Bacterial expression and refolding of human trypsinogen

Journal of Biotechnology 109 (2004) 3–11

Bacterial expression and refolding of human trypsinogen Hubertus Hohenblum, Karola Vorauer-Uhl, Hermann Katinger, Diethard Mattanovich∗ Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna, Austria Received 16 November 2002; accepted 14 October 2003

Abstract The expression of recombinant trypsinogens from different mammalian origins in Escherichia coli typically leads to the formation of insoluble aggregates. This work describes the high level expression of human trypsinogen 1 in E. coli using the T7 expression system. Direct expression of trypsinogen was not possible, but the N-terminal fusion of the first 11 amino acids of the T7 protein 10 resulted in an expression level of 200 mg g−1 bacterial dry mass. A refolding procedure was optimized, and a method using continuous feed of denatured product was developed. Thus the working concentration of trypsinogen could be raised four-fold, while the yield of active protein could be maintained at 20–35%. The refolded trypsinogen was converted to trypsin by autocatalytic activation, and the utility for the detachment of mammalian cells in culture was proven. © 2004 Elsevier B.V. All rights reserved. Keywords: Recombinant trypsinogen; Escherichia coli; Inclusion body; Refolding

1. Introduction Trypsin (EC 3.4.21.4) is a supplement to different biotechnological procedures, e.g. for the detachment of surface adherent cells (Merten, 2000), for the production of influenza virus (Kaverin and Webster, 1995) or for proteolytic processing of hormones and other protein products (Thim et al., 1986). The conventional source for trypsin is porcine or bovine pancreas. There is a high demand for recombinant substitutes to avoid in the future the risk of a contamination with any pathogen associated to the donor organism. ∗ Corresponding author. Tel.: +43-1-36006-6569; fax: +43-1-3697615. E-mail address: [email protected] (D. Mattanovich).

Vasquez et al. (1989) have expressed rat trypsin in Escherichia coli using the pho leader sequence, so that the active enzyme was secreted to the periplasm. However, even in an optimized fed batch process the yield was quite low (56 mg l−1 ; Yee and Blanch, 1993). An obvious explanation for this low yield could be a damage of the host cells by the proteolytic activity of the product. When bovine trypsin or trypsinogen was expressed in the cytoplasm of E. coli, the product formed insoluble aggregates, also termed inclusion bodies (Greaney and Rosteck, 1994). Similarly, Kopetzki et al. (1999) and Szilagyi et al. (2001) described the formation of inclusion bodies of human trypsinogen in E. coli, again with a low yield of 30–60 mg l−1 , while Peterson et al. (2001) expressed bovine trypsinogen as inclusion bodies in E. coli at a level of 40 mg l−1 . A different approach was taken by Wöldike and Kjeldsen (1997) by the secretion of

0168-1656/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2003.10.022

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porcine or human trypsinogen from Aspergillus oryzae which resulted in a soluble product. These authors describe that trypsinogen was partially activated during production in this system. However, except for the work of Yee and Blanch (1993) no data on the yields of active product are provided. Renaturing of proteins is a process that has to be optimized in any new case. Plenty of protocols for protein renaturing is available (for recent reviews, see De Bernardez Clark (2001) and Misawa and Kumagai (1999)), which can be divided into two classes (Jaenicke and Rudolph, 1989): first, direct refolding after denaturing (by dilution, dialysis or other buffer exchange methods), and second, procedures that employ an intermediate step to protect reduced thiol groups by formation of S-sulfonate derivatives or mixed disulfides. The latter step can prevent rapid aggregation that occurs frequently with proteins that contain several cystein residues (Light, 1985; De Bernardez Clark et al., 1999). Native bovine trypsinogen has been used by Light et al. (1986) as a model for the in vitro folding of proteins. Their optimized procedure for denaturing and refolding employs the treatment with guanidinium hydrochloride and dithioerythritol (DTE) followed by desalting with size exclusion chromatography and lyophilization. Then the free thiol groups are coupled to glutathione residues by incubation with oxidized glutathione. After desalting as above and dissolving in urea, the protein was refolded in Tris buffer. In principle this approach is identical to that described by Jaenicke and Rudolph (1989). A general concern with refolding procedures in the production scale is dilution, which should be kept as low as possible. The often published dilution protocols work with a final protein concentration of approximately 10 mg l−1 which demands very high volumes and can therefore be economically inviable, even more when the refolding yield is significantly below 100%. One way to overcome this limitation is the pulse-fed dilution procedure (Fischer et al., 1992). By stepwise addition of denatured protein to the renaturing buffer, the actual concentration of unfolded protein is kept low, while the final concentration of refolded product can be significantly increased. Buswell et al. (2002) described an optimized procedure for the pulse fed dilution refolding of recombinant bovine trypsinogen.

