Onestep refolding and purification of recombinant

Research article Received: 2 March 2014,

Revised: 12 May 2014,

Accepted: 21 May 2014

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bmc.3276

One-step refolding and purification of recombinant human tumor necrosis factor-α (rhTNF-α) using ion-exchange chromatography Yan Wang, Wenxuan Ren, Dong Gao, Lili Wang, Ying Yang and Quan Bai* ABSTRACT: Protein refolding is a key step for the production of recombinant proteins, especially at large scales, and usually their yields are very low. Chromatographic-based protein refolding techniques have proven to be superior to conventional dilution refolding methods. High refolding yield can be achieved using these methods compared with dilution refolding of proteins. In this work, recombinant human tumor necrosis factor-α (rhTNF-α) from inclusion bodies expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography with a DEAE Sepharose FF column. Several chromatographic parameters influencing the refolding yield of the denatured/reduced rhTNF-α, such as the urea concentration, pH value and concentration ratio of glutathione/oxidized glutathione in the mobile phase, were investigated in detail. Under optimal conditions, rhTNF-α can be renatured and purified simultaneously within 30 min by one step. Specific bioactivity of 2.18 × 108 IU/mg, purity of 95.2% and mass recovery of 76.8% of refolded rhTNF-α were achieved. Compared with the usual dilution method, the ion exchange chromatography method developed here is simple and more effective for rhTNF-α refolding in terms of specific bioactivity and mass recovery. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: recombinant human tumor necrosis factor-α; inclusion bodies; ion exchange chromatography; protein refolding; protein folding liquid chromatography

Introduction Many kinds of recombinant proteins have been produced through different hosts (Desai et al., 2010). Escherichia coli (E. coli) is one of the most used microbial expression systems in biotechnology owing to its well-characterized genetics, very high expression level and ease of manipulation (Graumann and Premstaller, 2006; Huang et al., 2012). However, the overexpression of these proteins in E. coli often results in the accumulation of the protein product to form inactive and insoluble deposits inside the cells, called the inclusion bodies (Singh and Panda, 2005). The challenge is how to convert the insoluble inclusion body proteins into soluble bioactive ones, or protein in native state, that is, solving the problem of protein refolding (renaturation). The typical strategy used to recover an active protein from inclusion bodies usually involves three key steps: first, isolation and washing of inclusion bodies; second, solubilization of the aggregated protein; and finally, refolding of the solubilized protein. The refolding process is even more significant, and should be considered carefully to improve recovery of the bioactive protein. The inclusion body is usually insoluble in water. The solubilization is possible through the introduction of high concentrations of denaturing and reducing agents, such as 7.0 mol/L guanidine hydrochloride (GuHCl) or 8.0 mol/L urea and dithiothreitol (DTT) to unfold the protein chain. Protein refolding is achieved by reducing the concentration of denaturant in the solubilized protein solution, allowing the soluble protein to refold its correct structure. The methods frequently used for the renaturation of inclusion body include dilution and dialysis. Although these methods are simple, the recoveries of the mass and bioactivity of target protein obtained are very low. Furthermore, the large volume of solution after dilution causes great difficulty for subsequent chromatographic purification steps.

Biomed. Chromatogr. (2014)

Recently, the refolding protein based on liquid chromatography (LC) has drawn great attention and several reviews can be found in the literature (Geng and Wang, 2007, 2008; Jungbauer and Kaar, 2007; Jungbauer et al., 2004). When it is used in protein folding, the bioactivity recovery increases, 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 the ideal refolding method, named as protein folding liquid chromatography (PFLC). The advantages of the 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. Today it has become a very popular technique for protein folding (Geng and Wang, 2007, 2008; Jungbauer and Kaar, 2007; Jungbauer et al., 2004). PFLC has been applied successfully for the * Correspondence to: Quan Bai, Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Institute of Modern Separation Science, Key Laboratory of Modern Separation Science in Shaanxi Province, Northwest University, Xi’an, 710069, China. Email: [email protected] Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Institute of Modern Separation Science, Key Laboratory of Modern Separation Science in Shaanxi Province, Northwest University, Xi’an, 710069, China Abbreviations used: DTT, dithiothreitol; GSH, glutathione; GSSG, oxidized glutathione; IEC, ion exchange chromatography; PFLC, protein folding liquid chromatography; rhTNF-α, recombinant human tumor necrosis factor-α; SDS, sodium dodecyl sulfate; TNF-α, tumor necrosis factor-α; WAX, weak anion exchange chromatography; OD600, the optical density at 600 nm; TFA, trifluoroacetic acid.

