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Improvement of expression level of polysaccharide lyases with new tag GAPDH in E. coli
Journal of Biotechnology 236 (2016) 159–165
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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Improvement of expression level of polysaccharide lyases with new tag GAPDH in E. coli Zhenya Chen a , Ye Li b,∗ , Xinxiao Sun a , Qipeng Yuan a,∗ a State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, No.15 East Road of North Third Ring, Chao Yang District, Beijing, 100029, China b Department of Biotechnology, Beijing Polytechnic, No.1 TaiyanggongShaoyaoju, Chao Yang District, Beijing, 100029, China
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Article history: Received 2 July 2016 Received in revised form 18 August 2016 Accepted 24 August 2016 Available online 25 August 2016 Keywords: Housekeeping gene GAPDH Expression level Activity
a b s t r a c t Escherichia coli (E. coli) is widely used to express a variety of heterologous proteins. Efforts have been made to enhance the expression level of the desired protein. However, problems still exist to regulate the level of protein expression and therefore, new strategies are needed to overcome those issues. Glyceraldehyde3-phosphate dehydrogenase (GAPDH) which is properly expressed in E. coli might play a leading role and increase the expression levels of the target proteins. In this study, GAPDH was fused with a target enzyme, ChSase ABC I, an endoeliminase and polysaceharide lyase. Our results confirmed this hypothesis and indicated that GAPDH boosted the expression level of ChSase ABC I with an increase of 2.25 times, while the enzymatic activity with an increase of 2.99 times. The hypothesis were also supported by RTPCR study and GAPDH was more effective in enhancing the expression level and enzymatic activity as compared to MBP, which is commonly used as fused tag and can improve the soluble expression of target protein. addition, the expression level and enzymatic activity of other polysaceharide lyases were also improved in the presence of GAPDH. The findings of this study prove that GAPDH has a strong effect on enhancing the expression level and enzymatic activity of the target proteins. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Escherichia coli (E. coli) expression system continues to dominate the bacterial expression systems and remains to be the first choice for laboratory investigations. The proteins production capability of E. coli has been extensively studied for several reasons that include easy growth, short generation time and genetic manipulation for the development of engineered strains. However, there are still many problems in the expression process, like low expression level, which might be caused by the forming of inclusion bodies. Inclusion bodies have negative effects on the high yield and purity of heterologous proteins, and complex process are required to solubilize and refold the deposition in inclusion bodies to functional or non-functional proteins (Schindler et al., 2016). The size of the proteins, the effect of the tags, and the fermentation conditions may influence the fold of soluble proteins. Therefore, many strategies has been used to overcome those issues and improve the production and activity of some enzymes (Sorensen and Mortensen,
∗ Corresponding authors. E-mail addresses: [email protected] (Y. Li), [email protected] (Q. Yuan). http://dx.doi.org/10.1016/j.jbiotec.2016.08.016 0168-1656/© 2016 Elsevier B.V. All rights reserved.
2005). Firstly, common method was using different tags like MBP (Chen et al., 2015b), which was used as fused tags for improving the solubility and purity of recombinant proteins because of the assist of correct fold of target protein like chaperones (Samuelsson et al., 1994), the effectiveness of capturing target protein in low concentrations, the beneficial effects on the thermostability of target protein, and the enzymatic removal of themselves. Secondly, mutagenesis was a way to vary the specific amino acids of target enzymes, and caused the increase of catalytic efficiency and enzymatic activity (Chen et al., 2013; Nazari-Robati et al., 2013; Susan et al., 2013). Though different method had varied advantages, they also caused negative influences. For instance, MBP caused the low productivity and the decrease of catalytic efficiency of target enzyme (Chen et al., 2015b). Therefore, a new effective tag is needed for increasing the accumulation of recombinant proteins in E. coli, while also assisting the fold of recombinant proteins. We hypothesized the housekeeping genes could play a leading role to improve the expression level of heterologous proteins that they fused. Housekeeping genes are always selected as internal reference genes for data normalization in real time-PCR (RT-PCR) experiments because of the expression stability (Bustin, 2000). Housekeeping genes display relatively stable expression in host strain though other genes were transformed in it. Glyceraldehyde-3-
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phosphate dehydrogenase (GAPDH), which is an essential enzyme in glycolytic pathway and catalyzes the oxidative phosphorylation of d-glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate, is one of the most commonly used housekeeping genes in RTPCR. GAPDH, encoded by gapA, is known to have a high level of expression and needed NAD+ as a cofactor (Nelson et al., 1991). In addition to the significance in glycolytic pathway, GAPDH can also catalyze microtubule polymerization, modify protein phosphorylation, participate in membrane fusion and transport, regulate protein expression, and repair DNA damage et al. (Hara et al., 2005). GAPDH was also used as target protein to detect the effects of other tags (Gschaedler et al., 1999; Raran-Kurussi and Waugh, 2014). Chondroitinases (ChSases) are one kind of polysaccharide lyases that can degrade chondroitin sulfate (CS) through spliting -1,4-galactosaminidic bonds between d-glucuronic acid and DN-acetyl-galactosamine to unsaturated disaccharides. ChSase ABC has a wide range of substrates and has been studied in many processes like the treatment of some diseases (Lee et al., 2000; Makris et al., 2014). As a tool enzyme to study glycosaminoglycan, a number of ChSase ABCs were identified in Proteus vulgaris (P. vulgaris) and divided into ChSase ABC I (endoeliminase) and ChSase ABC II (exoeliminase), with different catalytic sites and catalytic efficiency (Hamai et al., 1997; Prabhakar et al., 2005, 2009; Sato et al., 1994; Yamagata et al., 1968). ChSase ABC I has also been heterologously expressed in our previous study (Chen et al., 2015a, 2015b, 2015c) Because of the functions, as well as stable and correct expression of gapA, gapA was chosen as target gene and the expressed protein (GAPDH) fused with ChSase ABC I. The expression level and enzymatic activity of ChSase ABC I, were considered as significant parameters and detected from different aspects. In addition, the comparison of GAPDH and MBP, and the effect of GAPDH on other polysaceharide lyases were also summarized. 