The aim of this work was to evaluate the possibility and feasibility of producing recombinant human trypsin with E. coli. In contrast to the work of Vasquez et al. (1989) it was decided not to secrete the enzyme into the bacterial periplasm, but to express it in the cytoplasm and to renature it from inclusion bodies. As denatured trypsin is highly susceptible to degradation by trypsin activity (Walsh, 1970), it was decided to express the inactive precursor trypsinogen. An additional benefit of this strategy is the avoidance of any damages to the host cell by proteolytic activity. For a larger scale process the costs for reagents as well as the feasibility of the processing steps need a different consideration than in the lab scale. Therefore it was decided to use urea as a cheaper chaotrop, and to evaluate a procedure to increase the working concentration of the product during refolding.

2. Materials and methods Unless stated otherwise, all chemicals were purchased from Merck Eurolab (Darmstadt, Germany). 2.1. Strains and vectors The E. coli strain K12 HMS174 (DE3) was used in combination with the plasmid pET3a (Studier et al., 1990), where the trypsinogen 1 gene was inserted. The recombinant gene was inserted either directly downstream of the translation signals into the NdeI restriction site (leading to plasmid pET3atrpA), or after the first 11 codons of the T7 gene 10 into the BamHI site (leading to pET3atrpB). The nucleotide sequences around the cloning site and the deducted amino acid sequences are presented in Fig. 1. Transcription of the trypsinogen gene is controlled by the phage T7 φ10 promoter, and is activated by induction of T7 RNA polymerase, encoded in the host genome under control of the lacUV5 promoter, with isopropyl-␤-d-thiogalactoside (IPTG). 2.2. Media and cultivation E. coli was cultivated in baffled shake flasks in M9LB medium (contents/l: bactotryptone 10 g, yeast extract 5 g, NaCl 5 g, NH4 Cl 1 g, KH2 PO4 3 g,

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Fig. 1. Nucleotide and amino acid sequences of the two expression vectors, pET3atrpA and pET3atrpB. Cloning sites are underlined, Shine–Dalgarno sequences (SD), the T7 gene 10 downstream box (DB) and start codons are boxed. The arrows indicate the proteolytic activation site of trypsinogen.

Na2 HPO4 6 g, glucose 4 g, MgSO4 ·7H2 O 0.25 g). The temperature was 37 ◦ C. When the cultures reached an OD600 = 1.0, 0.4 mg ml−1 IPTG (Gerbu, Gailberg, Germany) was added for induction, and the cultivation was continued for 4 h. After cell lysis with lysozyme and Triton X-100 (0.4% in 10 mM Tris–HCl pH 8.2; 15 mM MgCl2 ), recombinant trypsinogen was either analyzed or harvested as described below. 2.3. Product analysis Recombinant trypsinogen was analyzed by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) on 15% polyacrylamide (Bio-Rad, Hercules, CA, USA) gels with 0.1% SDS (Fluka, Buchs, Switzerland). Samples were boiled for 10 min with 1% SDS and 0.15% dithioerythritol (DTE) before loading to the gel. Protein bands in the gels were visualized by silver staining or Coomassie Blue staining, and—in the latter case—quantified by densitometry. Where appropriate, the trypsin activity was measured with p-tosyl-l-arginine methyl ester (TAME) after activation with bovine enterokinase. Briefly, 50 ␮l sample was incubated with 5 ␮g enterokinase (Sigma, St. Louis, MO, USA) in 50 mM Tris–HCl buffer pH 8.6, containing 50 mM CaCl2 , to activate the trypsinogen, and finally the activity was determined with the TAME assay (Walsh and Wilcox, 1970). Therefore, aliquots of the samples were added to 40 mM Tris–HCl buffer pH 8.1, containing 10 mM CaCl2 and 1 mM TAME (Sigma), and the increase of absorbance at 247 nm was followed.

