Escherichia coli EDA is a novel fusion expression partner to improve solubility of aggregation-prone heterologous proteins

Journal of Biotechnology 194 (2015) 39–47

Contents lists available at ScienceDirect

Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Escherichia coli EDA is a novel fusion expression partner to improve solubility of aggregation-prone heterologous proteins Yoon-Sik Kang a,1 , Jong-Am Song b,1 , Kyung-Yeon Han c , Jeewon Lee a,∗ a

Department of Chemical and Biological Engineering, Korea University, Anam-Dong 5-1, Seongbuk-Gu, Seoul 136-713, Republic of Korea SolGent Co., Ltd. Plant and Biomedical Research Institute, 43-10, Techno 5-ro, Yuseong-Gu, Daejeon 305-504, Republic of Korea Emerging Technology Research Center, Corporate Technology Operations SAIT, Samsung Electronics Co., Ltd., Giheung-gu, Yongin-si, Gyeonggi-do, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 7 October 2014 Received in revised form 24 November 2014 Accepted 27 November 2014 Available online 5 December 2014 Keywords: Escherichia coli BL21 Proteome Stress-responsive protein KDPG aldolase (EDA) Solubility enhancer

a b s t r a c t Since the use of solubility enhancer proteins is one of the effective methods to produce active recombinant proteins within Escherichia coli, the development of a novel fusion expression partner that can be applied to various aggregation-prone proteins is of crucial importance. In our previous work, two-dimensional electrophoresis (2-DE) was employed to systematically analyze the E. coli BL21 (DE3) proteome profile in response to heat treatment, and KDPG aldolase (EDA) was identified as a heat-responsive and aggregation-resistant protein. When used as fusion expression partner, EDA significantly increased the solubility of seven aggregation-prone heterologous proteins in the E. coli cytoplasm. The efficacy of EDA as a fusion expression partner was evaluated through the analysis of bioactivity or secondary structure of several target proteins: EDA-fusion expression resulted in the synthesis of bioactive human ferritin light chain and bacterial arginine deiminase and the formation of correct secondary structure of human granulocyte colony stimulation factor. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Because of the high production yield, low manufacturing cost, and a well-characterized expression system, Escherichia coli has been widely used as a host in recent research involving biosynthesis of recombinant proteins (Chung-Jr et al., 2012; Rachel, 2012). However, its application has been restricted due to undesirable aggregation and low productivity of active recombinant proteins, especially in the synthesis of eukaryotic proteins (Natalia et al., 2013; Rogl et al., 1998), because of the lack of appropriate folding-assistant proteins and a system to carry out posttranslational modifications. Several approaches have been investigated to improve this performance, such as coexpression of folding-assistant proteins (Goulding and Perry, 2003; Nishihara et al., 1998), optimization of codons (Zhou et al., 2004), and optimization of growth conditions by modulating the temperature and the inducer concentration (Baneyx and Mujacic, 2004; Winograd et al., 1993). Recently, research has focused on recombinant protein expression

∗ Corresponding author. Tel.: +82 2 3290 3304; fax: +82 2 926 6102. E-mail addresses: [email protected] (Y.-S. Kang), [email protected] (J.-A. Song), [email protected] (K.-Y. Han), [email protected] (J. Lee). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jbiotec.2014.11.025 0168-1656/© 2014 Elsevier B.V. All rights reserved.

with fusion expression partners to resolve these challenges (Ahn et al., 2007; Han et al., 2007a,b; Park et al., 2008; Seo et al., 2009; Song et al., 2011). Fusion expression partners have been widely used to enhance the solubility and folding efficiency of recombinant proteins. Some of these fusion expression partners (e.g., maltose-binding protein (MBP) (Zhu et al., 2009), thioredoxin (Trx) (Tomala et al., 2010), N-utilization substance A (NusA) (Nallamsetty and Waugh, 2006), and glutathione-S-transferase (GST) (Shimada et al., 2005)), have proven their effectiveness by overcoming inclusion body formation and enhancing the protein expression levels. However, these fusion expression partners have proven their effectiveness only for some proteins, and current fusion expression partners still need to be improved in order to properly express many of the heterologous proteins in E. coli. Therefore, there is an urgent need to develop appropriate fusion tags that can be used as effective expression and solubility enhancers for recalcitrant proteins. E. coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EDA) (22.3 kDa, accession no. P0A955), which catalyzes the reversible cleavage of KDPG to pyruvate and glyceraldehyde-3phosphate in the Entner–Doudoroff pathway, was used in this study. In our previous studies, many stress-resistant and stable proteins were identified. Their native structures were retained even under protein denaturing conditions (Han et al., 2008). Those