2. Materials and methods 2.1. Strains, plasmids, and reagents P. vulgaris (KCTC 2579) was purchased from Korea KCTC storage. pMAL-c2x plasmid is stored in our laboratory. Q5TM High-Fidelity 2 × Master Mix, T4 DNA ligase, and the restriction enzyme were obtained from New England Biolabs. RiboPureTM RNA Purification Kit, FastQuant RT Kit and Power SYBR® GreenPCR Master Mix were obtained from Ambion, TIANGEN, and Applied Biosystems, respectively. The substrates CS A (MW:50,000) and CS B (MW:50,000) were purchased from Nanjing Oddo’s Biological Technology Co., Ltd. All the reagents used in this study are analytical grade. The medium of E. coli was Luria-Bertani (LB). 2.2. Construction of the recombinant plasmids The genes of ChSase ABC I and GAPDH were amplified by PCR using genomic DNA of P. vulgaris and E. coli JM109, respectively. The primers used are shown in Table S1. The amplified ChSase ABCI fragment was digested with BamH I/Xho I, while the amplified gapA was digested with Nde I/BamH I. The recombinant plasmid was constructed by ligating pMAL-c2x which was digested with Nde I/Xho I with digested ChSase ABCI and gapA. The positive plasmid pMAL-c2x-gapA-ChSase ABC I (cut MBP) was confirmed by digestion and DNA sequencing. Other recombinant plasmid were also constructed and verified using the same method as mentioned above. 2.3. Expression of ChSase ABC i and GAPDH-ChSase ABC I The recombinant plasmids pMAL-c2x-ChSase ABC I (cut MBP) and pMAL-c2x-gapA-ChSase ABC I (cut MBP) were transformed
into E. coli JM109 in order to express ChSase ABC I and GAPDHChSase ABC I. The transformants were cultured at 37 ◦ C (200 rpm) for 12 h as the seed culture. 500 L of seed culture was transferred into 50 mL LB media and cultured at 37 ◦ C until OD600 reached around 0.6. The bacterial cells were induced by the addition of IPTG (final concentration 0.5 mmol/L) and the cells culture was incubated (200 rpm) at 16 ◦ C for 20 h. After induction, the cells were harvested (8000 rpm at 4 ◦ C) and the obtained pellet was resuspended in 15 mL buffer A (20 mmol/L Tris-HCl, pH 7.4). Finally, the cells were disrupted by ultrasonic instrument, followed by centrifugation. The supernatant obtained was used as a source of crude enzyme. 2.4. Western blot and Q-TOF analysis The crude enzyme containing ChSase ABC I or GAPDH-ChSase ABC I (50–100 ng), which normalized with the same OD600 , was subjected to 12% SDS-PAGE, and transferred to a nitrocellulose membrane before immunoblotting with the relevant primary antibodies. The proteins ChSase ABC I and GAPDH-ChSase ABC I were probed with ChSase ABC I antibody (1000 × dilution, NOVUS Biologicals USA), followed by anti-mouse IgG (1000 × dilution, Santa Cruz), and developed with enhanced chemiluminescence (Tanon). Chemiluminescence on the membranes were detected by luminescent image analyzer (Tanon). Target proteins ChSase ABC I and GAPDH-ChSase ABC I were also confirmed by quadrupole time-offlight mass spectrometer (Q-TOF) in Beijing Protein Innovation. 2.5. Activity assay Enzymatic activity of ChSase ABC I was measured according to the UV 232 nm method. The enzymatic reaction was carried out at 37 ◦ C using CS A or CS B as substrate in buffer A. CS degradation was monitored by UV absorbance at 232 nm and the activity was calculated using a molar extinction coefficient of 3800 L/(mol cm) (Chen et al., 2011; Ye et al., 2009). Protein concentration was detected by Bradford Protein Assay Kit (Bio-rad). One international unit was defined as the amount of protein that can release 1 mol 4,5unsaturated uronic acid per minute at 37 ◦ C. 2.6. Detection of the level of mRNA by RT-PCR The samples were collected during the fermentation processes of E. coli JM109 (pMAL-c2x-ChSase ABC I (cut MBP)) and E. coli JM109 (pMAL-c2x-gapA-ChSase ABC I (cut MBP)). Cells were obtained from 4 mL fermentation in different times (0 h (before induction), 4 h, 8 h, 11 h, and 20 h (after induction)). Total RNA isolation was performed with RNA Purification Kit, then reversely transcripted to cDNA by Fast Quant RT Kit. The primers used are shown in Table S1. RT-PCR was performed on a EppendorfMastercycler in triplicate for each experimental sample, with threshold cycle values (Ct) averaged from the triplicates. Reactions were run with Power SYBR® Green PCR Master Mix mixed with cDNA and primers. The PCR program was maintained at 95 ◦ C for 4 min, followed by 40 cycles of 95 ◦ C for 30 s, 55 ◦ C for 30 s, 72 ◦ C for 30 s, followed by melting curve. 3. Results and discussion 3.1. Construction of the recombinant plasmids The genes of ChSase ABC I and GAPDH were amplified, digested and ligated to digested plasmid pMAL-c2x, which has tac promoter and MBP tag and is usually used to improve soluble expression of heterologous protein (Chen et al., 2013, 2011, 2015b; Wu et al., 2014). The positive plasmid pMAL-c2x-gapA-ChSase ABC I
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Fig. 1. Construction of the recombinant plasmids. Construction of recombinant ChSase ABC I with different tags (A). Digestion of recombinant pMAL-c2x-gapA-ChSase ABC I (cut MBP) with different enzymes. Lane M: 5000 bp DNA marker. Lane 1: digestion of recombinant pMAL-c2x-gapA-ChSase ABC I with BamH I and Xho I. Lane 2: digestion of recombinant pMAL-c2x-gapA-ChSase ABC I (cut MBP) with Nde I and Xho I. Lane M: 10,000 bp DNA marker (B). Construction of recombinant ChSase AC, ChSase B and Hep A with or without GAPDH tag (C).
(cut MBP) was digested with BamH I/Xho I and Nde I/Xho I, and it would appear band at 6000 bp/3000 bp and 5000 bp/4000 bp approximately. This result was verified in Fig. 1B. Subsequently, pMAL-c2x-gapA-ChSase ABC I (cut MBP) was sequenced in Beijing Genomics Institute and compared with the sequence of NCBI (GenBank: GQ996964.1 and CP001509.3) through NCBI sequence alignment analysis. It had 100% identity with the sequence of NCBI as mentioned above. Other recombinant plasmids were also constructed as shown in Fig. 1A and confirmed successfully (data not shown).
reasons of the impact of GAPDH on the expression level of its fusion partner might have similarity with other fused tag in the previous study (Raran-Kurussi and Waugh, 2014). In previous reports, 80% soluble GAPDH was achieved when gapA recombinantly expressed in exogenous cells at certain conditions (Branlant et al., 1983; Gschaedler et al., 1999) and the high-efficiency expression might explain the improvement of ChSase ABC I expression level.
