Protein Expression and Purification 99 (2014) 1–5

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High-level soluble expression of a thermostable xylanase from thermophilic fungus Thermomyces lanuginosus in Escherichia coli via fusion with OsmY protein Yilin Le ⇑, Huilei Wang School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China

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Article history: Received 11 February 2014 and in revised form 7 March 2014 Available online 18 March 2014 Keywords: Xylanase Fusion protein Soluble expression Escherichia coli Thermomyces lanuginosus

a b s t r a c t A thermostable xylanase is encoded by xynA from fungus Thermomyces lanuginosus. The problem emerged from overexpression of xynA in Escherichia coli has been the formation of inclusion bodies. Here we describe the xynA was fused with the hyperosmotically inducible periplasmic protein of E. coli, OsmY. The fusion protein OsmY-xynA was expressed as almost all soluble form. The soluble expression level of fusion protein reached 98 ± 6 U/ml when cells containing pET-OsmY-xynA were expressed without IPTG induction at 37 °C. The induction is probably due to auto-induction due to lactose in the medium (Studier (2005) [21]). The cells harboring pET-OsmY-xynA expressed an activity level about 24 times higher than that expressed from pET-20b-xynA. Xylanase activity was observed in the extracellular (36 ± 1.3 U/ml) and the periplasmic (42 ± 4 U/ml) when cells containing pET-OsmY-xynA were induced without IPTG addition. After the cold osmotic shock procedure followed by nickel affinity chromatography, the purified fusion protein showed a single band on SDS–PAGE gel with a molecular mass of 44 kDa. The purified fusion enzyme exhibited the highest activity at 65 °C and pH 6.0. Ó 2014 Elsevier Inc. All rights reserved.

Introduction Thermomyces lanuginosus produces a thermostable GF11 endoxylanase encoded by xynA gene. This xylanase is free of cellulase activity, and hydrolyses xylan to produce xylooligosaccharides with little xylose [1]. These properties make the enzyme attractive for the industrial application. Escherichia coli is one of the most extensively used prokaryotic organisms for the industrial production of enzyme because of its well-characterized genetics, and its ability to grow rapidly and at high density on inexpensive substrates [2]. The xylanase gene xynA has been sequenced and cloned into E. coli as a LacZ fusion protein, but efficient expression was not obtained [3]. Recently, the DNA sequence of xynA has been optimized, and the expression of the enzyme in E. coli has reached a high level by using recombinant plasmid pET-20b-xynA. However, the recombinant enzyme was mainly found in inclusion bodies, and only a small proportion was soluble and active [4]. It is a common problem that some recombinant proteins will aggregate to form inclusion bodies in the cytoplasm and/or

periplasm [5]. Inclusion body formation of eukaryotic proteins in E. coli with many contributing factors: insolubility of the product at the concentrations being produced, inability to fold correctly in the bacterial environment, or lack of appropriate bacterial chaperone proteins [6]. Many attempts have been made to improve the soluble expression of recombinant proteins in E. coli. The formation of inclusion bodies could be decreased by changing the promoter to regulate the level of expression, controlling the growth conditions (especially the pH of the culture), controlling fermentation medium, changing the temperature of induction and enabling secretion into the periplasm, fusing the target gene to another gene [7]. OsmY has been used as a fusion partner to excrete target proteins into the medium [8–10]. When fused to OsmY, E. coli alkaline phosphatase, Bacillus subtilis a-amylase, and human leptin could be secreted into the medium at high levels [11]. Here we report the construction of vector pET-OsmY-xynA, and the overexpression of soluble fusion protein OsmY-xynA in E. coli. Materials and methods Bacterial strains, plasmids and growth media

⇑ Corresponding author. Tel.: +86 511 88796122. E-mail address: [email protected] (Y. Le). http://dx.doi.org/10.1016/j.pep.2014.03.004 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.