40

Y.-S. Kang et al. / Journal of Biotechnology 194 (2015) 39–47

proteins have successfully been employed to produce many heterologous proteins in the soluble and active forms in E. coli (Han et al., 2007b; Song et al., 2011). Consequently, it can be reasonably assumed that the unusually stable structure of EDA can be applied as an effective fusion expression partner. Our findings indicate that EDA is a highly effective fusion expression partner that can enhance the solubility of heterologous proteins that have a strong tendency to be aggregated in inclusion bodies in E. coli. The EDA fusion protein was established by separately fusing seven aggregation-prone heterologous proteins to its C-terminus, and each fusion protein was expressed in E. coli. We also estimated the solubility and expression level of each fusion protein. The biological activities and functions of the purified recombinant fusion and fusion-free proteins were also demonstrated.

(A)

NdeI

T7

(B)

HindIII

Target protein (hG-CSF, hFTN-L, ppGRN, mpINS, IL2, EGF, and ADI) XhoI

NdeI

T7

(C)

H6

EDA

H6

2. Materials and methods

Target protein (hG-CSF, hFTN-L, ppGRN, mpINS, IL2, EGF, and ADI)

XhoI

NdeI

T7

HindIII

EDA

HindIII

hG-CSF or hFTN-L

Enterokinase cleavage site (DDDDK, D4K)

2.1. Bacterial strain and plasmids The target genes including human granulocyte colonystimulating factor (hG-CSF), human ferritin light chain (hFTN-L), human prepro-ghrelin (ppGRN), human minipro-insulin (mpINS), human interleukin-2 (hIL-2), human epidermal growth factor (EGF), and mycoplasma arginine deiminase (ADI) were cloned by PCR amplification using appropriate primers (Table 1). The direct expression vectors were constructed by inserting each of the recombinant genes into the NdeI-HindIII enzymatically cleavable site of the pT7-7 plasmid (Fig. 1A). The EDA gene was amplified by PCR from genomic DNA of E. coli strain BL21 (DE3). The fusion expression vector was constructed using the appropriate primers (Table 1) for PCR amplification, and EDA fusion proteins (EDAhG-CSF, EDA-hFTN-L, EDA-ppGRN, EDA-mpINS, EDA-IL2, EDA-EGF, and EDA-ADI) were ligated into the NdeI-HindIII site of the pT77 plasmid (Fig. 1B). The purification of fusion-free recombinant hG-CSF (or hFTN-L) was carried out by inserting an enterokinase cleavage sequence (D4 K) between the EDA and hG-CSF (or hFTNL) genes and adding a polyhistidine tag (H6 ) at the N-terminus of EDA (i.e. H6 -EDA-D4 K-hG-CSF (or hFTN-L)). This mutant gene was subsequently inserted into the NdeI-HindIII site of the pT7-7 plasmid (Fig. 1C). E. coli BL21 (DE3) [F− ompT hsdSB (rB− mB− )] was

Fig. 1. The plasmid expression vectors used for direct and fusion expression of heterologous proteins in E. coli. (A) Direct expression vector, (B) EDA fusion expression vector, (C) fusion expression vector for hG-CSF (or hFTN-L) that is designed for metal (Ni2+ ) affinity purification of H6 -EDA-D4 K-hG-CSF (or hFTN-L), followed by fusion expression partner (EDA) removal by enterokinase cleavage.

transformed with each of the hybrid plasmids after DNA sequencing of all gel-purified constructs. Subsequently, ampicillin-resistant transformants were selected using LB-agar plates supplemented with ampicillin (100 mg/L). 2.2. Recombinant gene expression Each E. coli transformant was incubated with 50 mL LB media containing 100 mg/L ampicillin at 37 ◦ C with 130 rpm shaking. IPTG (1 mM) was added to induce protein expression when the culture turbidity (OD600 ) reached 0.5–0.6. After the induction by IPTG addition began, the recombinant E. coli was cultivated either for an additional 5–6 h at 37 ◦ C or for an additional 12 h at 20 ◦ C. All of the recombinant cells were harvested after by centrifugation at 13,000 rpm (MICRO17TR, Hanil Science Industrial, Korea) for 5 min. The cell pellets were resuspended in

Table 1 The primer sequences for PCR amplification of various target genes. Heterologous proteins

GenBank accession no.