3.2. Expression of ChSase ABC I and GAPDH-ChSase ABC I The plasmid pMAL-c2x containing ChSase ABC I gene without MBP and the plasmid pMAL-c2x containing gapA and ChSase ABC I genes without MBP were expressed by IPTG inducing in E. coli. The crude enzymes were obtained after ultrasonic treatment and centrifugation of fermentation broth, and analyzed with western blotting. Q-TOF confirmed ChSase ABC I and GAPDH-ChSase ABC I were expressed successfully in JM109. After Image J2X software analysis and calculation (Li et al., 2015; Liu et al., 2014), the expression level of target protein GAPDH-ChSase ABC I (148.00 kDa) was about 2.25 times higher than that of ChSase ABC I (112.50 kDa) (Fig. 2). GAPDH-ChSase ABC I was the main composition of the total protein, indicating that GAPDH has a positive effect on the expression of ChSase ABC I. These results implied high-level gene expression was motivated by GAPDH, which as a fused tag. The
Fig. 2. Western blot analysis of the recombinant ChSase ABC I and GAPDH-ChSase ABC I. Lane M: 250 kDa protein molecular weight marker. Lane 1: total protein of control. Lane 2: crude enzyme containing ChSase ABC I. Lane 3: crude enzyme containing GAPDH-ChSase ABC I. Western blot analysis was performed by using ChSase ABC I antibody.
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Fig. 3. Enzymatic activities and specific activities of crude enzymes. The crude enzymes containing ChSase ABC I (A) or GAPDH-ChSase ABC I (B) with CS A and CS B as substrates. Data represent the mean of three determinations ± SD.
3.3. Enzymatic activities of ChSase ABC I and GAPDH-ChSase ABC I To investigate the influence of fused GAPDH on enzymatic activity of the total crude enzyme, we determined the enzymatic activities and specific activities of GAPDH-ChSase ABC I and ChSase ABC I using CS A and CS B as substrates. The crude enzyme containing protein GAPDH-ChSase ABC I had an enzymatic activity of 29.63 ± 3.25 IU/mL crude enzyme (8887.97 ± 973.89 IU/L fermentation liquor) with substrate CS A, while having a specific activity of 37.75 ± 3.49 IU/mg protein. The enzymatic activity of the crude enzyme containing GAPDH-ChSase ABC I was 2.99 times higher than that of another crude enzyme containing ChSase ABC I. As similar to use substrate CS A, the enzymatic activity was also enhanced using substrate CS B (Fig. 3). These findings confirmed that GAPDH improved the enzymatic activity of the total crude enzyme, consisted with the increase of expression level.
3.4. Detection of the amount of mRNA by RT-PCR In order to confirm gapA’s influence on the transcription level of ChSase ABC I, RT-PCR (Arezi and Hogrefe, 2007; Duarte et al., 2015) was used to detect the amount of mRNA with 16S rRNA as a reference gene. The variations of the levels of ChSase ABC I mRNAs were studied for a period of time following IPTG addition. The total RNAs of every sample were reversely transcripted to cDNA. The RT-PCR mix with cDNA as template, primers, and polymerase containing SYBR Green reacted as mentioned in 2.6. Ct values were presented after normalizing the Ct values of every sample, which displayed after qRT-PCR. Fig. 4 illustrated that the Ct values of GAPDH-ChSase ABC I were lower than that of ChSase ABC I in the whole fermentation process after adding IPTG, meaning that the cDNAs of GAPDH-ChSase ABC I were higher than that of ChSase ABC I. Meanwhile, it was observed that the ChSase ABC I mRNA levels of GAPDH-ChSase ABC I exceeded that of ChSase ABC I by 1.61, 5.21, 9.14 and 8.08 times at 4, 8, 11 and 20 h, respectively (Fig. 5). The time-dependent improvement in the transcription level of ChSase ABC I gene of GAPDH-ChSase ABC I as compared to that of ChSase ABC I was attributed to the stable transcription of gapA gene, which could in turn stabilize the transcription of ChSase ABC I gene. Because of the very high expression level of the cloned E. coli gapA gene, it also exhibited a strong influence on the transcription and translation (Fig. 2) level of its fused gene (ChSase ABC I) as compared to ChSase ABC I gene without gapA gene. These results were also compatible with the increase of the amount of GAPDH-ChSase ABC I protein.
3.5. Comparison of GAPDH and MBP The effect of GAPDH on the improved expression of heterologous soluble protein was compared with that of MBP, commonly used as fused tag (Chen et al., 2011; Raran-Kurussi and Waugh, 2014). The strains E. coli JM09, containing plasmids pMAL-c2x-ChSase ABC I and pMAL-c2x-gapA-ChSase ABC I respectively, were cultured to express recombinant proteins MBP-ChSase ABC I and MBP-GAPDHChSase ABC I. Fig. 6A presented the enzymatic activity of the crude enzyme with MBP-ChSase ABC I had an decrease of 1.67 times as compared with that of the crude enzyme with GAPDH-ChSase ABC I (Fig. 3B), as well as the specific activity reduced 1.41 times. On the other hand, the enzymatic activity and specific activity of the crude enzyme with MBP-GAPDH-ChSase ABC I showed 1.76 and 1.76 times lower than that of another crude enzyme containing MBPChSase ABC I with CS A as a substrate (Fig. 6B). MBP increased ChSase ABC I gene expression in the absence of gapA gene as described in the above results and Fig. 6C also confirmed. Conversely, the decrease of ChSase ABC I gene expression appeared in the presence of gapA gene. The high molecular weight of MBP-GAPDH-ChSase ABC I resulted in the difficulty of recombinant protein folding and the pressure of host strain expressing heterologous protein. The more effectivity in enhancing the expression level and enzymatic activity that GAPDH had was validated as compared to MBP in the above designed experiment and consequence.
Fig. 4. mRNA level of ChSase ABC I and GAPDH-ChSase ABC I. Ct of ChSase ABC I and GAPDH-ChSase ABC I (A). Data represent the mean of three determinations ± SD.
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Fig. 5. 2−Ct of control (ChSase ABC I) and GAPDH-ChSase ABC I. After inducing 4 h (A), 8 h (B), 11 h (C) and 20 h (D). Data represent the mean of three determinations ± SD.
