5.0 lg/mL, the binding ability of rhRnR to recombinant human renin was tested by injecting different concentrations of the purified rhRnR (0–0.13 lmol/L, i.e., 5.0 lg/mL) over a recombinant human renin-immobilized sensor chip (Figure 10).7 A surface lacking immobilized human renin was used as a control. It was observed that the response increased with the concentration of rhRnR solution, which clearly demonstrated that renatured rhRnR can bind specifically to renin even when the concentration is very low. The association constant33 of rhRnR to recombinant human renin was calculated to be 3.25 3 108 L/mol. It is well known that the binding ability is essential and crucial for the renin receptor to perform its biological function. Although there is no currently available standardized bioactivity assay, successful binding of rhRnR to renin can be a strong proof that rhRnR has already been renatured and simultaneously purified only by one step with WAX.
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Figure 10. Binding assay of rhRnR to recombinant human renin immobilized onto to a sensor chip using SPR. Solution of purified rhRnR with different concentrations (a: 0.0051 lmol/L, b: 0.013 lmol/L, c: 0.026 lmol/L, d: 0.13 lmol/L) were injected into a sensor chip immobilized with human renin, respectively. Unreacted carboxymethyl groups of a sensor chip lacking immobilized protein were blocked with ethanolamine as a control for non-specific binding. Sensorgram overlays of response difference between the left and right channel of the sensor chip of SPR are shown.
Conclusions rhRnR expressed by E. coli could be refolded and purified simultaneously with WAX. By using PFLC for the refolding and purification of rhRnR, the process became simpler and easier and could be achieved in 30 min by one step. The mass recovery was 71.8%. The purity of rhRnR increased greatly from 60.2% to 94.8%, which was further testified by western blotting analysis, and no multimers were observed after chemiluminescence. Although there are no currently suitable methods for the analysis of the bioactivity of rhRnR, the inactive and denatured inclusion bodies of rhRnR could be refolded to soluble forms with high purity by PFLC, and the specific binding ability of rhRnR to recombinant human renin was determined by SPR assay. The association constant of rhRnR to renin was calculated to be 3.25 3 108 L/ mol, which demonstrated that rhRnR could be renatured and purified simultaneously after this “one step” chromatographic process. Therefore, PFLC is a powerful tool not only for the separation but also for the refolding with simultaneously purification of recombinant proteins expressed in E. coli.
Acknowledgments The plasmid of rhRnR was donated by Prof. Weiming Gong (Institute of Biophysics, Chinese Academy of Science, Beijing, China). This work is supported by the National 863 Program (No. 2006AA02Z227), the National Natural Science Foundation in China (21006077), the Natural Science Foundation of Shaanxi Province (2011JZ002), the Foundation of Key Laboratory in Shaanxi Province (2010JS103, 11JS097).
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2. Nguyen G, Muller DN. The biology of the (pro)renin receptor. J Am Soc Nephrol. 2010; 21:18–23. 3. Nguyen G. The (pro)renin receptor: Pathophysiological roles in cardiovascular and renal pathology. Curr. Opin. Nephrol. Hypertens. 2007;16:129–133. 4. Ichihara A, Hayashi M, Kaneshiro Y, Suzuki F, Nakagawa T, Tada Y, Koura Y, Nishiyama A, Okada H, Uddin MN, Nurun Nabi AHM, Ishida Y, Inagami T, Saruta T. Inhibition of diabetic nephropathy by a decoy peptide corresponding to the “handle” region for nonproteolytic activation of prorenin. J Clin Invest. 2004;114:1128–1135. 5. Yang XH, Lu XZ. Research progress in prorenin in and prorenin in receptor. Chin. J. Pathophysiol. 2010;26:405–408. 6. Batenburg WW, Jan Danser AH. The (pro)renin receptor: A new addition to the renin–angiotensin system? Eur J Pharmacol. 2008;585:320–324. 7. Kato T, Kageshima A, Suzuki F, Park EY. Expression and purification of human (pro)renin receptor in insect cells using baculovirus expression system. Protein Exp Purif. 2008;58:242–248. 8. Davis JM, Narachi MA, Alton NK, Arakawa T. Structure of human tumor necrosis factor alpha. derived from recombinant DNA. Biochemistry 1987;26:1322–1326. 9. Guise AD, West SM, Chaudhuri JB. Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies. Mol Biotechnol. 