E. coli DH5a (TaKaRa, Dalian, China) was used as hosts for gene cloning. E. coli BL21(DE3) (TaKaRa, Dalian, China) was used as hosts

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for the expression of fusion protein. The cells harboring expression plasmids were cultured in Luria–Bertani (LB)1 medium supplemented with ampicillin (100 lg/ml). Construction of fusion expression plasmid pET-OsmY-xynA Based on the nucleotide sequences coding for the OsmY protein (GenBank accession No. NC_000913.3), the PCR amplification was carried out by using E. coli DH5a genome as template, with the following primers: 50 -GCCGAATTCATGACTATGACAAGACTGAAG-30 and 50 -GGCGGATCCCTTAGTTTTCAGATCATTTTTAAC-30 . PCR was carried out using the high-fidelity Pyrobest DNA polymerase (TaKaRa, Dalian, China). PCR products were purified using the QIAquick PCR purification kit and followed by digestion with BamH I and EcoR I restriction enzyme(s). The reverse PCR amplification was carried out by using plasmid pET-20b-xynA [4] as template, with primers X1 (50 -GCCGAATTCTATATCTCCTTCTTAAAGTTAAAC-30 ) and X2 (50 -GGCGGATCCCAGACTACCCCGAACTCTGAAG-30 ). PCR products were purified using the QIAquick PCR purification kit and followed by digestion with corresponding restriction enzyme(s). The digested PCR products were ligated to OsmY at BamH I/EcoR I sites. Expression and purification of fusion protein The plasmid pET-OsmY-xynA was transformed into the E. coli BL21(DE3) by electroporation. The cells carrying pET-OsmY-xynA were grown at 37 °C, and induced for gene expression by addition IPTG (isopropyl-b-D-thio galactopyranoside). The recombinant enzyme was isolated from the periplasm by cold osmotic shock according to a published protocol [12]. The cells (wet weight 1.5 g) harvested by centrifugation at 6000g for 5 min were resuspended in 12 ml of 100 mM Tris–HCl containing 20% sucrose and 1 mM EDTA (pH 8.0), and then pelleted by centrifugation at 8000g for 5 min followed by re-suspension in 5 ml of ice-cold water for 10 min. After the addition of MgCl2 to a final concentration of 1 mM, the cell suspension was incubated on ice for a further 10 min before being pelleted by centrifugation at 8000g for 10 min at 4 °C. The supernatant (5 ml) was loaded onto a 1 ml HisTrap HP columns (GE Healthcare), washed with 60 mM imidazole and 0.5 M NaCl in 20 mM Tris–HCl buffer (pH 7.9), and eluted with 1 M imidazole and 0.5 M NaCl in 20 mM Tris–HCl buffer (pH 7.9). The pooled fractions were dialyzed into storage buffer containing 1 mM EDTA, and 20% (v/v) glycerol before the enzyme was stored at 20 °C. The SDS–PAGE was performed according to standard procedures. Protein concentration was determined by the Bradford method using BSA as a standard [13].

Results Construction of expression plasmids The gene encoding the OsmY (including the signal sequence) was amplified from the genomic DNA of E. coli DH5a, and inserted into the plasmid pET-20b-xynA (pelB signal sequence was deleted) [4] at BamH I/EcoR I sites. Newly generated plasmid is designated as pET-OsmY-xynA (carried an N-terminal OsmY signal sequence). Target protein xylanase from fungus T. lanuginosus was linked to the C-terminus of OsmY by a BamH I site sequence. The fusion protein OsmY-xynA was expressed with a C-terminal His-tag.