Copyright © 2014 John Wiley & Sons, Ltd.

Y. Wang et al. renaturation and simultaneous purification of some recombinant proteins produced by E. coli (Bai et al., 2003, 2007; Geng et al., 2004; Wang et al., 2007, 2006) and the recovery of bioactivity of these renatured recombinant proteins was increased 2–3 times over that of other common methods. Tumor necrosis factor-α (TNF-α) is a leukocyte-derived protein that exhibits anti-tumor activity in vitro and in vivo. TNF-α not only plays a decisive role in cell proliferation, differentiation, immune regulation and programmed cell death, but also has a very powerful anti-tumor function. Zhang et al. (1999) purified rhTNF-α expressed in E. coli with hydrophobic interaction chromatography and ion exchange chromatography (IEC), the final specific activity of rhTNF-α reached 2.89 × 107 IU/mg and the total recovery was 40.6%. They also investigated the separation and purification of rhTNF-α in detail. Utilizing TNF’s heat stability, rhTNF-α by heating treatment was purified with IEC and size exclusion chromatography. The final specific activity of rhTNF-α was 1.7 × 107 IU/mg (Wang et al., 1999). Zhang et al. (2002) reported that rhTNF-α was purified by salting out and Q-Sepharose FF and S-Sepharose FF. The purity and specific activity of rhTNF-α were achieved 99% and 1 × 109 IU/mg, respectively. Papaneophytou and Kontopidis (2012) expressed human GST-TNF-α fusion protein in a soluble form by E. coli. GST-TNF-α was purified using a glutathione agarose affinity chromatography column. A mutated human TNF-α expressed in E. coli was purified by ammonia sulfate precipitation and two sequential chromatographic steps (Yan et al., 2006). IEC is a widely used chromatographic method for protein separation. It was reported that 70% of protocols for protein purification involved IEC, and IEC now has become one of the most frequently used methods in PFLC. The successful purification with simultaneous renaturation of recombinant proteins by IEC has achieved high mass and bioactivity recovery (Geng and Wang, 2007, 2008; Jungbauer and Kaar, 2007; Jungbauer et al., 2004). In this paper, the renaturation and simultaneous purification of rhTNF-α expressed in E. coli with weak anion exchange chromatography (WAX) is presented, and an alternative process for efficient and simple refolding of rhTNF-α has been developed.

Experimental

purchased from Xi’an Chemical Company (Xi’an, China). Molecular mass marker was obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Water used in the experiments was prepared using a Cascada LC ultra-pure water system (Pall, USA).

Expression of rhTNF-α The engineered pBV-TNF plasmid was transformed into BL21(DE3) E. coli strain. A single colony of the bacterium was inculated into LB medium containing 100 μg/mL ampicillin and grown overnight at 30°C with shaking at 200 rpm, named pre-cultures. The pre-cultures (5%, v/v) were used to seed M9 medium supplemented with 100 μg/mL ampicillin. The cultures were grown at 32°C in a shake flask at 200 rpm for 4 h until the OD600 reached 0.5. Thereafter, the cultures were induced by elevating the temperature to 42°C and further cultured for 5 h. After cultivation, cells were collected by centrifugation at 10,000 rpm for 15 min. Protein expression levels were monitored by SDS–PAGE.

Recovery of rhTNF-α inclusion bodies The harvested cells were washed three times at room temperature with cleaning buffer I (20 mmol/L Tris +1 mmol/L EDTA, pH 7.4), and disrupted by ultrasonication in an ice-water bath, followed by centrifugation at 9000 rpm for 15 min, 4°C. The isolated inclusion bodies were washed with cleaning buffers II (20 mmol/L Tris + 1 mmol/L EDTA + 2.0 mol/L urea + 1.0 mol/L NaCl, pH 7.4), III (20 mmol/L Tris + 1 mmol/L EDTA + 1.0 mol/L NaCl,pH 7.4) and I (20 mmol/L Tris + 1 mmol/L EDTA, pH7.4) three times. After each washing step, the suspension was centrifuged at 14,000 rpm and 4°C for 15 min, the supernatant was discarded. The isolated inclusion bodies were stored at 20°C.