1996;6:53–64. 10. Gao YG, Guan YX, Yao SJ, Cho MG. On-column refolding of recombinant human interferon-cwith an immobilized chaperone fragment. Biotechnol Prog. 2003;19:915–920. 11. Xie H, Chen Y, Sun E, Wang H. Applications of detergents in membrane proteins research. Biotechnol Bull. 2010;2:205–212. 12. Gohon Y, Popot J. Membrane protein–surfactant complexes. Curr Opin Colloid Interface Sci. 2003;8:15–22. 13. Geng XD, Bai Q. Mechanism of simultaneously refolding and purification of proteins by hydrophobic interaction chromatographic unit and applications. Sci China Ser. B 2002;45:655– 669. 14. Bai Q, Kong Y, Geng XD. Studies on the refolding of reduceddenaturated insulin with high performance hydrophobic interaction chromatography. Chem Res Chin Univ. 2002;23:1483–1488. 15. Geng XD, Wang CZ. Protein folding liquid chromatography and its recent developments. J Chromatogr. B. 2007;249:69–80. 16. Geng XD, Bai Q, Wang CZ. Protein Folding Liquid Chromatography (in Chinese). Beijing: Science Press; 2006:1–6. 17. Xu X, Hirpara J, Epting K, Jin M, Ghose S, Rieble S, Li ZJ. Clarification and capture of high-concentration refold pools for E. coli-based therapeutics using expanded bed adsorption chromatography. Biotechnol Prog. 2014;30:113–123. 18. Freydell EJ, van der Wielen LA, Eppink MH, Ottens M. Techno-economic evaluation of an inclusion body solubilization and recombinant protein refolding process. Biotechnol Prog. 2011;27:1315–1328. 19. Geng XD, Bai Q, Zhang YJ, Li X, Wu D. Refolding and purification of interferon-gamma in industry by hydrophobic interaction chromatography. J Biotechnol. 2004;113:137–149. 20. Wu D, Wang CZ, Geng XD. An approach for increasing the mass recovery of proteins derived from inclusion bodies in biotechnology. Biotechnol Prog. 2007;23:407–412. 21. Bai Q, Kong Y, Geng XD. Studies on renaturation with simultaneous purification of recombinant human proinsulin from E. coli with high performance hydrophobic interaction chromatography. J Liq Chromatogr Relat Technol. 2003;26:683–695. 22. Wang CZ, Liu JF, Geng XD. Refolding with simultaneous purification of recombinant human granulocyte colony-stimulating factor from Escherichia coli using strong anion exchange chromatography. Chin Chim Lett. 2005;16:389–392. 23. Bai Q, Chen G, Liu JB, Geng XD. Renaturation and Purification of rhGM-CSF with ion-exchange chromatography. Biotechnol Prog. 2007;23:1138–1142. 24. Wang LL, Wang CZ, Geng XD. Expression, renaturation and simultaneous purification of recombinant human stem cell factor in Escherichia coli. Biotechnol Lett. 2006;28:993–997. 25. Rane AM, Jonnalagadda S, Li ZY. On-column refolding of bone morphogenetic protein-2 using cation exchange resin. Protein Exp Purif. 2013;90:135–140.
Biotechnol. Prog., 2014, Vol. 30, No. 4 26. Sanchez-Arreola PB, Lopez-Uriarte S, Marichal-Gallardo PA, Gonzalez-Vazquez JC, Perez-Chavarrıa R, Soto-Vazquez P, Lopez-Pacheco F, Ramırez-Medrano A, Rocha-Piz~na MR, Alvarez MM. A baseline process for the production, recovery, and purification of bacterial influenza vaccine candidates. Biotechnol Prog. 2013;29:896–908. 27. Guo LA, Chang JH. Separation of Protein by Chromatography. Beijing: Chemical Industry Press; 2011. 28. Wang CZ, Wang LL, Geng XD. Renaturation with simultaneous purification of rhG-CSF from Escherichia coli by ion exchange chromatography. Biomed Chromatogr. 2007;21:1291–1296. 29. Wang CZ, Liu JH, Wang LL, Geng XD. Solubilization and refolding with simultaneous purification of recombinant human stem cell factor. Appl Biochem Biotechnol. 2008;144:181–189. 30. Bradford MM. A rapid and sensitive 332 method for the quantitation of microgram quantities of protein utilizing the
871 principle of protein-dye binding. Anal Biochem. 1976;72:248– 254. 31. Van den Eijnden-Van Raaij AJM, Koornneef I, van Oostwaard ThMJ, de Laat SW, van Zoelen EJJ. Cation-exchange highperformance liquid chromatography: Separation of highly basic proteins using volatile acidic solvents. Anal Biochem. 1987;163: 263–269. 32. De Bernardez Clark E, Hevehan D, Szela S, Maachupalli-Reddy J. Oxidative renaturation of hen egg-white lysozyme. Folding vs aggregation. Biotechnol Prog. 1998;14:47–54. 33. Karlsson R, Michaelsson A, Mattsson L. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J Immunol Methods. 1991;145:229–240. Manuscript received Aug. 9, 2013, and revision received Mar. 27, 2014.