Primer sequences for PCR amplification

Direct expression Human granulocyte colony-stimulating factor (hG-CSF)

NM172219



Fusion expression 

5 -cat atg act cca ctc gga cct g-3

5 -aag ctt tca tgg ctg tgc aag-3 Human ferritin light chain (hFTN-L)

NM000146

5 -cat atg agc tcc cag att cgt-3

5 -ctc gag acc ccc ctg ggc cct gcc-3 5 -ctc gag gac gat gac gat aaa acc ccc ctg ggc cct gcc-3 (for enterokinase digestion) 5 -aag ctt tca tgg ctg tgc aag-3

5 -aag ctt tta gtc gtg ctt gag agt-3

5 -ctc gag agc tcc cag att cgt-3 5 -ctc gag gat gac gat gac aag agc tcc cag att cgt-3 (for enterokinase digestion) 5 -aag ctt tta gtc gtg ctt gag agt-3

Human prepro-ghrelin (ppGRN)

NM016362

5 -cat atg ggc tcc agc ttc ctg-3 5 -aag ctt tca ctt gtc ggc t-3

5 -ctc gag ggc tcc agc ttc ctg-3 5 -aag ctt tca ctt gtc ggc t-3

Human minipro insulin (mpINS)

EF518215

5 -cat atg ttt gtc aac caa cat-3 5 -aag ctt tta gtt aca gta gtt c-3

5 -ctc gag ttt gtc aac caa cat-3 5 -aag ctt tta gtt aca gta gtt c-3

Human interleukin-2 (hIL-2)

NM000586

5 -cat atg gca cct act tca agt-3 5 -aag ctt tta tca agt cag tgt-3

5 -ctc gag gca cct act tca agt-3 5 -aag ctt tta tca agt cag tgt-3

Human epidermal growth factor (EGF)

M15672

5 -cat atg aac tct gac tcc gaa tgc-3 5 -aag ctt tta acg cag ttc cca cca-3

5 -ctc gag aac tct gac tcc gaa tgc-3 5 -aag ctt tta acg cag ttc cca cca-3

Mycoplasma arginine deiminase (ADI)

X54141

5 -cat atg tct gta ttt gac agt-3 5 -aag ctt cta tca ctt aac atc-3

5 -ctc gag tct gta ttt gac agt-3 5 -aag ctt cta tca ctt aac atc-3

Y.-S. Kang et al. / Journal of Biotechnology 194 (2015) 39–47

12 mL lysis buffer (10 mM Tris–HCl, pH 7.5, 10 mM EDTA) and disrupted with a Branson Sonifier (Branson Ultrasonics Corp., Danbury, CT). Centrifugation (13,000 rpm for 10 min, MICRO17TR, Hanil Science Industrial, Korea) was used to separate the cell-free supernatant and insoluble protein aggregates. For enterokinase treatment, 3 U of enterokinase (Invitrogen, CA) and 10 ␮L of 10× buffers were mixed with 87 ␮L of cell-free lysate, which contained about 300 ␮g of the fusion protein hG-CSF. One enterokinase activity unit (U) is defined as the enzyme activity that digests 20 ␮g of fusion protein by 90% in 20 min at 37 ◦ C. The

41

lysate-enterokinase mixture was then incubated for 10 h at 20 ◦ C and subsequently centrifuged at 13,000 rpm for 10 min to separate the aggregates. 2.3. Purification of recombinant protein Metal affinity chromatography was employed to purify recombinant hG-SCF or hFTN-L. Briefly, a ProBond resin (Ni2+ ) column was filled with the polyhistidine (H6 )-tagged hG-CSF (or hFTNL) fusion protein (H6 -EDA-D4 K-hG-CSF (or hFTN-L)). To minimize

Fig. 2. Results of SDS-PAGE analyses of E. coli cell lysates containing the directly expressed-, EDA-fused-, and GST-fused heterologous proteins. (The results of solubility assessment in Fig. 3 were based on SDS-PAGE of Figure) (M: see blue marker; S: soluble fractions; IS: insoluble fractions).

42

Y.-S. Kang et al. / Journal of Biotechnology 194 (2015) 39–47

Fig. 2. (Continued ).

Y.-S. Kang et al. / Journal of Biotechnology 194 (2015) 39–47

43

Fig. 2. (Continued ).