Solubilization of rhTNF-α inclusion bodies The inclusion bodies were suspended in solubilization buffer (20 mmol/L Tris + 8 mol/L urea + 100 mmol/L DTT + 1.0 mmol/L EDTA, pH 8.0) and stirred continuously to solubilize proteins at 4°C overnight. The suspension was centrifuged to remove insoluble material. The solubilized supernatant was recovered and stored at 4°C. Protein concentration in the supernatant was measured to be 8.75 mg/mL according to the Bradford method.

Refolding and purification rhTNF-α by IEC

Equipment A LC-20A high-performance liquid chromatographic instrument (Shimadzu, Japan), including two LC-20 AT νp pumps, a SCL-10A νp system controller, a Rheodyne 7725i manual sample injector, an N2000 chromatographic workstation and an SPD-10A vp UV detector were used. DEAE Sephorase Fast Flow was purchased from General Electric Company (GE Healthcare, USA) and packed into a simple plastic column (50 × 10 mm i.d.) purchased from Shaanxi Xida Kelin Gene-Pharmacy Co. Ltd (Xi’an, China). An ultrasonicator (VCF1500, Sonics, USA) was employed for disrupting the cell pellets. The electrophoresis apparatus was obtained from Bio-Rad Company (USA). A Sorvall RC28S centrifuge (Kendro, USA) was used for centrifugation, and a Cs-9310 dualwavelength thin-layer chromatographic scanner (Shimadzu, Japan) was used for the determination of the purity of rhTNF-α.

All chromatographic experiments were carried out on LC-20A HPLC with an IEC column packed with DEAE Sepharose Fast Flow gel (50 × 10 mm i.d.). The column was equilibrated with solution A for at least 15 min. A 500 μL aliquot of rhTNF-α extract solution (containing 8.75 mg/mL total protein) was directly injected into the column by cumulative sampling, and then a 30 min linear gradient elution of 0–100% buffer solution B was performed at a flow rate of 2.0 mL/min. The detection wavelength was 280 nm. The eluted fractions of the target protein were collected for the measurements of the mass recovery, bioactivity and purity of rhTNF-α. The mobile phase comprised: solution A, 20 mmol/L phosphatebuffered saline (PBS) + 1.0 mmol/L EDTA + 3.0 mol/L urea + 1.8 mmol/L GSH + 0.3 mmol/L GSSG, pH 8.0; solution B, 20 mmol/L PBS + 1.0 mmol/L EDTA + 3.0 mol/L urea + 1.8 mmol/L GSH + 0.3 mmol/L GSSG + 1.0 mol/L NaCl, pH 8.0.

Chemicals

Refolding of rhTNF-α by dilution

Acrylamide and bisacrylamide, reduced glutathione (GSH), oxidized glutathione (GSSG), DTT, triton X-100, sodium dodecyl sulfate (SDS) and tris (hydroxy-methyl) aminomethane (Tris) were purchased from Sigma (St Louis, MO, USA). All other chemicals were analytic grade and

A 500 μL aliquot of rhTNF-α extract was diluted with 10 times the volume of refolding buffer solution (20 mmol/L Tris, 1.0 mmol/L EDTA, 1.8 mmol/L GSH, 0.3 mmol/L GSSG and 20 mmol/L NaCl, pH 8.0). The procedure was performed at 4°C with mild stirring for 24 h.

wileyonlinelibrary.com/journal/bmc



One-step refolding and purification rhTNF-α using IEC Analytical procedure

Effect of pH

The purity of the rhTNF-α was determined by SDS–PAGE and reversedphase liquid chromatography (RPLC). Protein concentration was determined by the Bradford method (Bradford, 1976). The cytotoxic assay of rhTNF-α was measured by the cell lysis assay as described by Aggarwal et al. (1985) towards L929 cell line. The peak of aim protein was collected, and then the protein concentration of the fraction was determined and the purity of aim protein was assayed with SDS–PAGE as described above. The mass recovery was calculated by eqn (1):