Purification of the recombinant human renin receptor (rhRnR) is a major aspect of its biological or biophysical analysis, as well as structural research. A simple and efficient method for the refolding and purification of rhRnR expressed in Escherichia coli with weak anionexchange chromatography (WAX) was presented in this work. The solution containing denatured rhRnR in 8.0 mol/L urea extracted from the inclusion bodies was directly injected into the WAX column. The aggregation was prevented and the soluble form of renatured rhRnR in aqueous solution was obtained after desorption from the column. Effects of the extracting solutions, the pH values and urea concentrations in the mobile phase, as well as the sample size on the refolding and purification of rhRnR were investigated, indicating that the above mentioned factors had remarkable influences on the efficiency of refolding, purification and mass recovery of rhRnR. Under the optimal conditions, rhRnR was successfully refolded and purified simultaneously by WAX in one step within only 30 min. The result was satisfactory with mass recovery of 71.8% and purity of 94.8%, which was further tested by western blotting. The specific binding of the purified rhRnR to recombinant human renin was also determined using surface plasmon resonance (SPR). The association constant of rhRnR to recombinant human renin was calculated to be 3.25 3 108 L/mol, which demonstrated that rhRnR was already renatured and simultaneously purified in one step using WAX. All of the above demonstrate that protein folding liquid chromatography (PFLC) should be a powerful C 2014 American Institute of Chemical tool for the purification and renaturation of rhRnR. V Engineers Biotechnol. Prog., 30:864–871, 2014 Keywords: ion exchange chromatography, protein refolding, purification, protein folding liquid chromatography, recombinant renin receptor
Introduction The renin-angiotensin system (RAS)1 plays an important role in balancing normal human activities, especially in cardiovascular and renal physiology and disease. The (Pro)renin receptor binds specifically to (pro)renin and has critical connections with diseases caused by the activation of RAS, including hypertension and diabetes mellitus,2–4 which could be a perfect target for drugs for the cure of cardiovascular and renal disease. The (Pro)renin receptor is a type of transmembrane protein that consists of 350 amino acids with a nonglycosylation and hydrophobic N-terminus.5 The highly purified (pro)renin receptor not only facilitates the further study of (pro)renin receptor blockade but also provides a target for research on intracellular signal transduction.6 However, the separation and purification of RnR has met with great difficulties due to its extremely strong hydrophobicity. The (Pro)renin receptor can be expressed in the host cell through genetic engineering methods. 7 However, recombinant human (pro)renin receptor (rhRnR) expressed in Correspondence concerning this article should be addressed to Q. Bai at [email protected]. 864
Escherichia coli (E. coli) can form inactive inclusion bodies, which are insoluble in water and can only be dissolved in solutions containing high concentrations of denaturant, such as 7.0 mol/L guanidine hydrochloride (GuHCl) or 8.0 mol/L urea. Because it is inactive in the denaturant solution, it is necessary to refold or renature the denatured (Pro)renin receptor before further separation and purification. Although scientists have developed many renaturation methods to solve this problem, such as the frequently used dilution and dialysis,8 as well as the artificial molecular chaperon,9,10 the extremely strong hydrophobicity of the inclusion body of recombinant protein makes renaturation by dilution a difficult process because the aggregation and precipitation of the inclusion body become much serious when the concentration of the denaturation agents decreases, resulting in low mass recovery and renaturation efficiency. Although adding detergents into the extracting buffer is an effective way to address hydrophobic membrane proteins,11 the separation of detergents and target protein tends to be annoying and tedious, and the result is always not very satisfactory.12 Application of liquid chromatography (LC) to protein folding is one of the most interesting and exciting methods that have been developed in recent years.13–15 When it is used in protein folding, the bioactivity recovery increases, C 2014 American Institute of Chemical Engineers V
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the folded protein can be easily separated from misfolded forms, protein concentration after refolding is relatively high, and it is easy to scale up and automate; therefore, it is regarded as an efficient, and close to ideal refolding method referred to as protein folding liquid chromatography (PFLC).16 The advantages of PFLC are that it not only prevents the unfolded protein molecules from aggregating with each other, but it also simultaneously purifies or partially purifies the protein during the chromatographic process. Currently, it has become a very popular technique for protein folding, especially been applied to a large-scale industrial chromatographic technique, such as expanded bed adsorption chromatography15,17 and simulated moving bed chromatography.15,18 PFLC has been applied successfully for the renaturation and simultaneous purification of some recombinant proteins produced by E. coli, such as recombinant human interferon-c (rhINF-c),19,20 recombinant human proinsulin,21 recombinant human granulocyte colony-stimulating factor (rhG-CSF),22 recombinant human granulocyte macrophage colony stimulation factor (rhGM-CSF),23 recombinant human stem cell factor (rhSCF),24 and human bone morphogenetic protein-2,25 and the bioactivity recovery of these renatured recombinant proteins was two to three times that of other typical methods. In addition, PFLC was also used in the purification and refolding of bacterial influenza vaccine candidates.26 Ion-exchange chromatography (IEC) is a widely used chromatographic method for protein separation,27and has now become one of the most frequently used methods in PFLC. The successful purification with simultaneous renaturation of recombinant proteins by IEC have all achieved high mass and bioactivity recovery,23,25,28,29 proving that recombinant proteins that possess strong hydrophobicity really can be purified and renatured under IEC mode, which indeed has the potential to solve the purification problem of the recombinant human renin receptor (rhRnR) due to its hydrophobicity. The (Pro)renin receptor is an acid protein whose pI is 5.3 and molecular weight is 39 kDa without any disulfide bonds. In this article, the recombinant human renin receptor (rhRnR) was expressed in E. coli and then renatured and purified simultaneously with weak anion exchange chromatography (WAX). The influences of the inclusion body extraction buffer, pH, urea concentrations in the mobile phase and sample size on the purification with WAX were investigated in detail. The results indicated that rhRnR could be renatured and purified under optimal conditions only in one step with WAX.