2.4. Refolding of human trypsinogen from inclusion bodies After cell lysis with Triton X-100 (0.4% in 10 mM Tris–HCl pH 8.2; 15 mM MgCl2 ), the insoluble fraction was centrifuged and washed two times in 3–4 M urea (Gerbu). Then the pellet was resuspended in denaturing solution (9 M urea; 100 mM Tris–HCl pH 8.6; 1 mM EDTA; 10 mM DTE) and incubated for 2.5 h at 37 ◦ C. To remove DTE, the buffer of the supernatant was changed by ultradiafiltration under nitrogen with washing buffer (8 M urea; 100 mM Tris–HCl pH 8.6; 1 mM EDTA) using an Amicon YM10 membrane and an Amicon stirring chamber (Millipore, Bedford, MA, USA). A 10-fold dilution of DTE was sufficient to allow for the subsequent formation of mixed disulfides. Then an equal volume of 200 mM oxidized glutathione (GSSG; Sigma) in 8 M urea was added and the mixture incubated for 3 h. Finally, the buffer was changed again by ultradiafiltration with washing buffer. The optimum dilution of GSSG was 30-fold. The protocol for refolding was optimized based on the dilution method. The best conditions were: refolding buffer: 50 mM Tris–HCl pH 8.6; 50 mM CaCl2 ; 3 mM reduced glutathione (GSH; Sigma); 0.3 mM GSSG; final trypsinogen concentration 10 ␮g ml−1 ; final urea concentration 0.4 M; incubation at 5 ± 2 ◦ C for 3 h under nitrogen atmosphere. With the intention to increase the trypsinogen concentration in the refolding reaction, we designed a continuous dilution reactor based on cross flow ultradiafiltration. The reactor consists of following

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Fig. 2. Schematic setup of the refolding device.

features (Fig. 2): • • • • •

a device for mixing; an ultradiafiltration circuit; a device for feeding sample; a device for feeding buffer; an outlet for product.

As a prototype, a 500 ml glass vessel was adapted with glass nozzles for feeding and removing of solutions and mixed with a magnetic stirrer. Diafiltration was performed over an Amicon Miniplate Bioconcentrator with a YM10 membrane (Millipore). The concentration of denaturing agent is permanently reduced by ultradiafiltration. Therefore the concentration of denaturant can be maintained constant during the operation without the need to dilute the refolding reaction. The amount of filtrate removed from the system (F3 ) is replaced continuously by refolding buffer (F2 ), so that the volume in the reactor is kept constant. Generally, the concentration of denaturing agent will not be reduced to zero, but to an equilibrium level depending mainly on the flux levels. A certain low concentration of chaotropic agent is in most cases beneficial to the refolding rate (as described above). Based on the assumption of a constant volume and no retention of the denaturing agent at the diafiltration membrane (as it is the case for e.g. urea), the residual concentration of denaturing agent is a function of the fluxes of denatured protein and the refolding buffer, and of the initial concentration in the denatured

protein solution (Eqs. (1)–(3)): CF1 F1 + CF2 F2 + CF3 F3 + CF4 F4 = 0

(1)

where CFi is the concentration of denaturing agent at flux i (i = 1–4), Fi the fluxes as described in Fig. 1. In an equilibrium state: CF3 = CF4 = CR

(concentration in the reactor) (2)

If one assumes that F1 = −F4 and CF2 = 0, it follows that F3 CF − C R =− 1 (3) F1 CR Following Eq. (3), the relation of F3 to F1 can be calculated based on the given value of CF1 and the desired value of CR . 2.5. Activation of trypsinogen to trypsin To test whether autocatalytic activation can be initiated with intrinsic trypsin activity of the trypsinogen preparation, the buffer of an aliquot of the refolded material was changed to basic refolding buffer. The trypsinogen concentration of this sample (calculated from the activity measured after enterokinase activation of an aliquot) was 8 ␮g/ml. The sample was incubated and trypsin activity was measured at various time points. 2.6. Endotoxin content To measure the residual endotoxin content of the activated recombinant trypsin preparation, a Limulus