nonspecific binding of untagged protein contaminants, the resin was washed twice with 10 column volumes of binding buffer (50 mM potassium phosphate, 300 mM NaCl, and 20 mM imidazole, pH 8.0) prior to sample loading. Binding was conducted in batch mode at 4 ◦ C, and the Ni2+ -NTA resin was washed three times with 10 mL wash buffer (50 mM potassium phosphate, 300 mM NaCl, and 50 mM imidazole, pH 8.0) and subsequently washed twice with 10 mL Tris–HCl (10 mM Tris, pH 8.0). Then, enterokinase digestion was carried out in batch mode at 20 ◦ C for 16 h with 5 U of enterokinase (Invitrogen, CA). Both the fusion expression partner-free hG-CSF (or hFTN-L) and the enterokinase were gravitationally collected without using any elution buffer and centrifuged at 13,000 rpm (MICRO17TR, Hanil Science Industrial, Korea) for 10 min. The supernatant and insoluble fractions were analyzed by SDS-PAGE and Western blot respectively. The enterokinase was removed using the manufacturer’s protocol (EK-AwayTM resin, Invitrogen, Germany). 2.4. SDS-PAGE and Western blot analysis The cell-free supernatants and inclusion bodies were subjected to 12% reducing polyacrylamide gel electrophoresis, and Coomassie-stained protein bands were scanned and analyzed by densitometry (Duoscan T1200, Bio-Rad, Hercules, CA). The fusion expression partner-free hG-CSF analysis was conducted as follows: the cell-free supernatant soluble fraction and purified hG-CSF were subjected to reducing 12% SDS-PAGE using a tris–glycine gel, and subsequently transferred onto a nitrocellulose membrane (Schleicher and Schuell BioScience GmbH, Germany) for 2 h at 4 ◦ C in transfer buffer (glycine 2.9 g/L, tris base 5.8 g/L, SDS 0.37 g/L, MeOH 200 mL, distilled water 800 mL). Blocking buffer (1% nonfat skim milk in phosphate buffered saline, PBS) was employed to block nonspecific antibody binding on the membrane by incubating for 1 h at room temperature. After the membrane was incubated with 1:1000 diluted hG-CSF primary antibody (mouse antihuman G-CSF monoclonal antibody; Clone 3D1, Santa Cruz Biotechnology Inc., CA) for an additional 1 h at room temperature, it was washed three times with PBS and incubated with 1:1000 diluted goat anti-mouse IgG conjugated horseradish peroxidase (HRP) secondary antibody (Cat. No. SC-2005, Santa Cruz Biotechnology Inc., CA) for an additional 1 h before washing three times with PBS. The membrane was developed using an HRP conjugate substrate kit (Sigma–Aldrich, MO, USA).

2.5. Circular dichroism Cell lysate containing the fusion protein H6 -EDA-D4 K-hGCSF was loaded onto the Ni2+ -NTA column to avoid undesirable degradation, followed by removal of endogenous E. coli protein contaminants including proteases, by adding washing buffer (Song et al., 2009). To separate the N-terminal fusion expression partner (EDA) from the recombinant hG-CSF, enterokinase that specifically cleaves the D4K between EDA and hG-CSF was added inside the Ni2+ -NTA column containing the fusion protein. A JASCO J-710 spectropolarimeter (Korea Basic Science Center, Ochang, Korea) was employed to measure the circular dichroism (CD) spectra of the purified EDA-free hG-CSF (74 mg/L) and the commercial standard hG-CSF (Grasin Prefilled-Syringe (Filgrastim), Kirin, Japan) in PBS buffer with 1-mm path cell. 2.6. Transmission Electron Microscopy (TEM) analysis In order to conduct the TEM test, unstained samples of the purified protein, H6 -EDA-hFTN-L and EDA-free hFTN-L, were prepared. To obtain stained images of protein nanoparticles, small air-dried drops of a sample solution were placed onto carbon-coated copper electron microscopy grids, which were incubated with a 2% (w/v) aqueous uranyl acetate solution for 1 min at room temperature and washed 3 times with distilled water. Protein nanoparticle images were examined using the Philips Tecnai 20 (200 kV) electron microscope. In order to obtain high diffraction intensities, electron diffraction patterns were recorded from a selected area with a high protein nanoparticle density. 3. Results and discussion 3.1. Direct expression of aggregation-prone heterologous proteins in the E. coli cytoplasm Seven proteins were selected as research targets in this study because they are well known to form insoluble aggregates when expressed in E. coli (Lee et al., 2014; Park et al., 2007, 2008; Song et al., 2009): hG-CSF, hFTN-L, ppGRN, mpINS, IL2, EGF, and ADI. The target genes were inserted into the pT7-7 plasmid vectors to construct the plasmid vector, which was employed for direct expression of these seven aggregation-prone proteins in E. coli (Fig. 1A). The solubility of all the heterologous proteins that form insoluble aggregates was negligible (Fig. 2A).