Electrostatic interaction is the main interaction in IEC and proteins can be separated in solution with a certain pH based on the difference in net charges. Therefore, 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 (van den Eijnden-van Raaij et al., 1987). In addition, the rhTNF-α molecule has two disulfide bonds. The suitable pH value is important to the formation of disulfide bonds. The refolding buffer pH influences the ionic interactions and redox potential of the system and has been recognized as one of the most influential parameters in protein refolding (Willis et al., 2005). The rhTNF-α extract was separated with different pH of mobile phase on the WAX column, and the chromatograms are shown in Fig. 1. All of the eluted fractions were collected and detected by SDS–PAGE, in which the target protein of rhTNF-α was found as the peak marked with an asterisk in Fig. 1. Furthermore, the specific bioactivity and mass recovery of rhTNF-α at different pH value are shown in Fig. 2. From the two figures, the results indicate that a higher chromatographic peak was obtained and a better result was achieved when a pH of 8 was chosen. In Fig. 2, the specific bioactivity of the refolded rhTNF-α increased from pH 6.0 to 8.0 with a maximum value at pH 8.0, but decreased when the pH was increased further. In addition, the mass recovery of rhTNF-α increased to 58.2% from 35.3% in the range of pH from 6.0 to 8.0, and an obvious decrease was observed from 8.0 to 9.0 because a weak basic environment can enhance the ionization of the thiols present in the cysteines of rhTNF-α molecules and consequently the formation of disulfide bonds. When the mobile phase has a pH >8.0, the risk of forming mis-paired disulfide bonds increases and has little opportunity to rearrange by GSH/GSSG (Li et al., 2002). In Fig. 1, it can be seen that the retention of rhTNF-α becomes stronger with the increase in pH of the mobile phase. This is because rhTNF-α is an acidic protein, and its pI is 5.3. However,

R¼

C 1 V 1 P1 C 2 V 2 P2

(1)

where R is the mass recovery (%), C1 and C2 are protein concentrations of the collected chromatographic fraction and rhTNF-α extract solution (mg/mL), respectively, V1 is the volume of chromatographic fractions (mL), V2 is the sample loading volume (mL), and P1 and P2 are the purities of target proteins in the collected chromatographic fractions and rhTNF-α extract solution (%). Chromatographic condition of RPLC. The C18 column (VP-ODS, 150 × 4.6 mm i.d.; Shimadzu, Japan) was equilibrated with mobile phase A (H2O + 0.1% TFA), and then the target protein was eluted with a linear gradient from 0 to 65% of mobile phase B (CH3OH + 0.1% TFA) for 20 min with a delay for 10 min. The flow rate was 1.0 mL/min and the detection wavelength was 280 nm.

Results and discussion As mentioned above, PFLC is one of the most interesting and exciting protein refolding methods developed in recent years. IEC, hydrophobic interaction chromatography, affinity chromatography and size exclusion chromatography have been employed for PFLC at laboratory or industrial scales. In this work, because rhTNF-α is an acid protein with an isoelectric point (pI) of 5.3, WAX with a stationary phase DEAE Sepharose FF was used to refold and purify simultaneously rhTNF-α expressed in E. coli. When the sample solution containing rhTNF-α in 8.0 mol/L urea was loaded onto the IEC column, the denatured rhTNF-α was captured by the stationary phase. The nonretained 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 rhTNF-α molecules reduced, initiating the collapse of rhTNF-α molecules. The electrostatic interaction between the denatured rhTNF-α molecules and the IEC stationary phase suppressed the nonspecific association of the unfolded molecules or incompletely folded molecules, preventing rhTNF-α molecules from aggregation with each other. Actually, the operation of both PFLC and normal LC is 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 should be considered by PFLC, the chromatographic conditions must be carefully optimized. Therefore, several factors influencing the refolding yield of rhTNF-α, including urea concentration in mobile phase, pH and the concentration ratio of GSH/GSSG, were investigated in detail in subsequent experiments.