Materials and Methods Instruments LC-20A high-performance liquid chromatographic instrument (Shimadzu, Japan), consisting of two LC-20AT vp pumps, one SPD-20A vp UV-vis detector, one SCL-20A vp system controller, and one Rheodyne 7725 valve was used. All chromatographic data were collected and evaluated using the class-VP data system. DEAE Sepharose Fast Flow was purchased from General Electric Company (America) and packed into a simple plastic column (50 3 10 mm I.D.) from Shaanxi Xida Kelin Gene-Pharmacy Co., Ltd. A Mini Protean II (Bio-Rad, Hercules, CA) apparatus was used for electrophoresis. An SORVALL RC28S centrifuge (KENDRO) was used for centrifugation. Enhanced
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chemiluminescence system (Clinx Science Instruments, Shanghai, China) was used to develop the blots in western blotting. In surface plasmon resonance (SPR) assay, specific binding was detected using Reichert SR7500DC Dual Channel SPR System (Reichert Technology). Chemicals Potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate, sodium chloride, and urea are of analytical grade. Acrylamide and bis-acrylamide were of electrophoresis grade and were obtained from Sigma-Aldrich (St Louis, MO). Tris(hydroxymethyl)aminomethane, glycine, sodium dodecyl sulfate (SDS), EDTA(ethylene diamine tetraacetic acid), Triton X-100, sodium lauroyl sareosine(SLS), and Tween 20 were obtained from Amersco. Bovine serum albumin (BSA) and molecular mass marker were purchased from Sigma-Aldrich (St Louis, MO). Anti-FLAG M2 antibody (Sigma-Aldrich, St Louis, MO) and HRP-conjugated secondary anti-mouse IgG antibody (Millipore, Billerica, MA) were used in western blotting. Purified recombinant human renin (Sino Biological Inc., China) was used in SPR assay; 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ethanolamine were obtained from Sigma-Aldrich (St Louis, MO). All other chemicals were of analytical grade and purchased from Xi’an Chemicals Co. Expression of rhRnR in E. coli The expression vector PET22B 1 RenR was transformed into Rosetta cells to obtain the E. coli system used to produce rhRnR. The bacteria were cultured in LB medium for 12 h under 37 C in a shake flask, then in M9 medium for 4 h followed by the addition of isopropyl b-D-1-Thiogalactopyranoside (IPTG, 1.0 mmol/L) which induced the production of rhRnR. The culture was harvested at an OD600 of 0.8 and then resuspended in a 0.05 mol/L NaH2PO4/NaOH, pH 7.4 by centrifugation for 10 min at 10,000 rpm, 4 C and treated as follows. Recovery of rhRnR inclusion bodies The harvested cells were washed three times at room temperature with cleaning buffer I (20 mmol/L Tris 1 1 mmol/L EDTA, pH 8.3) and were disrupted by ultrasonication in an ice-water bath, followed by centrifugation at 9,000 rpm for 15 min, 4 C. The isolated inclusion bodies were washed with cleaning buffer II (20 mmol/L Tris 1 1 mmol/L EDTA 1 2.0 mol/L urea 1 1.0 mol/L NaCl, pH 7.4), III (20 mmol/L Tris 1 1 mmol/L EDTA 1 1.0 mol/L NaCl, pH 7.4) and IV (20 mmol/L Tris 1 1 mmol/L EDTA, pH 7.4) for three times, respectively. After each washing step, the suspension was centrifuged at 14,000 rpm and 4 C for 15 min, the supernatant was discarded. Then, the pellet fraction containing rhRnR inclusion bodies were obtained and stored at 220 C. Extraction and solubilization of rhRnR inclusion bodies The clean rhRnR inclusion bodies23 were dissolved in the following extracting solutions: solution I (8.0 mol/L urea 1 0.1 mol/L Tris 1 0.01 mol/L EDTA, pH 5 8.0), solution II (5.0 mol/L urea10.1 mol/L Tris 1 0.01 mol/L EDTA 1 1% SLS, pH 5 8.0) and solution III (8.0 mol/L urea 1 0.1 mol/L Tris 1 0.01 mol/L EDTA 1 0.1% Triton X-100,
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pH 5 8.0). After incubation at 4 C for 12 h with full shaking, the supernatant containing rhRnR was obtained by centrifuging at 12,000 rpm. Chromatographic conditions The WAX column (50 3 10 mm I.D.) packed with DEAE Sepharose Fast Flow media was initially equilibrated with buffer solution A for at least 15 min. The extract solution of rhRnR (containing 10.9 mg/mL total protein) was directly injected into the column by cumulative sampling, and then a 30 min linear gradient elution of 0 to 100% buffer solution B was performed at a flow rate of 1.0 mL/min. The detection wavelength was 280 nm. The eluted fractions of the target protein were collected for the measurements of the mass recovery and purity of rhRnR. Mobile phase A: 20 mmol/L Tris 1 0 to 4.0 mol/L urea, pH 8.0; mobile phase B: 20 mmol/L Tris 1 0 to 4.0 mol/L urea 1 1.0 mol/L NaCl, pH 8.0.