Fig. 6. Enzymatic activities, specific activities and SDS-PAGE of crude enzymes. Enzymatic activities and specific activities of crude enzymes containing MBP-ChSase ABC I (A) or MBP-GAPDH-ChSase ABC I (B). SDS-PAGE of crude enzymes containing MBP-ChSase ABC I or MBP-GAPDH-ChSase ABC I. Lane M: 250 kDa protein molecular weight marker. Lane 1-2: supernatant and precipitation of MBP-ChSase ABC I after ultrasonic treatment and centrifugation. Lane 3-4: supernatant and precipitation of MBP-GAPDH-ChSase ABC I after ultrasonic treatment and centrifugation (C).
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3.6. Influence of GAPDH on other polysaccharide lyases
References
To further verify the general applicability of GAPDH, we used other polysaccharide lyases as target proteins, including Chondroitinase AC (ChSase AC), Chondroitinase B (ChSase B) and Heparinase I (Hep A). These lyases have been heterologously expressed, and site-directed mutagenesis was used to enhance the productions of ChSase B and Hep A with a same source of Flavobacterium heparinum in recombinant E. coli (Chen et al., 2013; Pojasek et al., 2001; Tkalec et al., 2000). However, low productivity and inclusion bodies still exist in the process of the recombinant expression. Hence, GAPDH was considered as a new fused tag to attempt to solve these problems. The recombinant plasmids pMALc2x containing ChSase AC, ChSase B or Hep A, were transformed to E. coli JM109 to express ChSase AC (77.20 kDa), ChSase B (53.60 kDa) and Hep A (41.36 kDa). GAPDH-ChSase AC (112.80 kDa), GAPDHChSase B (89.10 kDa) and GAPDH-Hep A (76.90 kDa) were obtained after E. coli JM109 (pMAL-c2x-gapA-ChSase AC (cut MBP)), E. coli JM109 (pMAL-c2x-gapA-ChSase B (cut MBP)) and E. coli JM109 (pMAL-c2x-gapA-Hep A (cut MBP)) fermentation and treatment of ultrasonication and centrifugation (Fig. 1C). GAPDH increased the expression levels of ChSase AC, ChSase B and Hep A by 2.17, 1.43 and 1.64 times, respectively after Image J2X software calculation and normalization (data not shown). Enzymatic activity of the crude enzyme with GAPDH-ChSase AC improved 4.16 times as agreed with the increase of expression level. Moreover, enzymatic activity of the crude enzyme with GAPDH-ChSase B was 2.69 times higher than another crude enzyme with ChSase B. The impact of GAPDH on the enzymatic activity of Hep A presented the same trend and enzymatic activity raised 2.57 times (Table S2). All these results confirmed our hypothesis and indicate GAPDH has the potential to increase the expression levels and enzymatic activities of other proteins with various degree.
Arezi, B., Hogrefe, H.H., 2007. Escherichia coli DNA polymerase III epsilon subunit increases Moloney murine leukemia virus reverse transcriptase fidelity and accuracy of RT-PCR procedures. Anal. Biochem. 360, 84–91. Branlant, G., Flesch, G., Branlant, C., 1983. Molecular cloning of the glyceraldehyde-3-phosphate dehydrogenase genes of Bacillus stearothermophilus and Escherichia coli, and their expression in Escherichia coli. Gene 25, 1–7. Bustin, S.A., 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–193. Chen, S., Ye, F., Chen, Y., Zhao, H., Yatsunami, R., Nakamura, S., Arisaka, F., Xing, X.H., 2011. Biochemical analysis and kinetic modeling of the thermal inactivation of MBP-fused heparinase I: implications for a comprehensive thermostabilization strategy. Biotechnol. Bioeng. 108, 1841–1851. Chen, S., Huang, Z., Wu, J., Chen, Y., Ye, F., Zhang, C., Yatsunami, R., Nakamura, S., Xing, X.H., 2013. Combination of site-directed mutagenesis and calcium ion addition for enhanced production of thermostable MBP-fused heparinase I in recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 97, 2907–2916. Chen, Z., Li, Y., Feng, Y., Chen, L., Yuan, Q., 2015a. Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis. RSC Adv. 5, 76040–76047. Chen, Z., Li, Y., Yuan, Q., 2015b. Expression, purification and thermostability of MBP-chondroitinase ABC I from Proteus vulgaris. Int. J. Biol. Macromol. 72, 6–10. Chen, Z., Li, Y., Yuan, Q., 2015c. Study the effect of His-tag on chondroitinase ABC I based on characterization of enzyme. Int. J. Biol. Macromol. 78, 96–101. Duarte, M.D., Carvalho, C.L., Barros, S.C., Henriques, A.M., Ramos, F., Fagulha, T., Luis, T., Duarte, E.L., Fevereiro, M., 2015. A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2). J. Virol. Methods 219, 90–95. Gschaedler, A., Robas, N., Boudrant, J., Branlant, C., 1999. Effects of pulse addition of carbon sources on continuous cultivation of Escherichia coli containing a recombinant E coli gapA gene. Biotechnol. Bioeng. 63, 712–720. Hamai, A., Hashimoto, N., Mochizuki, H., Kato, F., Makiguchi, Y., Horie, K., Suzuki, S., 1997. Two distinct chondroitin sulfate ABC lyases. An endoeliminase yielding tetrasaccharides and an exoeliminase preferentially acting on oligosaccharides. J. Biol. Chem. 272, 9123–9130. Hara, M.R., Agrawal, N., Kim, S.F., Cascio, M.B., Fujimuro, M., Ozeki, Y., Takahashi, M., Cheah, J.H., Tankou, S.K., Hester, L.D., Ferris, C.D., Hayward, S.D., Snyder, S.H., Sawa, A., 2005. S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat. Cell Biol. 7, 665–674. Lee, M.C., Sung, K.L.P., Kurtis, M.S., Akeson, W.H., Sah, R.L., 2000. Adhesive force of chondrocytes to cartilage effects of chondroitinase ABC. Clin. Orthop. Relat. Res. 370, 286–294. Li, L., Khan, M.N., Li, Q., Chen, X., Wei, J., Wang, B., Cheng, J.-W., Gordon, J.R., Li, F., 2015. G31P, CXCR1/2 inhibitor, with cisplatin inhibits the growth of mice hepatocellular carcinoma and mitigates high-dose cisplatin-induced nephrotoxicity. Oncol. Rep. 33, 751–757. Liu, K., Zhang, G., Wang, Z., Liu, Y., Dong, J., Dong, X., Liu, J., Cao, J., Ao, L., Zhang, S., 2014. The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800 MHz radiofrequency electromagnetic radiation. Toxicol. Lett. 228, 216–224. Makris, E.A., Macbarb, R.F., Paschos, N.K., Hu, J.C., Athanasiou, K.A., 2014. Combined use of chondroitinase-ABC, TGF-1, and collagen crosslinking agent lysyl oxidase to engineer functional neotissues for fibrocartilage repair. Biomaterials 35, 6787–6796. Nazari-Robati, M., Khajeh, K., Aminian, M., Mollania, N., Golestani, A., 2013. Enhancement of thermal stability of chondroitinase ABC I by site-directed mutagenesis: an insight from Ramachandran plot. BBA Proteins Proteom. 1834, 479–486. Nelson, K., Whittam, T.S., Selander, R.K., 1991. Nucleotide polymorphism and evolution in the glyceraldehyde-3-phosphate dehydrogenase gene (gapA) in natural populations of Salmonella and Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 88, 6667–6671. Pojasek, K., Shriver, Z., Kiley, P., Venkataraman, G., Sasisekharan, R., 2001. Recombinant expression, purification, and kinetic characterization of chondroitinase AC and chondroitinase B from Flavobacterium heparinum. Biochem. Biophys. Res. Commun. 286, 343–351. Prabhakar, V., Capila, I., Bosques, C.J., Sasisekharan, R., 2005. Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification. Biochem. J. 386, 103–112. Prabhakar, V., Capila, I., Soundararajan, V., Raman, R., Sasisekharan, R., 2009. Recombinant expression, purification, and biochemical characterization of chondroitinase ABC II from Proteus vulgaris. J. Biol. Chem. 284, 974–982. Raran-Kurussi, S., Waugh, D.S., 2014. Unrelated solubility-enhancing fusion partners MBP and NusA utilize a similar mode of action. Biotechnol. Bioeng. 111, 2407–2411. Samuelsson, E., Moks, T., Nilsson, B., Uhlen, M., 1994. Enhanced in vitro refolding of insulin-like growth factor I using a solubilizing fusion partner. Biochemistry 33, 4207–4211. Sato, N., Shimada, M., Nakajima, H., Oda, H., Kimura, S., 1994. Cloning and expression in E: coli of the gene encoding the Proteus vulgaris chondroitin ABC lyase. Appl. Microbiol. Biotechnol. 41, 39–46.