Figure 1. Effects of the different pH of mobile phase on the separation of recombinant human tumor necrosis factor-α (rhTNF-α) with weak anion exchange chromatography (WAX). Conditions: stationary phase, DEAE Sepharose F F; mobile phase A, 20 mmol/L KH2PO4 + 1.0 mmol/L EDTA; mobile phase B, mobile phase A containing 1.0 mol/L NaCl; linear gradient elution, 100% A–100% B for 30 min; flow rate, 2.0 mL/min; detection wavelength, 280 nm. 1, pH 6.0; 2, pH 7.0; 3, pH 8.0; 4, pH 9.0. * Target protein.



Y. Wang et al.

Figure 2. Effect of the different pH in mobile phase on the specific activity, purity and mass recovery of rhTNF-α renatured by ion exchange chromatography (IEC). (▲) Specific bioactivity; (■) mass recovery; (●) purity.

too high a pH results in an irreversible adsorption and may cause the degradation of peptides chains (Wetlaufer and Xie, 1995). Therefore, the two figures illustrate that a higher chromatographic peak can be obtained and a better result is achieved when a pH of 8.0 is chosen.

Figure 3. Chromatograms of the renatured and purified rhTNF-α under different concentration ratios of glutathione/oxidized glutathione (GSH/GSSG) by IEC. Conditions: stationary phase, DEAE Sepharose F F; mobile phase A, 20 mmol/L KH2PO4 + 1.0 mmol/L EDTA + 0.3–3.0 mmol/L GSH + 0.3 mmol/L GSSG, pH 8.0; mobile phase B, mobile phase A containing 1.0 mol/L NaCl; linear gradient elution, 100% A–100% B for 30 min; flow rate, 2.0 mL/min; detection wavelength, 280 nm. GSH/GSSG ratio, 1, 1:1; 2, 3:1; 3, 6:1; 4, 10:1. * Target protein.

The effect of the different concentration ratios of GSH/GSSG in mobile phase The rhTNF-α molecule contains two disulfide bonds. When the inclusion body is dissolved by the buffer containing DTT, the disulfide bond in rhTNF-α is reduced and becomes two hydrosulfide groups. Thus the native rhTNF-α with high bioactivity can be obtained with correct formation of the disulfide bonds in the molecule. With provision of the appropriate redox surroundings by adding oxidant and reductant with certain concentration in the refolding buffer solution, such as GSH/GSSG, the active sulfhydryls will form disulfide bonds quickly and correctly; this will also promote the refolding of proteins greatly (Clark, 2001). Therefore, the effects of the concentration and ratio of GSH/GSSG on refolding and purification of rhTNF-α were studied in detail. The chromatograms of rhTNF-α with various concentration ratios of GSH/GSSG in mobile phase are shown in Fig. 3, and the specific bioactivity and mass recovery of rhTNF-α, measured after one step renaturation and simultaneous purification by WAX on the above optimal pH is also shown in Fig. 4. The results indicate that both the specific bioactivity and mass recovery of refolded rhTNF-α by WAX increased with the increased ratio of GSH to GSSG from 1:1 to 10:1. When GSH/GSSG was added in the mobile phase with a concentration ratio of 6:1, the best renaturation efficiency of rhTNF-α was obtained. The specific bioactivity and mass recovery of rhTNF-α were increased to 2.16 × 108 IU/mg and 61.2% from 1.48 × 107 IU/mg and 55.1%, respectively. This result demonstrates that rhTNF-α can be renatured completely with the correct disulfide bonds when GSH/GSSG is added to the mobile phase. It also proves that the addition of GSH/GSSG in the appropriate ratio can help the formation of disulfide bonds and the refolding of rhTNF-α.


Figure 4. Effect of the different concentration ratios of GSH/GSSG on the specific activity, purity and mass recovery of rhTNF-α by IEC. (▲) Specific bioactivity; (■) mass recovery; (●) purity.

The effect of urea concentration in mobile phase Although PFLC has so many advantages, several of 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, perhaps even blocking it. Additionally, the mass and bioactivity recoveries of the target protein will decrease. The urea is not only a strong protein denaturing agent, but also an effective agent for inhibiting the aggregation of protein molecules. When urea is added in the mobile phase, it can diminish the aggregation of denatured protein so that a higher mass recovery can be achieved (De Bernardez Clark et al., 1998; Hagen et al., 1990). It was reported that the refolding efficiency of recombinant protein could be dramatically increased by gradually changing the concentration of urea in the mobile phase (Freydell et al., 2010). The effects of various concentrations of urea in the mobile phase on the refolding with simultaneous