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amoebocyte lysate (LAL) assay was performed (Friberger et al., 1982). 2.7. Functionality/cell detachment After activation the sample was concentrated and the buffer changed to PBS def. (contents/l: NaCl 10 g, KCl 0.25 g, Na2 HPO4 1.44 g, KH2 PO4 0.25 g; pH = 7.2). The final activity was adjusted to that of the porcine trypsin routinely used in the cell culture lab (equal to 31.6 ␮g ml−1 pure trypsin). Afterwards, 0.02% EDTA was added to the recombinant trypsin, and the activity decrease somewhat (equal to 25.7 ␮g ml−1 trypsin). CHO-K1 cells were grown in 25 cm2 Roux bottles. The supernatant was discarded and the cell layer was rinsed with 5 ml PBS def. Then 1 ml of recombinant trypsin, porcine trypsin (Sigma) or PBS def., respectively, was added to each Roux bottle, equally distributed on the cell layer and discarded. The release of the cells from the support was controlled under the microscope and the time was recorded. The released cells were suspended in 10 ml culture medium. Two milliliter were used for viability staining, and another 2 ml were transferred into a new 25 cm2 Roux bottle with 8 ml culture medium and incubated at 37 ◦ C to record the growth after treatment.

3. Results 3.1. Expression levels After inserting the trypsinogen gene directly after the T7 φ10 promoter and the translation initiation signals, no expression of trypsinogen could be observed (Fig. 3). This phenomenon has been observed for other proteins, and could be attributed in some cases to problems with translation initiation due to secondary structure of the mRNA or unfavorable codons downstream of the initiation codon (Looman et al., 1987; De Boer and Hui, 1990). One option to circumvent this problem is the N-terminal fusion of an easily translated gene fragment (Studier et al., 1990). Therefore the trypsinogen gene was fused in frame with its N-terminus to the first 11 amino acids of protein 10

Fig. 3. SDS-PAGE of lysates of E. coli clones carrying trypsinogen expression vectors. Lane 1: pET3atrpA (plasmid construct with trypsinogen gene directly after the T7 promoter); insoluble fraction. Lane 2: pET3atrpB (plasmid construct with trypsinogen gene inserted after the first 11 amino acid codons of T7 protein 10); insoluble fraction. Lane 3: molecular weight standard (kDa): 26.6; 19; 14.4; 6.5; 3.5; 1.4. Lane 4: same as lane 1, soluble fraction. Lane 5: same as lane 2, soluble fraction. Arrow: trypsinogen.

(the major coat protein) of phage T7. A strong positive band of expressed trypsinogen could be observed in the SDS-PAGE then (Fig. 3). The protein appears in the insoluble fraction of the cell lysates and could not be solubilized by treatment with 5% Triton X-100 or up to 5 M urea, but only with high concentrations of chaotropic agents like 8 M urea or 6 M guanidinium hydrochloride. Therefore we concluded that human trypsinogen forms inclusion bodies in E. coli, as it is described for bovine trypsinogen. The amount of produced trypsinogen was measured via densitometry of Coomassie Blue stained SDS-PAGE gels with bovine trypsinogen (Sigma) as a standard. The analysis of total cell lysates resulted in 200 mg g−1 trypsinogen per bacterial dry mass, which is a very high expression level as compared to most E. coli systems (Makrides, 1996), and more than five-fold higher than the expression of human trypsinogen described by Szilagyi et al. (2001). Therefore it was decided to produce the material for the following refolding studies in shake flasks rather than in a fermenter.

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Fig. 4. SDS-PAGE of insoluble and soluble fractions of washing steps. Lane 1: cell lysate, insoluble fraction. Lane 2: after first wash (with 3 M urea), insoluble fraction. Lane 3: after second wash (with 4 M urea), insoluble fraction. Lane 4: cell lysate, soluble fraction. Lane 5: after first wash (with 3 M urea), soluble fraction. Lane 6: after second wash (with 4 M urea), soluble fraction. Arrow: trypsinogen.