4. Conclusions In conclusion, the role of GAPDH was investigated on the expression level and enzymatic activity of ChSase ABC I. It is interesting to note that GAPDH enhanced the expression level and enzymatic activity of ChSase ABC I by 2.25 and 2.99 times, respectively, as compared to ChSase ABC I. These results were also supported by RT-PCR study which revealed that the transcription level of ChSase ABC I was improved in the presence of GAPDH. Furthermore, GAPDH was found more potent than MBP in enhancing the expression level and enzymatic activity of ChSase ABC I. The findings of this study clearly demonstrate that GAPDH could be an attractive fusion protein for the successful expression of other proteins and might play a leading role to increase the expression levels of the fused protein. Acknowledgements The research was funded by the National Natural Science Foundation of China (grant number 21306002 and 21176018), Academic Leader of Beijing Polytechnic (DTR201601), the Fundamental Research Funds for the Central Universities (buctrc201613), the Key Project of Beijing Polytechnic (YZK2016028), the Science-Technology Foundation of Beijing Municipal Commission of Education (grant number KM201510858001), and Scientific Research Team of Beijing Polytechnic (TD201602). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec.2016.08. 016.
Z. Chen et al. / Journal of Biotechnology 236 (2016) 159–165 Schindler, S., Missbichler, B., Walther, C., Sponring, M., Cserjan-Puschmann, M., Auer, B., Schneider, R., Durauer, A., 2016. N(pro) fusion technology: on-column complementation to improve efficiency in biopharmaceutical production. Protein Expr. Purif. 120, 42–50. Sorensen, H.P., Mortensen, K.K., 2005. Advanced genetic strategies for recombinant protein expression in Escherichia coli. J. Biotechnol. 115, 113–128. Susan, M., Jasmina, N.R., Martin, L.B., Hermann, H., Francisco, S., Heinz, D., O’Connor, K.E., 2013. Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches. Biotechnol. Bioeng. 110, 1849–1857. Tkalec, A.L., Fink, D., Blain, F., Zhang-Sun, G., Laliberte, M., Bennett, D.C., Gu, K., Zimmermann, J.J., Su, H., 2000. Isolation and expression in Escherichia coli of
165
cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum. Appl. Environ. Microbiol. 66, 29–35. Wu, J., Chong, Z., Xiang, M., Ye, L., Xing, X.H., 2014. Controllable production of low molecular weight heparins by combinations of heparinase I/II/III. Carbohydr. Polym. 101, 484–492. Yamagata, T., Saito, H., Habuchi, O., Suzuki, S., 1968. Purification and properties of bacterial chondroitinases and chondrosulfatases. J. Biol. Chem. 243, 1523–1535. Ye, F., Kuang, Y., Chen, S., 2009. Characteristics of low molecular weight heparin production by an ultrafiltration membrane bioreactor using maltose binding protein fused heparinase I. Biochem. Eng. J. 46, 193–198.
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Improvement of expression level of polysaccharide lyases with new tag GAPDH in E. coli Zhenya Chen a , Ye Li b,∗ , Xinxiao Sun a , Qipeng Yuan a,∗ a State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, No.15 East Road of North Third Ring, Chao Yang District, Beijing, 100029, China b Department of Biotechnology, Beijing Polytechnic, No.1 TaiyanggongShaoyaoju, Chao Yang District, Beijing, 100029, China
a r t i c l e
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Article history: Received 2 July 2016 Received in revised form 18 August 2016 Accepted 24 August 2016 Available online 25 August 2016 Keywords: Housekeeping gene GAPDH Expression level Activity
a b s t r a c t Escherichia coli (E. coli) is widely used to express a variety of heterologous proteins. Efforts have been made to enhance the expression level of the desired protein. However, problems still exist to regulate the level of protein expression and therefore, new strategies are needed to overcome those issues. Glyceraldehyde3-phosphate dehydrogenase (GAPDH) which is properly expressed in E. coli might play a leading role and increase the expression levels of the target proteins. In this study, GAPDH was fused with a target enzyme, ChSase ABC I, an endoeliminase and polysaceharide lyase. Our results confirmed this hypothesis and indicated that GAPDH boosted the expression level of ChSase ABC I with an increase of 2.25 times, while the enzymatic activity with an increase of 2.99 times. The hypothesis were also supported by RTPCR study and GAPDH was more effective in enhancing the expression level and enzymatic activity as compared to MBP, which is commonly used as fused tag and can improve the soluble expression of target protein. addition, the expression level and enzymatic activity of other polysaceharide lyases were also improved in the presence of GAPDH. The findings of this study prove that GAPDH has a strong effect on enhancing the expression level and enzymatic activity of the target proteins. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Escherichia coli (E. coli) expression system continues to dominate the bacterial expression systems and remains to be the first choice for laboratory investigations. The proteins production capability of E. coli has been extensively studied for several reasons that include easy growth, short generation time and genetic manipulation for the development of engineered strains. However, there are still many problems in the expression process, like low expression level, which might be caused by the forming of inclusion bodies. Inclusion bodies have negative effects on the high yield and purity of heterologous proteins, and complex process are required to solubilize and refold the deposition in inclusion bodies to functional or non-functional proteins (Schindler et al., 2016). The size of the proteins, the effect of the tags, and the fermentation conditions may influence the fold of soluble proteins. Therefore, many strategies has been used to overcome those issues and improve the production and activity of some enzymes (Sorensen and Mortensen,
∗ Corresponding authors. E-mail addresses: [email protected] (Y. Li), [email protected] (Q. Yuan). http://dx.doi.org/10.1016/j.jbiotec.2016.08.016 0168-1656/© 2016 Elsevier B.V. All rights reserved.