One-step refolding and purification rhTNF-α using IEC purification of rhTNF-α were also investigated in detail, and the result is shown in Fig. 5. The specific bioactivity and mass recovery of rhTNF-α with the different urea concentrations in the range from 1.0 to 5.0 mol/L are presented in Fig. 6. The results indicate that the chromatographic peak of rhTNF-α becomes higher with the increase in urea concentration in mobile phase. With the presence of 3.0 mol/L of urea in the mobile phase, the peak of rhTNF-α becomes higher and sharper (shown as curve 3 in Fig. 5). Figure 6 shows the specific bioactivity and mass recovery of rhTNF-α after adding urea to the mobile phase. It produces the same result as shown in Fig. 5. The lower concentration of urea can not only inhibit the aggregation of protein molecules, but also increases the mass recovery of protein. In contrast, the protein can be denatured in the higher concentration of urea solution. Therefore, the peak height of rhTNF-α decreases when the concentration of urea in mobile phase is >3.0 mol/L. Under the optimal chromatographic condition with

3.0 mol/L urea, the mass recovery of rhTNF-α increases from 62.4 to 76.8%, and the specific bioactivity can reach 2.18 × 108 IU/mg.

The renaturation and purification of rhTNF-α under optimal conditions Based on the discussion above, the optimal conditions of the renaturation and purification of rhTNF-α extracted with 8.0 mol/L urea solution by WAX were as follows: stationary phase, DEAE Sepharose FF; mobile phase A, 20 mmol/L KH2PO4 + 1.0 mmol/L EDTA + 1.8 mmol/L GSH + 0.3 mmol/L GSSG + 3.0 mol/L urea, pH 8.0; mobile phase B, 20 mmol/L KH2PO4 + 1.0 mmol/L EDTA + 1.8 mmol/L GSH + 0.3 mmol/L GSSG + 1.0 mol/L NaCl + 3.0 mol/L urea, pH 8.0; linear gradient elution, 100% A–100%B for 30 min; flow rate, 2.0 mL/min; detection wavelength, 280 nm. Under optimal chromatographic conditions, rhTNF-α extracted with 8.0 mol/L urea solution could be renatured and purified simultaneously by WAX in 30 min in one step (shown in Fig. 7). The purity of rhTNF-α was measured to be 95.8% with SDS–PAGE (shown in Fig. 8). The fractions containing the rhTNF-α protein were collected, followed by purification using RPLC. As shown in Fig. 9, the eluted rhTNF-α protein showed a single peak after RPLC, suggesting that the purified product had a purity of 95.9%. Its specific bioactivity and mass recovery were 2.18 × 108 IU/mg and 76.8%, respectively.

Figure 5. Chromatograms of the renatured and purified rhTNF-α under different urea concentrations by IEC. Conditions: stationary phase, DEAE Sepharose F F; mobile phase A, 20 mmol/L KH2PO4 + 1.0 mmol/L EDTA + 1.8 mmol/L GSH + 0.3 mmol/L GSSG + 1.0–5.0 mol/L urea, pH 8.0; mobile phase B, mobile phase A containing 1.0 mol/L NaCl; linear gradient elution, 100% A–100%B for 30 min; flow rate, 2.0 mL/min; detection wavelength, 280 nm. Urea concentration (mol/L): 1, 1.0; 2, 2.0; 3, 3.0; 4, 4.0; 5, 5.0. * Target protein. Figure 7. The chromatography of rhTNF-α refolding with simultaneous purification by WAX. Conditions: stationary phase, DEAE Sepharose F F; mobile phase A, 20 mmol/L KH2PO4 + 1.0 mmol/L EDTA + 1.8 mmol/L GSH + 0.3 mmol/L GSSG + 3.0 mol/L urea, pH 8.0; mobile phase B, mobile phase A containing 1.0 mol/L NaCl; linear gradient elution, 100% A–100% B for 30 min; flow rate, 2.0 mL/min; detection wavelength, 280 nm. * Target protein.

Figure 6. Effect of the different concentrations of urea in mobile phase on the specific bioactivity, purity and mass recovery of rhTNF-α renatured by IEC. (▲) Specific bioactivity; (■) mass recovery; (●) purity.