Figure 1.
Analysis of SDS-PAGE of rhRnR inclusion bodies extracted with three types of solutions. Lane: 1: 8.0 mol/L urea; 2: 5.0 mol/L urea 1 1% SLS; 3: 8.0 mol/L urea 1 1% Triton X-100.
Determination of protein concentration The protein concentration was estimated according to the Bradford method.30 SDS-PAGE and western blotting analysis The purity of rhRnR was determined by SDS-PAGE followed by western blotting. SDS-PAGE was performed using the Mini-protean II system (Bio-Rad, Hercules, CA). The purified protein was visualized on the SDS-PAGE gel by staining with Coomassie brilliant blue (CBB) R-250. After SDS-PAGE, protein was electroblotted onto polyvinylidene difluoride (PVDF) membrane. After blocking in phosphate buffered saline containing 0.1% Tween 20 and 10% nonfat dry milk, the membrane was incubated in 1:1,000 diluted anti-FLAG M2 antibody solution overnight at 4 C. After washing, the membrane was incubated for 1 h at 37 C in 1:3,000 diluted HRP-conjugated secondary anti-mouse IgG antibody. Blots were developed using the enhanced chemiluminescence system. Binding assay of rhRnR using SPR analysis SPR was applied to detect the specific binding of the purified rhRnR to recombinant human renin, which was immobilized onto a 10% COOH-PEG self-assembled monolayer sensor chip. The desalting process of the IEC fraction of rhRnR was necessary before the injection of the sample. After pumping an adequate amount of running buffer (PBST), a mixture of 0.20 mol/L EDC and 0.05 mol/L NHS was injected to activate the carboxyl group on the surface of the sensor chip. Then, rhRnR dissolved in PBST was injected until the desired amount was immobilized. Unreacted carboxymethyl groups on the sensor chip were blocked with ethanolamine to prevent non-specific binding. Then, the solution with different concentrations of purified rhRnR was injected into the sensor chip, respectively. Data analysis was performed with SPRAutolink post-processing system and Clamp98 software.
Results and Discussion Extraction and solubilization of rhRnR inclusion bodies Because of the extremely strong hydrophobicity, rhRnR expressed in E. coli formed inactive inclusion bodies, which
Figure 2. Chromatograms of rhRnR inclusion bodies extracted with three different solutions separated by WAX. The rhRnR inclusion bodies were dissolved in a: 8.0 mol/L urea; b: 5.0 mol/L urea containing 1% SLS; c: 8.0 mol/L urea containing 1% TritonX-100, respectively. Chromatographic conditions: stationary phase: DEAE Sepharose FF; mobile phase A: 20 mmol/L Tris 1 4.0 mol/L urea, pH 7.5; mobile phase B: mobile phase A containing 1.0 mol/L NaCl, pH 7.5; linear gradient elution for a and b: 0 to 5 min, 0%B; 5 to 35 min, 0%B to 100%B; linear gradient elution for c: 0 to 10 min, 0%B; 10 to 40 min, 0%B to 100%B; Flow rate: 1.0 mL/min; Detection wavelength: 280 nm. (*): Target protein.