2005). Firstly, common method was using different tags like MBP (Chen et al., 2015b), which was used as fused tags for improving the solubility and purity of recombinant proteins because of the assist of correct fold of target protein like chaperones (Samuelsson et al., 1994), the effectiveness of capturing target protein in low concentrations, the beneficial effects on the thermostability of target protein, and the enzymatic removal of themselves. Secondly, mutagenesis was a way to vary the specific amino acids of target enzymes, and caused the increase of catalytic efficiency and enzymatic activity (Chen et al., 2013; Nazari-Robati et al., 2013; Susan et al., 2013). Though different method had varied advantages, they also caused negative influences. For instance, MBP caused the low productivity and the decrease of catalytic efficiency of target enzyme (Chen et al., 2015b). Therefore, a new effective tag is needed for increasing the accumulation of recombinant proteins in E. coli, while also assisting the fold of recombinant proteins. We hypothesized the housekeeping genes could play a leading role to improve the expression level of heterologous proteins that they fused. Housekeeping genes are always selected as internal reference genes for data normalization in real time-PCR (RT-PCR) experiments because of the expression stability (Bustin, 2000). Housekeeping genes display relatively stable expression in host strain though other genes were transformed in it. Glyceraldehyde-3-
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phosphate dehydrogenase (GAPDH), which is an essential enzyme in glycolytic pathway and catalyzes the oxidative phosphorylation of d-glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate, is one of the most commonly used housekeeping genes in RTPCR. GAPDH, encoded by gapA, is known to have a high level of expression and needed NAD+ as a cofactor (Nelson et al., 1991). In addition to the significance in glycolytic pathway, GAPDH can also catalyze microtubule polymerization, modify protein phosphorylation, participate in membrane fusion and transport, regulate protein expression, and repair DNA damage et al. (Hara et al., 2005). GAPDH was also used as target protein to detect the effects of other tags (Gschaedler et al., 1999; Raran-Kurussi and Waugh, 2014). Chondroitinases (ChSases) are one kind of polysaccharide lyases that can degrade chondroitin sulfate (CS) through spliting -1,4-galactosaminidic bonds between d-glucuronic acid and DN-acetyl-galactosamine to unsaturated disaccharides. ChSase ABC has a wide range of substrates and has been studied in many processes like the treatment of some diseases (Lee et al., 2000; Makris et al., 2014). As a tool enzyme to study glycosaminoglycan, a number of ChSase ABCs were identified in Proteus vulgaris (P. vulgaris) and divided into ChSase ABC I (endoeliminase) and ChSase ABC II (exoeliminase), with different catalytic sites and catalytic efficiency (Hamai et al., 1997; Prabhakar et al., 2005, 2009; Sato et al., 1994; Yamagata et al., 1968). ChSase ABC I has also been heterologously expressed in our previous study (Chen et al., 2015a, 2015b, 2015c) Because of the functions, as well as stable and correct expression of gapA, gapA was chosen as target gene and the expressed protein (GAPDH) fused with ChSase ABC I. The expression level and enzymatic activity of ChSase ABC I, were considered as significant parameters and detected from different aspects. In addition, the comparison of GAPDH and MBP, and the effect of GAPDH on other polysaceharide lyases were also summarized. 2. Materials and methods 2.1. Strains, plasmids, and reagents P. vulgaris (KCTC 2579) was purchased from Korea KCTC storage. pMAL-c2x plasmid is stored in our laboratory. Q5TM High-Fidelity 2 × Master Mix, T4 DNA ligase, and the restriction enzyme were obtained from New England Biolabs. RiboPureTM RNA Purification Kit, FastQuant RT Kit and Power SYBR® GreenPCR Master Mix were obtained from Ambion, TIANGEN, and Applied Biosystems, respectively. The substrates CS A (MW:50,000) and CS B (MW:50,000) were purchased from Nanjing Oddo’s Biological Technology Co., Ltd. All the reagents used in this study are analytical grade. The medium of E. coli was Luria-Bertani (LB). 2.2. Construction of the recombinant plasmids The genes of ChSase ABC I and GAPDH were amplified by PCR using genomic DNA of P. vulgaris and E. coli JM109, respectively. The primers used are shown in Table S1. The amplified ChSase ABCI fragment was digested with BamH I/Xho I, while the amplified gapA was digested with Nde I/BamH I. The recombinant plasmid was constructed by ligating pMAL-c2x which was digested with Nde I/Xho I with digested ChSase ABCI and gapA. The positive plasmid pMAL-c2x-gapA-ChSase ABC I (cut MBP) was confirmed by digestion and DNA sequencing. Other recombinant plasmid were also constructed and verified using the same method as mentioned above. 2.3. Expression of ChSase ABC i and GAPDH-ChSase ABC I The recombinant plasmids pMAL-c2x-ChSase ABC I (cut MBP) and pMAL-c2x-gapA-ChSase ABC I (cut MBP) were transformed