Figure 8. SDS–PAGE analysis of rhTNF-α refolded by WAX under optimal conditions. Lane 1, marker, lysozyme; lane 2, rhTNF-α expressed in E. coli; lane 3, rhTNF-α inclusion bodies extract; lane 4 and 5, rhTNF-α refolded with simultaneous purification by WAX.



Y. Wang et al. Province (2011JZ002) and the Foundation of Key Laboratory in Shaanxi Province (2010JS103, 11JS097, 2013SZS18-K01).

References

Figure 9. RPLC analysis of rhTNF-α refolded and purified with WAX. Conditions: stationary phase, C18 column (150 × 4.6 mm, i.d.); mobile phase A, H2O + 0.1% TFA; mobile phase B, CH3OH + 0.1% TFA; linear gradient elution, 0–65% B for 20 min; flow rate, 1.0 mL/min; detection wavelength, 280 nm. * Target protein.

Comparison between dilution and PFLC methods for the refolding of rhTNF-α Dilution is the usual method for the refolding of denatured proteins. It is performed in a large volume of refolding buffer solution to decrease the concentration of denaturants, so that denatured proteins can fold spontaneously. In this work, a comparison between dilution refolding and PFLC method was conducted. The PFLC refolding was carried out with a WAX column under the optimal conditions, and the dilution refolding was carried out following the procedures described above. When rhTNF-α was refolded with WAX, higher specific bioactivity of 2.18 × 108 IU/mg and a mass recovery 76.8% were achieved. However, for the dilution method, the amount of rhTNF-α extract loaded onto the WAX column was diluted with 10 times the volume of refolding buffer solution at 4°C with mild stirring for 24 h. The specific bioactivity and mass recovery of rhTNF-α were obtained as 5.45 × 106 IU/mg and 32.2%, respectively. This proves that the PFLC method for the renaturation of rhTNF-α has advantages over the dilution method, and higher specific bioactivity and mass recovery of rhTNF-α can be achieved.

Conclusion Based on the results obtained in this work, the conclusion can be drawn that rhTNF-α expressed by E. coli can be refolded and purified simultaneously with WAX. Using PFLC for the renaturation and purification of rhTNF-α, the process becomes simpler and easier, and can be achieved in 30 min in one step. The refolded rhTNF-α had a purity of 95.2% and a bioactivity of 2.18 × 108 IU/mg, and a mass recovery of 76.8% could be achieved. The moderate concentration of urea, an appropriate pH and the ratio of GSH/GSSG are very important for this refolding process. The PFLC method presented was found to be better than the dilution method in terms of the level of specific bioactivity and mass recovery. Therefore, PFLC is a powerful tool not only for separation, but also for the renaturation of recombinant proteins.

Acknowledgments This work was supported by the National 863 Program (no. 2006AA02Z227), the National Natural Science Foundation in China (21006077), the Natural Science Foundation of Shaanxi