44

Y.-S. Kang et al. / Journal of Biotechnology 194 (2015) 39–47

3.2. Expression of aggregation-prone heterologous proteins with the fusion of EDA The previous studies (Manoj et al., 2007; Stephen et al., 2006) revealed the core sequences affecting the catalytic activity of EDA (i.e. the reversible cleavage of KDPG) through site-directed mutagenesis: the mutation of 45E to 45N caused 1000-fold decrease of EDA activity, while the mutation of 184S to 184A, 184L, or 184D also reduced the EDA activity by up to 10%, explaining that both of 45E and 184S are the sequences that are responsible for KDPG cleavage by EDA and KDPG access to EDA, respectively. Since both of the N- and C-terminal parts of EDA are important in forming the active conformation, we used the full sequence of EDA as fusion expression partner for the 7 aggregation-prone heterologous proteins. To construct expression vectors, each of the target proteins was fused to the C-terminus of EDA, resulting in the synthesis of EDA fusion proteins (Fig. 1B), and expressed in E. coli. Compared with directly expressed proteins, the solubility of all fusion proteins had significantly increased (Fig. 3A). In particular, 4 of the 7 EDA fusion proteins showed an increase greater than 85% (Fig. 2B). The expression level of all the recombinant fusion proteins was dramatically improved compared to the directly expressed proteins (Fig. 2B). The enhanced solubility and expression level of the EDA fusion proteins indicate the suitability of EDA as an effective fusion expression partner for the synthesis of aggregation-prone heterologous proteins in E. coli.

Because of the nonspecific hydrophobic interactions between the partially folded intermediates of recombinant proteins or the host proteins in the E. coli cytoplasm, inclusion bodies may be consequent products when nascent polypeptide chains are continuously synthesized. When EDA was employed as an N-terminal fusion expression partner, the solubility of foreign proteins significantly increased (Fig. 2B). It is considered that EDA shielded surface interactions between heterologous proteins to prevent nonspecific protein-protein interactions, which otherwise lead to the formation of inclusion bodies. Intermolecular aggregation and misfolding can be caused by broadly exposed hydrophobic surfaces of newly synthesized polypeptides. To prevent aggregation and misfolding, molecular chaperones (Dnak and its Hsp 70 homologs) recognize the hydrophobic patches of nascent proteins and interact with them (Maier et al., 2005). Hydrophobic amino acids are only exposed during the folding of polypeptide intermediates, although they are usually buried within the core of the folded proteins (Suh et al., 1999; Rüdiger et al., 1997), and the exposed heterologous polypeptide may interact along aggregation-associated hydrophobic surfaces. It is reasonable to believe that misfolding and aggregation were avoided due to the interaction between EDA and the hydrophobic surfaces of heterologous polypeptides, which is functionally similar to the role played by chaperones in eukaryotic cells. Furthermore, in the protein synthesis process, aggregation and folding that are caused by similar molecular interactions, are competitive (Jeffrey et al., 2006). In EDA, 64% of the polar and charged residues are located on the surface, while nonpolar residues are

80

80

Solubility(%)

(B) 100

Solubility(%)

(A) 100

60

40

60

40

20

20

0

0

(C) 100

Solubility(%)

80

60

40

20

0

Fig. 3. (A) EDA fusion expression was compared with direct expression. (B) EDA fusion expression was compared with GST fusion expression at 37 ◦ C. (C) EDA fusion expression was compared with GST fusion expression at 20 ◦ C. (Black and gray bars in (A) to (C) represent the solubility of EDA- and GST-fusion expressed recombinant proteins, respectively.).

Y.-S. Kang et al. / Journal of Biotechnology 194 (2015) 39–47

45

Fig. 4. Results of SDS-PAGE (A) and Western blot (B) analyses for total cell lysates (lane 1) containing the EDA-fused hG-CSF (indicated by black arrow), soluble (lane 2) and insoluble (lane 3) fractions of total cell lysates containing EDA-free hG-CSF (indicated by white arrow) that is released by enterokinase cleavage, and soluble (lane 4) and insoluble (lane 5) fractions of purified hG-CSF. (C) CD spectra of the purified EDA-free hG-CSF and commercial hG-CSF standard.

located roughly equally on the surface (Mavridis et al., 1982). EDA is expected to improve protein synthesis by acting as a nucleus of folding that prevents aggregation and allows the target protein to undergo its folding process. This allows the solubility of the target to be increased [“molten globule hypothesis” (Alexei et al., 2007)]. Because of these advantageous properties, it is highly feasible that EDA is an effective fusion expression partner in the synthesis of recombinant proteins, especially for proteins with a tendency to form insoluble aggregates in E. coli. Compared with direct expression (Fig. 3A), the solubility was enhanced by using EDA as a fusion expression partner, although the fusion proteins for mpINS, IL2, and EGF still had relatively low solubility compared to the other fusion proteins. In this study, low temperature expression was considered to improve the effective conditions for fusion expression in order to further increase the solubility of these proteins. When the EDA fusion proteins were expressed at 20 ◦ C, the solubility of mpINS, IL2, and EGF dramatically increased to over 85% (Fig. 3C). Compared with the GST fusion (Fig. 3B and C), EDA is a highly effective solubility enhancer. Mostly, using EDA resulted in higher yields of soluble protein than using GST at 37 ◦ C (Fig. 3B). Also at 20 ◦ C the EDA-constructs were in some cases (hFTN-L, mpINS, and EGF) also better soluble than the GST-constructs (Fig. 3C). When estimated using the pre-determined correlation between E. coli total proteins and OD600 of E. coli culture, the concentration of soluble EDA fusion proteins was apparently higher compared with that of soluble GST fusion proteins (Supplementary Fig. S1). Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec.2014.11.025. 3.3. Purification and characterization of EDA fusion-expressed heterologous proteins Fusion expression is one of the most attractive methods to produce active recombinant proteins, but the unexpected misfolding