were insoluble in water and could only be dissolved with solutions containing high concentrations of denaturant, such as 7.0 mol/L guanidine hydrochloride (GuHCl) or 8.0 mol/L urea. To purify rhRnR from the inclusion bodies formed in E. coli, we first determined the conditions for dissolving the inclusion bodies. Firstly, the inclusion bodies of rhRnR were dissolved in 8.0 mol/L urea. In general, the detergents are always used to solubilize the membrane proteins.12 To enhance the solubility of the inclusion bodies and to inhibit the aggregation of rhRnR, two types of detergents were added to the urea solution as the additives for dissolving the inclusion bodies: Triton X100 and sodium lauroyl sareosine (SLS). The SDS-PAGE analysis of the three extractions of rhRnR inclusion bodies is shown in Figure 1. In addition, the three types of extractions of rhRnR inclusion bodies were injected directly into the column and separated with WAX, respectively. The chromatograms are shown in Figure 2. The comparisons of the purity,
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Table 1. Comparison of the Protein Content, Purity, and Mass Recovery of rhRnR Extracted with Three Different Extractions Between Before and After Purification with WAX
Solutions Aim protein concentration (mg/mL) Purity of rhRnR (%) Purity of rhRnR after purification (%) Mass recovery (%)
8.0 mol/L Urea
5.0 mol/L Urea 1 1% SLS
8.0 mol/L Urea 1 1% Triton X-100
10.9
13.3
13.5
60.2 94.7
79.3 97.0
56.2 68.4
43.1
31.3
21.1
the content and mass recovery of the aim protein before and after purification with WAX are also listed in Table 1. From Figure 1 and Table 1, to compare the three extraction methods for dissolving the inclusion bodies, the result indicated that the least impurities of the extraction with 5.0 mol/L urea containing 1% SLS was observed, and the purity of rhRnR was 79.3% (Lane 2). On the contrary, when 1% Triton X-100 was added to 8 mol/L urea as the additive to dissolve the rhRnR inclusion bodies, much more impurities were extracted together with aim protein and the purity of rhRnR was only 56.2% (Lane 3). From Figure 2, it could be observed that when the detergents were added in the extraction solutions as the additives, the peak shapes of the target protein (marked with the asterisk) became distorted and multiple peaks appeared in Figures 2b,c. Only when 8.0 mol/L urea was used to dissolve the inclusion bodies did the peak shape become more symmetric and the highest mass recovery (43.1%) and purity (94.7%) of the target protein were obtained after purification with WAX. As a result, 8.0 mol/L urea was used to extract the rhRnR inclusion bodies.
Application of IEC to simultaneous refolding and purification of rhRnR Many types of recombinant proteins have been produced in E. coli resulting in the formation of inclusion bodies. The challenge is how to convert the insoluble inclusion body proteins into soluble and bioactive proteins. However, activity recovery in general is only 5% to 20% when refolding of denatured proteins using traditional dilution and dialysis methods. In addition, the impure proteins were difficult to separate. Therefore, protein refolding or renaturation has become the bottleneck during the production of recombinant proteins by biotechnology. As mentioned above, LC should be a powerful tool for protein refolding with high yield and is becoming one of the most interesting and exciting protein refolding methods developed in recent years; it is referred to as protein folding liquid chromatography (PFLC).14 It was reported that ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), affinity chromatography (AFC) and size exclusion chromatography (SEC) have been employed for PFLC at laboratory or industrial scales.15 The target proteins may be purified simultaneously during the protein refolding by PFLC. Now IEC has become one of the most frequently used methods in PFLC. In this work, refolding rhRnR with simultaneous purification with relatively high yield was set as the aim. Due to its isoelectric point at approximately 5.3, anion exchange chromatography (AEC) should be used to obtain an effective retention. In the
presented work, weak anion exchange chromatography (WAX) with a stationary phase DEAE Sepharose FF was used to refold rhRnR expressed in E. coli with simultaneous purification. When the sample solution containing rhRnR in 8.0 mol/L urea was loaded onto the IEC column, the denatured rhRnR was captured by the stationary phase. The nonretarded or very weakly retained proteins were eluted out the column directly. With the mobile phase flowing through the column, the urea in the denatured protein solution flowed out of the column without any retention. Thus the urea concentration surrounding the rhRnR molecules reduced, initiating the collapse of rhRnR molecules. The electrostatic interaction between the denatured rhRnR molecules and the IEC stationary phase suppressed the non-specific association of the unfolded molecules or incompletely folded molecules, preventing rhRnR molecules from aggregating with each other. Actually, the operation of both PFLC and normal LC are essentially the same. A sample solution containing the recombinant protein is directly injected into a suitable chromatographic column, and then the fractions containing the renatured target protein are collected. Because both refolding and purification are accomplished by PFLC, the chromatographic conditions must be carefully optimized. Therefore, several factors that influence the refolding yield of rhRnR, including urea concentration in the mobile phase, pH and sample size, were investigated in detail in subsequent experiments. Effect of pH The pH of the mobile phase is the significant factor in the IEC system because the pH value of the system strongly affects the retention of proteins by altering the net charge on both the protein and the stationary phase.31 rhRnR can be separated with the WAX column because it is an acid protein with an isoelectric point (pI) of 5.3. As is well-known, electrostatic interaction is the main interaction in ion exchange chromatography, proteins can be separated in the solution with a certain pH based on the difference of net charges. Therefore, the pH value of the mobile phase solution has a great effect on the retention behaviors of proteins in a chromatographic process. As a result, the refolding and purification of rhRnR depends on the pH of the mobile phase solution. The rhRnR extract was separated with different pHs of the mobile phase on the WAX column, and the chromatograms were shown in Figure 3. All of the eluted fractions were collected and analyzed by SDS-PAGE, in which the target rhRnR protein was found as the peak indicated with the asterisk in Figure 3. Furthermore, the mass recovery and the purity of rhRnR at different pH are shown in Figure 4. The two figures illustrated that a higher chromatographic peak was obtained and a better result was achieved when a pH of 7.5 was used. The purity of rhRnR rose to 94.8% from that of 60.2% in rhRnR inclusion body extract, and mass recovery was obtained to be 43.2%. Effect of urea concentration in mobile phase Although PFLC has so many advantages, several problems still exist. If proteins have strong hydrophobicity, some aggregates may form when loading the denatured protein solutions onto an LC column, resulting in increased back pressure of the employed column, which may even block it.