into E. coli JM109 in order to express ChSase ABC I and GAPDHChSase ABC I. The transformants were cultured at 37 ◦ C (200 rpm) for 12 h as the seed culture. 500 L of seed culture was transferred into 50 mL LB media and cultured at 37 ◦ C until OD600 reached around 0.6. The bacterial cells were induced by the addition of IPTG (final concentration 0.5 mmol/L) and the cells culture was incubated (200 rpm) at 16 ◦ C for 20 h. After induction, the cells were harvested (8000 rpm at 4 ◦ C) and the obtained pellet was resuspended in 15 mL buffer A (20 mmol/L Tris-HCl, pH 7.4). Finally, the cells were disrupted by ultrasonic instrument, followed by centrifugation. The supernatant obtained was used as a source of crude enzyme. 2.4. Western blot and Q-TOF analysis The crude enzyme containing ChSase ABC I or GAPDH-ChSase ABC I (50–100 ng), which normalized with the same OD600 , was subjected to 12% SDS-PAGE, and transferred to a nitrocellulose membrane before immunoblotting with the relevant primary antibodies. The proteins ChSase ABC I and GAPDH-ChSase ABC I were probed with ChSase ABC I antibody (1000 × dilution, NOVUS Biologicals USA), followed by anti-mouse IgG (1000 × dilution, Santa Cruz), and developed with enhanced chemiluminescence (Tanon). Chemiluminescence on the membranes were detected by luminescent image analyzer (Tanon). Target proteins ChSase ABC I and GAPDH-ChSase ABC I were also confirmed by quadrupole time-offlight mass spectrometer (Q-TOF) in Beijing Protein Innovation. 2.5. Activity assay Enzymatic activity of ChSase ABC I was measured according to the UV 232 nm method. The enzymatic reaction was carried out at 37 ◦ C using CS A or CS B as substrate in buffer A. CS degradation was monitored by UV absorbance at 232 nm and the activity was calculated using a molar extinction coefficient of 3800 L/(mol cm) (Chen et al., 2011; Ye et al., 2009). Protein concentration was detected by Bradford Protein Assay Kit (Bio-rad). One international unit was defined as the amount of protein that can release 1 mol 4,5unsaturated uronic acid per minute at 37 ◦ C. 2.6. Detection of the level of mRNA by RT-PCR The samples were collected during the fermentation processes of E. coli JM109 (pMAL-c2x-ChSase ABC I (cut MBP)) and E. coli JM109 (pMAL-c2x-gapA-ChSase ABC I (cut MBP)). Cells were obtained from 4 mL fermentation in different times (0 h (before induction), 4 h, 8 h, 11 h, and 20 h (after induction)). Total RNA isolation was performed with RNA Purification Kit, then reversely transcripted to cDNA by Fast Quant RT Kit. The primers used are shown in Table S1. RT-PCR was performed on a EppendorfMastercycler in triplicate for each experimental sample, with threshold cycle values (Ct) averaged from the triplicates. Reactions were run with Power SYBR® Green PCR Master Mix mixed with cDNA and primers. The PCR program was maintained at 95 ◦ C for 4 min, followed by 40 cycles of 95 ◦ C for 30 s, 55 ◦ C for 30 s, 72 ◦ C for 30 s, followed by melting curve. 3. Results and discussion 3.1. Construction of the recombinant plasmids The genes of ChSase ABC I and GAPDH were amplified, digested and ligated to digested plasmid pMAL-c2x, which has tac promoter and MBP tag and is usually used to improve soluble expression of heterologous protein (Chen et al., 2013, 2011, 2015b; Wu et al., 2014). The positive plasmid pMAL-c2x-gapA-ChSase ABC I
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Fig. 1. Construction of the recombinant plasmids. Construction of recombinant ChSase ABC I with different tags (A). Digestion of recombinant pMAL-c2x-gapA-ChSase ABC I (cut MBP) with different enzymes. Lane M: 5000 bp DNA marker. Lane 1: digestion of recombinant pMAL-c2x-gapA-ChSase ABC I with BamH I and Xho I. Lane 2: digestion of recombinant pMAL-c2x-gapA-ChSase ABC I (cut MBP) with Nde I and Xho I. Lane M: 10,000 bp DNA marker (B). Construction of recombinant ChSase AC, ChSase B and Hep A with or without GAPDH tag (C).
(cut MBP) was digested with BamH I/Xho I and Nde I/Xho I, and it would appear band at 6000 bp/3000 bp and 5000 bp/4000 bp approximately. This result was verified in Fig. 1B. Subsequently, pMAL-c2x-gapA-ChSase ABC I (cut MBP) was sequenced in Beijing Genomics Institute and compared with the sequence of NCBI (GenBank: GQ996964.1 and CP001509.3) through NCBI sequence alignment analysis. It had 100% identity with the sequence of NCBI as mentioned above. Other recombinant plasmids were also constructed as shown in Fig. 1A and confirmed successfully (data not shown).
reasons of the impact of GAPDH on the expression level of its fusion partner might have similarity with other fused tag in the previous study (Raran-Kurussi and Waugh, 2014). In previous reports, 80% soluble GAPDH was achieved when gapA recombinantly expressed in exogenous cells at certain conditions (Branlant et al., 1983; Gschaedler et al., 1999) and the high-efficiency expression might explain the improvement of ChSase ABC I expression level.
3.2. Expression of ChSase ABC I and GAPDH-ChSase ABC I The plasmid pMAL-c2x containing ChSase ABC I gene without MBP and the plasmid pMAL-c2x containing gapA and ChSase ABC I genes without MBP were expressed by IPTG inducing in E. coli. The crude enzymes were obtained after ultrasonic treatment and centrifugation of fermentation broth, and analyzed with western blotting. Q-TOF confirmed ChSase ABC I and GAPDH-ChSase ABC I were expressed successfully in JM109. After Image J2X software analysis and calculation (Li et al., 2015; Liu et al., 2014), the expression level of target protein GAPDH-ChSase ABC I (148.00 kDa) was about 2.25 times higher than that of ChSase ABC I (112.50 kDa) (Fig. 2). GAPDH-ChSase ABC I was the main composition of the total protein, indicating that GAPDH has a positive effect on the expression of ChSase ABC I. These results implied high-level gene expression was motivated by GAPDH, which as a fused tag. The
Fig. 2. Western blot analysis of the recombinant ChSase ABC I and GAPDH-ChSase ABC I. Lane M: 250 kDa protein molecular weight marker. Lane 1: total protein of control. Lane 2: crude enzyme containing ChSase ABC I. Lane 3: crude enzyme containing GAPDH-ChSase ABC I. Western blot analysis was performed by using ChSase ABC I antibody.