3.2. Optimization of inclusion body preparation, denaturing and renaturing of trypsinogen Every step from cell lysis over washing and solubilization of the inclusion bodies to mixed disulfide formation and refolding of the product was optimized. The optimized protocol is described in Section 2. After washing, only one dominant contamination remained visible, and the major fraction of the protein preparation was trypsinogen (Fig. 4). When the denatured, GSSG treated protein was diluted 20-fold in refolding buffer to give a final concentration of 10 ␮g ml−1 , the typical yield of active trypsin was 30–40%. However, the rest remained also in solution, as judged from SDS-PAGE analysis of the insoluble fraction after refolding (Fig. 5).

Fig. 5. SDS-PAGE of soluble and insoluble fractions of refolding by dilution. Lane 1: denatured trypsinogen before refolding; insoluble fraction. Lane 2: refolding step with GSH/GSSG; dilution 1:20, 3 h; insoluble fraction. Lane 3: molecular weight standard (kDa): 26.6; 19; 14.4; 6.5; 3.5; 1.4. Lane 4: denatured trypsinogen before refolding; soluble fraction. Lane 5: refolding step with GSH/GSSG; dilution 1:20, 3 h; soluble fraction. Arrow: trypsinogen.

unit. Filtrate was removed at a rate of 128 ml h−1 , and hence the concentration of urea was kept constant at 0.5 M. Samples were withdrawn every hour. After 5 h the total concentration of trypsinogen in the reactor was 40 ␮g ml−1 , and the yield of active trypsin was 20.3% (Table 1). The same denatured solution was refolded by dilution as a control (final concentration 10 ␮g ml−1 ), and resulted in 19.8% active trypsin (Fig. 6). 3.4. Activation In order to obtain active trypsin without adding an additional enzyme, the refolded trypsinogen solution was incubated at 37 ◦ C for 21 h. Autoactivation of trypsinogen initiated the autocatalytic process

3.3. Continuous feed refolding With the aim to increase the protein concentration in the refolded preparation, we evaluated the replacement of the pulse-fed dilution procedure by a continuous fed process. The refolding vessel was filled with 150 ml refolding buffer, and denatured trypsinogen (120 ␮g ml−1 ) was fed at a rate of 10 ml h−1 , while the reaction mixture was filtered over a diafiltration

Table 1 Total trypsinogen concentration and achievable active trypsin after 3–5 h of continuous flow refolding Time (h)

Total trypsinogen (␮g ml−1 )

Active trypsin (␮g ml−1 )

Yield (%)

3 4 5

24.0 32.0 40.0

3.6 6.1 8.1

15.0 19.1 20.3

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detachment is achieved. The recombinant trypsin treatment needed about three times longer to completion, but the viability of the cells after treatment was significantly higher with recombinant trypsin compared to porcine trypsin or the controls.

4. Discussion

Fig. 6. SDS-PAGE of soluble fractions from continuous feed refolding. Lane 1: denatured trypsinogen before refolding. Lane 2: after 1 h. Lane 3: after 2 h. Lane 4: after 3 h. Lane 5: after 4 h. Lane 6: after 5 h. Arrow: trypsinogen.

of trypsin activation, and trypsin activity reached a plateau after 19 h. 3.5. Endotoxin concentration Prior to application to animal cell culture, the endotoxin concentration of the refolded trypsinogen sample was tested. The result of the LAL test was 224 pg ml−1 , whereas the refolding buffer was below the detection limit. From this result it was concluded that the reduction of the initial endotoxin load from the lysed bacterial cells was satisfactory and that the product could be applied to cell culture. 3.6. Cell detachment The results of the assay for detachment of CHO cells are presented in Table 2. It should be noted that under the assay conditions significant detachment takes place also without enzymatic treatment, but it is not complete even after significantly longer time. One can clearly see that only with trypsin treatment 100% Table 2 Detachment of CHO cells with recombinant human trypsin and with control substances (porcine trypsin, PBS def. as buffer control) Sample

Time (s)

Detachment (%)

Viability (%)

Growth

Recombinant trypsin Porcine trypsin PBS def.