Expression level and solubility of the fusion protein OsmY-xynA The recombinant plasmid pET-OsmY-xynA was isolated, which was then transformed into E. coli BL21(DE3) for the production of fusion protein OsmY-xynA using IPTG induction. The xylanase activity of fusion protein was obtained after induction at different IPTG concentrations (Fig. 1). Interestingly, xylanase activity produced by cells containing pET-OsmY-xynA with 0 mM, 0.1 mM, 0.3 mM, 0.5 mM IPTG, was 98 ± 6 U/ml, 6.4 ± 0.2 U/ml, 6.3 ± 0.15 U/ml, 6 ± 0.1 U/ml, respectively. There was a gradual decrease in xylanase activity upon increasing the IPTG concentration (Fig. 1). The recombinant cells induced without IPTG addition produced an activity level 16 times higher than that expressed with 0.5 mM IPTG induction. Previously, intracellular expression of the xylanase was improved by sequence optimization by site-directed mutagenesis without changing the protein sequence [4]. But the recombinant xylanase mainly appeared as inclusion bodies [4]. In the current study, the expression levels and solubility of the fusion protein expressed from pET-OsmY-xynA were shown in Fig. 2. The fusion protein expression level is very high when the fusion gene expression was induced by the addition of IPTG at 37 °C (Fig. 2, line 3–5), and only a small proportion was soluble and active (Fig. 2 line 7–9). However, the fusion protein was expressed as almost soluble form when the fusion gene expression was induced without IPTG addition (Fig. 2 line 2 and 6).

Secretion of fusion protein in E. coli When pET-OsmY-xynA vector was used to express fusion protein, the xylanase activities in the extracellular, periplasmic and cytoplasmic fractions were monitored (Fig. 3). Protein expression was initiated by the addition of IPTG to a final concentration of 0.5 mM when the optical density at 600 nm (OD600) reached 0.8.

Enzyme assays Xylanase activity was determined by the 4-hydroxybenzoic acid hydrazide method [14]. Xylan from birch wood (Sigma Aldrich, Munich, Germany) was used as the substrate. The reaction mixture comprised of 100 ll 1% (w/v) birch wood xylan in water, 90 ll phosphate buffer (50 mM, pH 6.0) and 10 ll properly diluted enzyme. The reaction was conducted at 65 °C for 10 min, and stopped when 600 ll of 4-hydroxybenzoic acid hydrazide solution were added into the reaction mixture. The reducing sugar was determined by reading the absorbance at 410 nm after the test tubes were incubated for 10 min in boiling water bath and cooled down on ice. One unit of xylanase activity was defined as the amount of enzyme releasing 1 lmol reducing sugar per min.

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Abbreviations used: LB, Luria–Bertani; IPTG, isopropyl-b-D-thio galactopyranoside.

Fig. 1. Total expresion levels of fusion protein OsmY-xynA in cytoplasm, periplasm and culture medium were observed after induction at different IPTG concentrations.

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Fig. 2. SDS–PAGE analysis for the expression of fusion protein in pET-OsmY-xynA after induction at different IPTG concentrations. Lanes: M, protein markers; 1, total protein of E. coli containing pET-20b(+); 2–5, total protein of E. coli containing pETOsmY-xynA induction with 0 mM, 0.1 mM, 0.3 mM, 0.5 mM IPTG, respectively; 6–9, soluble protein of E. coli containing pET-OsmY-xynA induction with 0 mM, 0.1 mM, 0.3 mM, 0.5 mM IPTG, respectively.

When E. coli BL21(DE3) was used as host to express fusion protein in LB medium, a maximal cell density (OD600 5.3 ± 0.1) was obtained without IPTG addition (Fig. 3). In comparison, a significantly higher level of xylanase activity was observed in the extracellular, periplasmic and cytoplasmic fractions when cells induced without IPTG addition (Fig. 3). As shown in Fig. 3a, when the fusion protein was expressed by the addition of IPTG to a final concentration of 0.5 mM, most of the xylanase activity (5.7 ± 0.2 U/ml) was observed in the extracellular fraction at 24 h post-induction. However, when the fusion protein was expressed without IPTG induction, most of the activity was observed in the extracellular (36 ± 1.3 U/ml) and the periplasmic (42 ± 4 U/ml) (Fig. 3b). Purification of the fusion protein The fusion protein was purified in two steps: cold osmotic shock and nickel affinity chromatography (Table 1). The periplasm fusion proteins were extracted by the cold osmotic shock procedure and furthermore purified with nickel affinity chromatography. An analysis by SDS–PAGE showed that the recombinant