that causes malfunction of the target proteins still restricts its application. The influence of EDA on the structure or function of the expressed protein (hG-CSF, hFTN-L, and ADI) was investigated. To obtain EDA-free hG-CSF (or hFTN-L), the enterokinase cleavage sequence (D4 K) was inserted between EDA and hG-CSF (or hFTN-L) (Fig. 1C), and a polyhistidine tag (H6 ) was also added to the N-terminus of EDA for metal (Ni2+ ) affinity purification (Fig. 1B and C). The first lane in Fig. 4A indicates that the fusion protein, H6 EDA-D4 K-hG-CSF, was well expressed as a soluble protein and was recognized by the anti-hG-CSF antibody by western blot (lane 1 in Fig. 4B). According to SDS-PAGE and Western blot analysis, the recombinant cell lysate containing the fusion protein (N-H6 EDA-D4 K-hG-CSF-C) reacted with enterokinase to release hG-CSF from the fusion expression partner EDA. The two main split bands in SDS-PAGE on lane 2 (Fig. 4A) correspond to the N-terminal portion (N-H6 -EDA-D4 K-C) and released hG-CSF. The insoluble fraction (lane 3 in Fig. 4A) of released hG-CSF was almost negligible. EDA-free hG-CSF was analyzed by SDS-PAGE (lane 4 in Fig. 4A). This result indicated that EDA-free hG-CSF still existed as a water-soluble form, and it was clearly identified by Western blot analysis (lane 4 in Fig. 4B). The recombinant hG-CSF that was released from the fusion expression partner and purified was analyzed by CD spectroscopy, showing that purified EDA-free hG-CSF has the same secondary structure as that of commercial hG-CSF standard (Fig. 4C). Fig. 4C demonstrates that the recombinant hGCSF expressed as an EDA fusion protein was folded to have a correct secondary structure. Also, the self-assembly function of hFTN-L to form nanoscale (around 12 nm in diameter) ferritin particles was analyzed using the fusion protein N-H6 -EDA-hFTN-L-C (Fig. 1B). After purification, the EDA-fused (lane 1 in Fig. 5A) and EDA-free hFTN-L (lane 3 in Fig. 5A) were analyzed by TEM. The formation of spherical ferritin nanoparticles in the TEM images clearly indicates that the fusionexpressed hFTN-L (Fig. 5B) had the same biological activity as the

46

Y.-S. Kang et al. / Journal of Biotechnology 194 (2015) 39–47

Fig. 5. Results of SDS-PAGE (A) analyses for soluble (lane 1) and insoluble (lane 2) fractions of purified EDA-fused hFTN-L. Soluble (lane 3) and insoluble (lane 4) fractions of purified EDA-free hFTN-L. TEM analyses for the purified (B) EDA-hFTN-L and (C) EDA-free hFTN-L. (In (B,C) the scale bars on the left and right TEM images correspond to 100 and 20 nm, respectively.).

native hFTN-L (Fig. 5C). According to the colorimetric determination based on the estimation of amount of l-citrulline produced from l-arginine, the bioactivity of fusion-expressed ADI (Fig. 6A) was also assayed (Noh et al., 2002; Misawa et al., 1994). The average specific activity of EDA-ADI was 14.01 U/mg, while the specific activity of ADI that was refolded after expressed in the form of inclusion body in E. coli was 11.3 U/mg (Noh et al., 2002) [U of ADI activity is defined as l-citrulline production rate (␮mol/min) per mg of ADI, and the amount of produced l-citrulline (␮mol) was estimated using the pre-determined correlation: 1.07 × OD530 ].

The biological activity of EDA-ADI undoubtedly proved that during the time-course increase in absorbance (OD530 nm ), l-arginine was converted to l-citrulline (Fig. 6B). The efficacy of EDA as a fusion expression partner to enhance protein solubility in the E. coli cytoplasm was evaluated. The solubility of seven fusion proteins was compared after each aggregation-prone heterologous protein was fused to EDA and expressed in E. coli. Some of the EDA fusion proteins still showed a high degree of aggregation, although their overall solubility did increase. To increase the aggregation-prone recombinant proteins’

Fig. 6. Results of SDS-PAGE (A) and colorimetric enzyme assay (B) for the purified H6 -EDA-ADI (lane 1, indicated by black arrow for (A), () for (B)). In (B), the symbol, (䊉) represents negative control (PBS buffer only).