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Figure 3.
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The effects of pH on the separation of rhRnR with WAX. Chromatographic conditions: mobile phase A:20 mmol/L Tris14.0 mol/L urea; mobile phase B: 20 mmol/L Tris 1 4.0 mol/L urea 1 1.0 mol/L NaCl; (1) pH 6.5; (2) pH 7.0; (3) pH 7.5; (4) pH 8.0; detection wavelength: 280 nm; linear gradient: 100% A to 100% B; sample size: 100 lL; flow rate: 1.0 mL/ min; (*): target protein.
Figure 5. Comparisons of the chromatograms of rhRnR separated with WAX under the different concentrations of urea in the mobile phase. Chromatographic conditions: mobile phase A: 20 mmol/L Tris 1 1 to 5.0 mol/L urea, pH 5 7.5; mobile phase B: 20 mmol/L Tris 1 1 to 5.0 mol/L urea 1 1.0 mol/L NaCl, pH 5 7.5; urea concentration: 1, 1.0 mol/L; 2, 2.0 mol/L; 3, 3.0 mol/L; 4, 4.0 mol/L; 5, 5.0 mol/L; linear gradient: 100% A to 100% B, 30 min; sample size: 180 lL; flow rate: 1.0 mL/min; detection wavelength: 280 nm. (*): Target protein.
Figure 4. Effects of the different pH of the mobile phase on the mass recovery and purity of rhRnR separated with WAX. 䊏: mass recovery; ~: purity.
Figure 6. Purity and mass recovery of rhRnR separated by WAX with different urea concentrations in mobile phase. 䊏: mass recovery; ~: purity.
Additionally, mass and bioactivity recoveries of the target protein would decrease. rhRnR was refolded and purified in 30 min by one step on the WAX column, but its mass recovery was lower. This decrease might be caused by the aggregation of denatured rhRnR on the column. It is well known that urea is a strong denaturant for proteins as well as an effective blocking agent against the aggregation of protein molecules. It was reported that the refolding efficiency of recombinant protein could be dramatically increased by a gradually changing concentration of urea in the mobile phase,32 because the addition of urea is able to effectively prevent the serious aggregation of protein caused by the sudden separation from the denaturant. The effects of various concentrations of urea in the mobile phase on the refolding with simultaneous purification of rhRnR were also investigated in detail, and the result is shown in Figure 5. The mass recovery and the purity of rhRnR with the different urea concentration in the range from 1.0 to 5.0 mol/L are presented in Figure 6. The results indicated that rhRnR could not be eluted or partially eluted with lower urea
concentrations in the mobile phase leading to the lower mass recovery. With the increase in urea concentration in the mobile phase from 1.0 to 5.0 mol/L, the chromatographic peak of rhRnR became higher and the mass recovery increased. With the presence of 4.0 mol/L of urea in the mobile phase, the peak of rhRnR was the highest, and the mass recovery reached 41.0%. However, when the urea concentration was increased to 5.0 mol/L, a lower purity of 87.1% of rhRnR was obtained because of the co-eluting of impurities. As a result, 4.0 mol/L urea was the best choice. Effect of sample size The effects of sample sizes on the purification and refolding of rhRnR were also investigated. The different sample sizes, such as 100 lL, 200 lL, 300 lL, 400 lL and 500 lL of rhRnR extracts were loaded directly onto the column, and a linear gradient elution was performed. The chromatograms
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Figure 7.
The effects of sample sizes of rhRnR extract loaded onto the column on the separation of rhRnR with WAX. Chromatographic conditions: mobile phase A: 20 mmol/L Tris 1 4.0 mol/L urea, pH 5 7.5; mobile phase B: 20 mmol/L Tris 1 4.0 mol/L urea 1 1.0 mol/L NaCl, pH57.5; sample size: 1, 100 lL; 2, 200 lL; 3, 300 lL; 4, 400 lL; 5, 500 lL; linear gradient: 100% A to 100% B, 30 min; flow rate: 1.0 mL/min; detection wavelength: 280 nm. (*): target protein.