Fig. 4. SDS–PAGE analysis for the expression of fusion protein. lanes: M, protein markers; 1, total protein of E. coli containing pET-20b(+); 2, total protein and 3, soluble protein of E. coli containing pET-OsmY-xynA; 4, periplasmic protein obtained by cold osmotic shock; 5, fusion protein purified after a His-tagged affinity chromatography.

fusion OsmY-xynA had a molecular mass of about 44 kDa (Fig. 4). After the enzymes were purified, the specific activity of fusion protein OsmY-xynA was 739 ± 45 U/mg. In comparison, the purified xylanase from Aspergillus niger exhibited a specific activity of 808.5 U/mg towards birch wood xylan [15]. The specific enzyme activity of xylanase from Thermotoga thermarum was up to 145.8 U/mg [16]. XynA from Clostridium cellulovorans had a high specific activity with birch wood xylan (825 U/mg) [17].

Effects of pH and temperature on enzyme activity and stability When birch wood xylan was used as substrate, the optimal reaction of fusion protein occurred at 65 °C, pH 6.0 (Fig. 5a and b). The purified enzyme retained over 80% of its activity after holding at a pH ranging from 5.8 to 7.8 for 1 h at 60 °C (Fig. 5c). The thermostability was evaluated by determination of the residual ligating activity after incubating the mixtures (0.042 mg/ml OsmY-xynA, 50 mM pH 6.0) for various times at 60 °C, 65 °C, 70 °C. Fig. 5d shows the residual activity assayed under standard reaction conditions.

Fig. 3. The effect of IPTG induction on the cell density and the production of recombinant protein in the E. coli cells harboring pET-OsmY-xynA. When the OD600 reached 0.8 (denoted as time zero), IPTG was added to cell cultures. (a) with 0.5 mM IPTG induction, (b) without IPTG addition. Symbols: -j- cell density; -D- xylanase activities in the extracellular; -h- xylanase activities in the periplasmic; -s- xylanase activities in the cytoplasmic fractions.

Table 1 Purification of fusion protein OsmY-xynA from E. coli harboring pET-OsmY-xynA. Purification step

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Purification fold

Periplasmic fraction Nickel affinity chromatography

3.3 0.27

442.2 199.5

134 739

1.0 5.5

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Fig. 5. The effects of temperature and pH on the xylanase activity and enzyme stability of OsmY-xynA. (a) The optimal temperature determined by standard assay at various temperatures (pH 6.0). (b) The optimal pH determined at 65 °C in citrate buffer buffer, or phosphate buffer. (c) The pH stability of OsmY-xynA. The remaining activity was determined after purified enzyme (0.042 mg/ml) was incubated at 60 °C for 1 h in 50 mM in citrate buffer buffer, or phosphate buffer. (d) The thermostability of the OsmYxynA. The purified enzyme (0.042 mg/ml) in 50 mM phosphate buffer (pH 6.0) was incubated for various durations at 60 °C (-N-), 65 °C (-d-), 70 °C (-j-). Residual activities were assayed at 65 °C in 50 mM phosphate buffer (pH 6.0). The initial activity (739 ± 45 U/mg) was defined as 100%. Data are means ± standard deviations from three replications.

Discussion To facilitate enzyme production and the industrial use of the thermophilic xylanase preparations, several xylanase genes from thermophilic fungi have been cloned and expressed in different heterologous systems [18,19]. The mature xylanase from T. lanuginosus has 196 amino acids, with 31 amino acids encoded by the codons rarely used in E. coli [3]. In previous study, the xylanase gene from T. lanuginosus had been optimized for high level expression in the cytoplasm of E. coli by using pET-20b-xyA, but the enzyme was produced at a largely insoluble state, the soluble expression level of recombinant enzyme was very poor, with a highest activity of 4.1 U/ml [4]. OsmY of E. coli is the hyperosmotically inducible periplasmic protein. Many studies have been focused on the excretion of target proteins into the medium. When fused to OsmY, many recombinant proteins of different origin could be excreted into the medium [8–11]. Here, we constructed a vector pET-OsmY-xynA to export fusion protein. OsmY-xynA was successfully expressed at a high level in a soluble form when the fusion gene expression was induced without IPTG addition. Interestingly, the majority of the recombinant proteins were in the insoluble fraction when cells were induced by the addition of different IPTG concentrations (Fig. 2). SDS–PAGE analysis revealed that the fusion proteins expressed from pETOsmY-xynA after induction at different IPTG concentrations were