Y.-S. Kang et al. / Journal of Biotechnology 194 (2015) 39–47

solubility, a low temperature expression system was developed by decreasing the expression temperature from 37 ◦ C to 20 ◦ C. Under these conditions, highly soluble proteins with negligible aggregation were obtained, and the biological activity of fusion-expressed target proteins (hFTN-L and ADI) was also confirmed. Moreover, the CD spectrum indicated that the secondary structure of EDAfree and purified hG-CSF was the same as the commercial standard for hG-CSF (Fig. 4C). 4. Conclusions EDA as a fusion expression partner was able to dramatically increase the cytoplasmic solubility of aggregation prone heterologous proteins. EDA-fused ADI was active in the hydrolysis of PNB. Even after the removal of EDA, hFTN-L was biologically active and hG-CSF correctly formed native secondary structure. The smaller size of EDA (22.3 kDa), compared to other well-known fusion expression partners [MBP (40.7 kDa) or NusA (54.9 kDa)], is advantageous in increasing production yield of target proteins. Compared with GST with similar molecular mass (25.6 kDa), EDA was evidently a more effective solubility enhancer. Consequently, E. coli EDA is a potent cis-acting solubility enhancer and can produce soluble and active heterologous proteins, indicative that it is an attractive candidate for fusion expression of a variety of aggregation-prone heterologous proteins in E. coli. Acknowledgments This study was supported by the 2012 NLRL (National Leading Research Lab.) Project (grant no. 2012R1A2A1A01008085) (the main project that supported this work) and the Basic Science Research Program (ERC program, grant no. 2010-0029409) of the National Research Foundation of Korea (NRF). We also appreciate the kind donation of cDNA clone of Mycoplasma arginine deiminase from Prof. Bon Hong Min (College of Medicine, Korea University). References Alexei, F., Dmitry, I., Sergiy, G., Oxana, G., 2007. Understanding the folding rates and folding nuclei of globular proteins. Curr. Protein Pept. Sci. 8, 521–536. Ahn, K.Y., Song, J.A., Hah, K.Y., Park, J.S., Seo, H.S., Lee, J., 2007. Heterologous protein expression using a novel stress-responsive protein of E-coli RpoA as fusion expression partner. Enzyme Microb. Technol. 41, 859–866. Baneyx, F., Mujacic, M., 2004. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol. 22, 1399–1408. Chung-Jr, H., Henry, L., Xiaoming, Y., 2012. Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements. J. Ind. Microb. Biotechnol. 39, 383–399. Goulding, C.W., Perry, L., 2003. Protein production in Escherichia coli for structural studies by X-ray crystallography. J. Struct. Biol. 142, 133–143. Han, K.Y., Park, J.S., Seo, H.S., Ahn, K.Y., Lee, J., 2008. Multiple stressor-induced proteome responses of Escherichia coli BL21 (DE3). J. Proteome Res. 7, 1891–1903. Han, K.Y., Seo, H.S., Song, J.A., Ahn, K.Y., Park, J.S., Lee, J., 2007a. Transport proteins PotD and Crr of Escherichia coli, novel fusion partners for heterologous protein expression. Biochim. Biophys. Acta 1774, 1536–1543. Han, K.Y., Song, J.A., Ahn, K.Y., Park, J.S., Seo, H.S., Lee, J., 2007b. Solubilization of aggregation-prone heterologous proteins by covalent fusion of stressresponsive Escherichia coli protein, SlyD. Protein Eng. Des. Sel. 20, 543–549. Jeffrey, M., Suzanne, E., Yiting, L., Peter, L., Xun, Z., Tauseef, B., 2006. Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci. 15, 182–189.