160 50 600

100 100 70

94 86 68

Normal Normal Normal

Recombinant human trypsinogen 1 could be produced at very high expression levels of 200 mg g−1 bacterial dry mass in E. coli. However, this high yield could only be obtained when the trypsinogen gene was fused with its N-terminus to the 11 N-terminal amino acid codons of the phage T7 gene 10, while direct cloning of the trypsinogen gene after the start codon did not lead to any detectable expression. Sprengart et al. (1996) have described that the downstream box (DB) of T7 gene 10, a sequence complementary to 16S rRNA, serves as a strong enhancer of translation, both in the presence and absence of the Shine–Dalgarno sequence. Etchegaray and Inouye (1999) suggested that the DB can strongly enhance translation initiation in the presence of a SD sequence. While the DB can be an enhancer for translation, it is not a prerequisite, which may also be deducted from the large number of non-bacterial genes successfully expressed in the T7 expression system. Therefore it is plausible to conclude that the N-terminal sequence of the human trypsinogen 1 gene is especially unfavorable to initiate translation in E. coli, which is alleviated when the N-terminus of T7 gene 10 is fused to it. For many applications, especially in the biopharmaceuticals field, it is desirable to produce unfused proteins to avoid any non-native structures added to the product. A solution would be the use of the two-cistron system developed by Schoner et al. (1986). In the present work, this problem is elegantly solved as the activation peptide including the fusion partner needs to be cleaved off and removed anyhow. This work shows that human trypsinogen 1 forms inclusion bodies in the cytoplasm of E. coli, as observed for bovine trypsinogen before. However, this comes not unexpected due to the closely related structure of the different trypsinogens. In order to obtain active material, an approach as outlined by Jaenicke and Rudolph (1989) was followed. A similar approach was described by Light et al. (1986), and encompasses

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the formation of mixed disulfides between protein and glutathione to prevent the protein from aggregation. Direct refolding after solubilization proved to be not practicable in this case, as no activity could be obtained in any attempt of direct refolding. A general aim of improving refolding procedures is to increase the working concentration. Most published attempts use either a stepwise or semi-continuous addition of denatured protein solution into the refolding buffer (Fischer et al., 1992; Katoh and Katoh, 2000), which both reach there limit with the rising concentration of chaotropic agent in the refolding mixture. The advantage of the method described here lies in the constant concentration of chaotrop, which can be adjusted to the optimum concentration supporting refolding by adapting the flow rate of the filtrate to that of denatured protein solution. Consequently the denatured protein solution can be fed continuously while maintaining a beneficial concentration of chaotrop, which enables, contrary to the above mentioned methods, a real continuous process. The refolding yields achieved in this study are comparable to the yields described by other authors. While optimizing the pulse-fed refolding of recombinant bovine trypsinogen, Buswell et al. (2002) observed yields in the range between 12 and 20%. As an alternative to dilution methods, chromatographic methods have been described for refolding, e.g. by Batas and Chaudhuri (1995). It would be of interest in the future to evaluate such protocols for the refolding of trypsinogen. Advantages can be seen in the possibility to work with comparatively high concentrations, but generally chromatographic methods work mainly discontinuously. The next important step towards producing applicable recombinant trypsin is the activation of trypsinogen under conditions that avoid the use of an animal derived enzyme like enterokinase. The ability for autoactivation of the refolded trypsinogen was evaluated and found to be practicable. Autoactivation seems to follow the principles described by Kay and Kassell (1971), who ascribe bovine trypsinogen the ability to form dimers that hydrolyze each other and so remove the activation peptide. A recombinant trypsin preparation was diluted to the activity routinely used in mammalian cell culture and the endotoxin content was determined. It could be shown that the endotoxin content was low enough to be directly used in cell culture, indicating that the

depletion of endotoxins during the preparation of the material was effective enough for this application. Finally the functional activity of recombinant protein was evaluated as the ability to detach CHO cells from the surface of Roux bottles. Compared to conventional porcine trypsin, recombinant trypsin needed longer time of incubation, but resulted in a higher viability of the cells. After passaging into fresh medium, normal growth was observed after both trypsin treatments. Acknowledgements The authors wish to thank Ali Assadian for endotoxin analysis and Susanne Wiederkum for the cell detachment experiments, as well as Andreas Wagner and Günter Kreismayr for support in the construction of the refolding vessel. This project was supported by a grant from the Österreichische Nationalbank.

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