Aggarwal BB, Kohr WJ, Hass PE, Moffat B, Spencer SA, Henzel WJ, Bringman TS, Nedwin GE, Goeddel DV and Harkins RN. Human tumor necrosis factor. Production, purification, and characterization. Journal of Biological Chemistry 1985; 260: 2345–2354. Bai Q, Kong Y and Geng XD. Studies on renaturation with simultaneous purification of recombinant human proinsulin from E. coli with high performance hydrophobic interaction chromatography. Journal of Liquid Chromatography and Related Technologies 2003; 26: 683–695. Bai Q, Chen G, Liu J and Geng XD. Renaturation and purification of rhGM-CSF with ion-exchange chromatography. Biotechnology Progress 2007; 23: 1138–1142. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein– dye binding. Analytical Biochemistry 1976; 72: 248–254. Clark EDB. Protein refolding for industrial processes. Current Opinion in Biotechnology 2001; 12: 202–207. De Bernardez Clark E, Hevehan D, Szela S and Maachupalli-Reddy J. Oxidative renaturation of hen egg-white lysozyme. Folding vs aggregation. Biotechnology Progress 1998; 14: 47–54. Desai PN, Shrivastava N and Padh H. Production of heterologous proteins in plants: strategies for optimal expression. Biotechnology Advances 2010; 28: 427–435. Freydell EJ, van der Wielen L, Eppink M and Ottens M. Ion-exchange chromatographic protein refolding. Journal of Chromatography A 2010; 1217: 7265–7274. Geng XD and Wang CZ. Protein folding liquid chromatography and its recent developments. Journal of Chromatography B 2007; 849: 69–80. Geng XD and Wang LL. Liquid chromatography of recombinant proteins and protein drugs. Journal of Chromatography B 2008; 866: 133–153. Geng XD, Bai Q, Zhang YJ, Li X and Wu D. Refolding and purification of interferon-gamma in industry by hydrophobic interaction chromatography. Journal of Biotechnology 2004; 113: 137–149. Graumann K and Premstaller A. Manufacturing of recombinant therapeutic proteins in microbial systems. Biotechnology Journal 2006; 1: 164–186. Hagen AJ, Hatton TA and Wang DIC. Protein refolding in reversed micelles. Biotechnology and Bioengineering 1990; 35: 955–965. Huang H, Lin Z, Lin Y, Sun X, Xie Y, Zhang L and Chen G. Preparation and evaluation of poly(4-vinylphenylboronic acid-co-pentaerythritol triacrylate) monolithic column for capillary liquid chromatography of small molecules and proteins. Journal of Chromatography A 2012; 1251: 82–90. Jungbauer A and Kaar W. Current status of technical protein refolding. Journal of Biotechnology 2007; 128: 587–596. Jungbauer A, Kaar W and Schlegl R. Folding and refolding of proteins in chromatographic beds. Current Opinion in Biotechnology 2004; 15: 487–494. Li M, Zhang GF and Su ZG. Dual gradient ion-exchange chromatography improved refolding yield of lysozyme. Journal of Chromatography A 2002; 959: 113–120. Papaneophytou CP and Kontopidis GA. Optimization of TNF-α overexpression in Escherichia coli using response surface methodology: purification of the protein and oligomerization studies. Protein Expression and Purification 2012; 86: 35–44. Singh SM and Panda AK. Solubilization and refolding of bacterial inclusion body proteins. Journal of Bioscience and Bioengineering 2005; 99: 303–310. van den Eijnden-van Raaij AJM, Koornneef I, van Oostwaard TMJ, de Laat SW and van Zoelen EJJ. Cation-exchange high-performance liquid chromatography: separation of highly basic proteins using volatile acidic solvents. Analytical Biochemistry 1987; 163: 263–269. Wang CZ, Wang LL and Geng XD. Renaturation with simultaneous purification of rhG-CSF from Escherichia coli by ion exchange chromatography. Biomedical Chromatography 2007; 21: 1291–1296. Wang GH, Feng XL, Zhang XJ, Su ZG and Wu SH. Separation and purification of recombinant tumor necrosis factor from Escherichia coli. Chinese Journal of Biotechnology 1999; 15: 270–273.



One-step refolding and purification rhTNF-α using IEC Wang LL, Wang CZ and Geng XD. Expression, renaturation and simultaneous purification of recombinant human stem cell factor in Escherichia coli. Biotechnology Letters 2006; 28: 993–997. Wetlaufer DB and Xie Y. Control of aggregation in protein refolding: a variety of surfactants promote renaturation of carbonic anhydrase II. Protein Science 1995; 4: 1535–1543. Willis MS, Hogan JK, Prabhakar P, Liu X, Tsai K, Wei Y and Fox T. Investigation of protein refolding using a fractional factorial screen: a study of reagent effects and interactions. Protein Science 2005; 14: 1818–1826.


Yan Z, Zhao N, Wang Z, Li B, Bao C, Shi J, Han W and Zhang Y. A mutated human tumor necrosis factor-alpha improves the therapeutic index in vitro and in vivo. Cytotherapy 2006; 8: 415–423. Zhang YQ, Zhao N, Li B, Liu L, Wang ZL, Zhu BE, Yan Z and Su CZ. Preparation of a novel recombinant human tumor necrosis factor-α by gene engineering technology. Chinese Journal of Cellular and Molecular Immunology 2002; 18: 402–405. Zhang ZQ, Feng XL and Su ZG. Seperation and purification of recombinant human tumor necrosis factor-α. Chinese Journal of Biochemical Pharmaceutics 1999; 20: 163–166.