separated into two adjacent bands, suggesting that some of the enzyme molecules were still carrying signal peptide in the periplasm (Fig. 2). These observations suggested that recombinant fusion xylanase was not correctly folded when the heterogeneous proteins were expressed in a very high rate after induction with IPTG. The pET system is the most powerful system used for the cloning and expression of recombinant proteins in E. coli [20]. Induction is probably due to auto-induction due to lactose in the medium [21]. It is known that LB medium may be contaminated with lactose, and lactose contamination increases background level of protein expression. Supplementing culture media with glucose could maintain very low basal expression levels of T7 RNA polymerase in the kDE3 lysogenic expression hosts used in the pET System [22,23]. Much lower expression of the target protein was observed (4.5 ± 0.5 U/ml) when the strain harboring pET-OsmY-xynA was grown in LB medium supplemented with 1% glucose by incubation overnight at 37 °C without IPTG. In our previous study, the xylanase gene from T. lanuginosus was overexpressed in the cytoplasm, but the enzyme was produced at a largely insoluble state [4]. The improvement of soluble protein is primarily attributed to the fact that the periplasmic spac provides a more oxidative environment than the cytoplasm [24]. The fusion proteins were translocated into the periplasmic space, and enzyme activity analysis shown that about 43% of enzyme activity was localized in the periplasmic space (Fig. 3a). A simple osmotic shock was used to obtain the products (Fig. 4).

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Previous results have shown that the signal peptide of OsmY is responsible for the secretion into the periplasm, while OsmY is responsible for the excretion from the periplasm to the medium [11]. On the basis of xylanase activity, about 37% of enzyme activity was in the medium when cells were induced without IPTG addition (Fig. 3). In summary, while the mechanism for the pET-OsmY-xynA mediated high-level soluble expression of an aggregation-prone xylanase in E. coli is not completely understood, this paper offers an alternative protocol to prevent the inclusion body formation of a thermostable xylanase from thermophilic fungus. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 31300088), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (Grant No. 12KJB180002) and Doctoral Scientific Research Foundation of Jiangsu University (Grant No. 10JDG117). References [1] J. Gomes, I. Gomes, W. Kreiner, H. Esterbauer, M. Sinner, W. Steiner, Production of high level xylanase by a wild strain of Thermometers lanuginosus using beechwood xylan, J. Biotechnol. 30 (1993) 283–297. [2] F. Baneyx, Recombinant protein expression in Escherichia coli, Curr. Opin. Biotechnol. 10 (1999) 411–421. [3] A. Schlacher, K. Holzmann, M. Hayn, W. Steiner, H. Schwab, Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus, J. Biotechnol. 49 (1996) 211–218. [4] E. Yin, Y. Le, J. Pei, W. Shao, Q. Yang, High-level expression of the xylanase from Thermomyces lanuginosus in Escherichia coli, World J. Microbiol. Biotechnol. 24 (2008) 275–280. [5] K.L. Pan, H.C. Hsiao, C.L. Weng, M.S. Wu, C.P. Chou, Roles of DegP in prevention of protein misfolding in the periplasm upon overexpression of penicillin acylase in Escherichia coli, J. Bacteriol. 185 (2003) 3020–3030. [6] S.M. Singh, A.K. Panda, Solubilization and refolding of bacterial inclusion body proteins, J. Biosci. Bioeng. 99 (2005) 303–310. [7] L. Strandberg, S.O. Enfors, Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli, Appl. Environ. Microbiol. 57 (1991) 1669–1674.

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