47

Lee, J.H., Lee, J.Y., Song, J.A., Han, K.Y., Lee, D.S., Lee, J., 2014. A stress-responsive Escherichia coli protein, CysQ is a highly effective solubility enhancer for aggregation-prone heterologous proteins. Protein Expr. Purif. 101, 91–98. Maier, T., Ferbitz, L., Deuerling, E., Ban, N., 2005. A cradle for new proteins: trigger factor at the ribosome. Curr. Opin. Struct. Biol. 15, 204–212. Manoj, C., Eric, T., Carol, F., 2007. Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates. Protein Sci. 16, 2368–2377. Mavridis, I.M., Hatada, M.H., Tulinsky, A., Lebioda, L., 1982. Structure of 2-keto3-deoxy-6-phosphogluconate aldolase at 2.8A resolution. J. Mol. Biol. 162, 419–444. Misawa, S., Aoshima, A., Takaku, H., Matsumoto, M., Hayashi, H., 1994. High-level expression of mycoplasma arginine deiminase in Escherichia coli and its efficient renaturation as an anti-tumor enzyme. J. Biotechnol. 36, 145–155. Nallamsetty, S., Waugh, D.S., 2006. Solubility-enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners. Protein Expr. Purif. 45, 175–182. Natalia, B., Joost, S., Frederic, R., Johan, E., 2013. Protein aggregation in bacteria: the thin boundary between functionality and toxicity. Microbiology 159, 1795–1806. Nishihara, K., Kanemori, M., Kitagawa, M., Yanagi, H., Yura, T., 1998. Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl. Environ. Microbiol. 64, 1694–1699. Noh, E.J., Kang, S.W., Shin, Y.J., Kim, D.C., Park, I.S., Kim, M.Y., Chun, B.G., Min, B.H., 2002. Characterization of mycoplasma arginine deiminase expressed in E. coli and its inhibitory regulation of nitric oxide synthesis. Mol. Cells 13, 137–143. Park, J.S., Han, K.Y., Lee, J.H., Song, J.A., Ahn, K.Y., Seo, H.S., Sim, S.J., Kim, S.W., Lee, J., 2008. Solubility enhancement of aggregation-prone heterologous proteins by fusion expression using stress-responsive Escherichia coli protein, RpoS. BMC Biotechnol. 8, 15. Park, J.S., Han, K.Y., Song, J.A., Ahn, K.Y., Seo, H.S., Lee, J., 2007. Escherichia coli malate dehydrogenase, a novel solubility enhancer for heterologous proteins synthesized in Escherichia coli. Biotechnol. Lett. 29, 1513–1518. Rachel, C., 2012. Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnol. Adv. 30, 1102–1107. Rogl, H., Kosemund, K., Kuhlbrandt, W., Collinson, I., 1998. Refolding of Escherichia coli produced membrane protein inclusion bodies immobilised by nickel chelating chromatography. FEBS Lett. 432, 21–26. Rüdiger, S., Buchberger, A., Bukau, B., 1997. Interaction of Hsp70 chaperones with substrates. Nat. Struct. Biol. 4, 342–349. Seo, H.S., Kim, S.E., Han, K.Y., Park, J.S., Kim, Y.H., Sim, S.J., Lee, J., 2009. Functional fusion mutant of Candida antarctica lipase B (CalB) expressed in Escherichia coli. Biochim. Biophys. Acta 1794, 519–525. Shimada, K., Nagano, M., Kawai, M., Koga, H., 2005. Influences of amino acid features of glutathione S-transferase fusion proteins on their solubility. Proteomics 5, 3859–3863. Song, J.A., Han, K.Y., Ahn, K.Y., Park, J.S., Seo, H.S., Lee, J., 2009. Proteolysis and synthetic strategy of human G-CSF in Escherichia coli BL21 (DE3). Enzyme Microb. Technol. 45, 7–14. Song, J.A., Lee, D.S., Park, J.S., Han, K.Y., Lee, J., 2011. A novel Escherichia coli solubility enhancer protein for fusion expression of aggregation-prone heterologous proteins. Enzyme Microb. Technol. 49, 124–130. Stephen, F., Jennifer, G., Alexandra, M., Manoj, C., Nathan, W., Michael, H., Carol, F., Eric, T., James, N., 2006. Mechanism of the Class I KDPG aldolase. Bioorg. Med. Chem. 14, 3002–3010. Suh, W.C., Lu, C.Z., Gross, C.A., 1999. Structural features required for the interaction of the Hsp70 molecular chaperone DnaK with its cochaperone DnaJ. J. Biol. Chem. 274, 30534–30539. Tomala, M., Lavrentieva, A., Moretti, P., Rinas, U., Kasper, C., Stahl, F., Schambach, A., Warlich, E., Martin, U., Cantz, T., Scheper, T., 2010. Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner. Protein Expr. Purif. 73, 51–57. Winograd, E., Pulido, M.A., Wasserman, M., 1993. Production of DNA-recombinant polypeptides by tac-inducible vectors using micromolar concentrations of IPTG. Biotechniques 14, 886–890. Zhou, Z., Schnake, P., Xiao, L., Lal, A.A., 2004. Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization. Protein Expr. Purif. 34, 87–94. Zhu, S., Yang, G., Yang, X., Zhao, Y., Li, X., Deng, P., Xie, Y., Gan, Z., Liu, Y., Li, Z., Liao, J., Yu, M., Liao, F., 2009. Soluble expression in Escherichia coli of active human cyclic nucleotide phosphodiesterase isoform 4B2 in fusion with maltose-binding protein. Biosci. Biotechnol. Biochem. 73, 968–970.