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Figure 9. SDS-PAGE and western blot analysis of rhRnR separated by WAX. Purified rhRnR was analyzed by SDS-PAGE (a) and western blot (b). Lane 1: molecular weight marker (from bottom to top 14,400, 20,100, 31,000, 43,000, 66,200, 97,400 Da); 2: rhRnR extract; 3: rhRnR after refolding and purification by WAX.
phase was 4.0 mol/L, the pH was 7.5 and the sample size was 400 lL. The chromatogram and the results of SDSPAGE analysis are shown in Figures 8 and 9a, respectively. Western blot analysis indicated that the purified rhRnR can bind successfully to anti-FLAG M2 antibody and the purity of renatured rhRnR was further demonstrated as no higher molecular weight multimers were observed in Figure 9b. As a result, rhRnR could be purified successfully within 30 min by one step, and the purity of 94.8% and mass recovery of 71.8% were achieved. Binding assay using SPR
Figure 8. Chromatograms of rhRnR under the optimal conditions by WAX. Chromatographic conditions: mobile phase A: 20 mmol/L Tris 1 4.0 mol/L urea, pH 5 7.5; mobile phase B: 20 mmol/L Tris 1 4.0 mol/L urea 1 1.0 mol/L NaCl; pH 5 7.5; sample size: 400 lL; (*): target protein; detection wavelength: 280 nm; liner gradient: 100% A to 100% B; flow rate: 1.0 mL/min.
of rhRnR separated by WAX with loading different sample sizes are shown in Figure 7. Although the purities of all cases above were obtained to be more than 94%, the mass recoveries of rhRnR with different sample sizes were determined to be 43.2%, 39.6%, 69.8%, 71.8% and 69.2%, respectively. The result indicated that the mass recovery of rhRnR separated with WAX increased with increasing of the sample size loaded on the column. The average mass recovery of rhRnR was 70%. Thus, the sample size was selected to be 400 lL.
Purification of rhRnR under optimal conditions rhRnR was finally purified and refolded by WAX under optimal conditions when the urea concentration in the mobile
Unfortunately, there is no suitable and standardized bioactivity assay method for rhRnR, the specific activity of rhRnR renatured with WAX cannot currently be determined. However, the specific binding assay was available for purified rhRnR using SPR. After the desalting process, rhRnR was dissolved in PBST to make an aqueous solution of purified rhRnR with different concentrations. Considering the fact that aggregation and precipitation will occur in aqueous rhRnR solution at concentrations higher than 5.0 lg/mL, the binding ability of rhRnR to recombinant human renin was tested by injecting different concentrations of the purified rhRnR (0–0.13 lmol/L, i.e., 5.0 lg/mL) over a recombinant human renin-immobilized sensor chip (Figure 10).7 A surface lacking immobilized human renin was used as a control. It was observed that the response increased with the concentration of rhRnR solution, which clearly demonstrated that renatured rhRnR can bind specifically to renin even when the concentration is very low. The association constant33 of rhRnR to recombinant human renin was calculated to be 3.25 3 108 L/mol. It is well known that the binding ability is essential and crucial for the renin receptor to perform its biological function. Although there is no currently available standardized bioactivity assay, successful binding of rhRnR to renin can be a strong proof that rhRnR has already been renatured and simultaneously purified only by one step with WAX.
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Figure 10. Binding assay of rhRnR to recombinant human renin immobilized onto to a sensor chip using SPR. Solution of purified rhRnR with different concentrations (a: 0.0051 lmol/L, b: 0.013 lmol/L, c: 0.026 lmol/L, d: 0.13 lmol/L) were injected into a sensor chip immobilized with human renin, respectively. Unreacted carboxymethyl groups of a sensor chip lacking immobilized protein were blocked with ethanolamine as a control for non-specific binding. Sensorgram overlays of response difference between the left and right channel of the sensor chip of SPR are shown.
Conclusions rhRnR expressed by E. coli could be refolded and purified simultaneously with WAX. By using PFLC for the refolding and purification of rhRnR, the process became simpler and easier and could be achieved in 30 min by one step. The mass recovery was 71.8%. The purity of rhRnR increased greatly from 60.2% to 94.8%, which was further testified by western blotting analysis, and no multimers were observed after chemiluminescence. Although there are no currently suitable methods for the analysis of the bioactivity of rhRnR, the inactive and denatured inclusion bodies of rhRnR could be refolded to soluble forms with high purity by PFLC, and the specific binding ability of rhRnR to recombinant human renin was determined by SPR assay. The association constant of rhRnR to renin was calculated to be 3.25 3 108 L/ mol, which demonstrated that rhRnR could be renatured and purified simultaneously after this “one step” chromatographic process. Therefore, PFLC is a powerful tool not only for the separation but also for the refolding with simultaneously purification of recombinant proteins expressed in E. coli.
Acknowledgments The plasmid of rhRnR was donated by Prof. Weiming Gong (Institute of Biophysics, Chinese Academy of Science, Beijing, China). This work is supported by the National 863 Program (No. 2006AA02Z227), the National Natural Science Foundation in China (21006077), the Natural Science Foundation of Shaanxi Province (2011JZ002), the Foundation of Key Laboratory in Shaanxi Province (2010JS103, 11JS097).
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