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Fig. 3. Enzymatic activities and specific activities of crude enzymes. The crude enzymes containing ChSase ABC I (A) or GAPDH-ChSase ABC I (B) with CS A and CS B as substrates. Data represent the mean of three determinations ± SD.
3.3. Enzymatic activities of ChSase ABC I and GAPDH-ChSase ABC I To investigate the influence of fused GAPDH on enzymatic activity of the total crude enzyme, we determined the enzymatic activities and specific activities of GAPDH-ChSase ABC I and ChSase ABC I using CS A and CS B as substrates. The crude enzyme containing protein GAPDH-ChSase ABC I had an enzymatic activity of 29.63 ± 3.25 IU/mL crude enzyme (8887.97 ± 973.89 IU/L fermentation liquor) with substrate CS A, while having a specific activity of 37.75 ± 3.49 IU/mg protein. The enzymatic activity of the crude enzyme containing GAPDH-ChSase ABC I was 2.99 times higher than that of another crude enzyme containing ChSase ABC I. As similar to use substrate CS A, the enzymatic activity was also enhanced using substrate CS B (Fig. 3). These findings confirmed that GAPDH improved the enzymatic activity of the total crude enzyme, consisted with the increase of expression level.
3.4. Detection of the amount of mRNA by RT-PCR In order to confirm gapA’s influence on the transcription level of ChSase ABC I, RT-PCR (Arezi and Hogrefe, 2007; Duarte et al., 2015) was used to detect the amount of mRNA with 16S rRNA as a reference gene. The variations of the levels of ChSase ABC I mRNAs were studied for a period of time following IPTG addition. The total RNAs of every sample were reversely transcripted to cDNA. The RT-PCR mix with cDNA as template, primers, and polymerase containing SYBR Green reacted as mentioned in 2.6. Ct values were presented after normalizing the Ct values of every sample, which displayed after qRT-PCR. Fig. 4 illustrated that the Ct values of GAPDH-ChSase ABC I were lower than that of ChSase ABC I in the whole fermentation process after adding IPTG, meaning that the cDNAs of GAPDH-ChSase ABC I were higher than that of ChSase ABC I. Meanwhile, it was observed that the ChSase ABC I mRNA levels of GAPDH-ChSase ABC I exceeded that of ChSase ABC I by 1.61, 5.21, 9.14 and 8.08 times at 4, 8, 11 and 20 h, respectively (Fig. 5). The time-dependent improvement in the transcription level of ChSase ABC I gene of GAPDH-ChSase ABC I as compared to that of ChSase ABC I was attributed to the stable transcription of gapA gene, which could in turn stabilize the transcription of ChSase ABC I gene. Because of the very high expression level of the cloned E. coli gapA gene, it also exhibited a strong influence on the transcription and translation (Fig. 2) level of its fused gene (ChSase ABC I) as compared to ChSase ABC I gene without gapA gene. These results were also compatible with the increase of the amount of GAPDH-ChSase ABC I protein.
3.5. Comparison of GAPDH and MBP The effect of GAPDH on the improved expression of heterologous soluble protein was compared with that of MBP, commonly used as fused tag (Chen et al., 2011; Raran-Kurussi and Waugh, 2014). The strains E. coli JM09, containing plasmids pMAL-c2x-ChSase ABC I and pMAL-c2x-gapA-ChSase ABC I respectively, were cultured to express recombinant proteins MBP-ChSase ABC I and MBP-GAPDHChSase ABC I. Fig. 6A presented the enzymatic activity of the crude enzyme with MBP-ChSase ABC I had an decrease of 1.67 times as compared with that of the crude enzyme with GAPDH-ChSase ABC I (Fig. 3B), as well as the specific activity reduced 1.41 times. On the other hand, the enzymatic activity and specific activity of the crude enzyme with MBP-GAPDH-ChSase ABC I showed 1.76 and 1.76 times lower than that of another crude enzyme containing MBPChSase ABC I with CS A as a substrate (Fig. 6B). MBP increased ChSase ABC I gene expression in the absence of gapA gene as described in the above results and Fig. 6C also confirmed. Conversely, the decrease of ChSase ABC I gene expression appeared in the presence of gapA gene. The high molecular weight of MBP-GAPDH-ChSase ABC I resulted in the difficulty of recombinant protein folding and the pressure of host strain expressing heterologous protein. The more effectivity in enhancing the expression level and enzymatic activity that GAPDH had was validated as compared to MBP in the above designed experiment and consequence.
Fig. 4. mRNA level of ChSase ABC I and GAPDH-ChSase ABC I. Ct of ChSase ABC I and GAPDH-ChSase ABC I (A). Data represent the mean of three determinations ± SD.
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Fig. 5. 2−Ct of control (ChSase ABC I) and GAPDH-ChSase ABC I. After inducing 4 h (A), 8 h (B), 11 h (C) and 20 h (D). Data represent the mean of three determinations ± SD.
Fig. 6. Enzymatic activities, specific activities and SDS-PAGE of crude enzymes. Enzymatic activities and specific activities of crude enzymes containing MBP-ChSase ABC I (A) or MBP-GAPDH-ChSase ABC I (B). SDS-PAGE of crude enzymes containing MBP-ChSase ABC I or MBP-GAPDH-ChSase ABC I. Lane M: 250 kDa protein molecular weight marker. Lane 1-2: supernatant and precipitation of MBP-ChSase ABC I after ultrasonic treatment and centrifugation. Lane 3-4: supernatant and precipitation of MBP-GAPDH-ChSase ABC I after ultrasonic treatment and centrifugation (C).
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3.6. Influence of GAPDH on other polysaccharide lyases
References
To further verify the general applicability of GAPDH, we used other polysaccharide lyases as target proteins, including Chondroitinase AC (ChSase AC), Chondroitinase B (ChSase B) and Heparinase I (Hep A). These lyases have been heterologously expressed, and site-directed mutagenesis was used to enhance the productions of ChSase B and Hep A with a same source of Flavobacterium heparinum in recombinant E. coli (Chen et al., 2013; Pojasek et al., 2001; Tkalec et al., 2000). However, low productivity and inclusion bodies still exist in the process of the recombinant expression. Hence, GAPDH was considered as a new fused tag to attempt to solve these problems. The recombinant plasmids pMALc2x containing ChSase AC, ChSase B or Hep A, were transformed to E. coli JM109 to express ChSase AC (77.20 kDa), ChSase B (53.60 kDa) and Hep A (41.36 kDa). GAPDH-ChSase AC (112.80 kDa), GAPDHChSase B (89.10 kDa) and GAPDH-Hep A (76.90 kDa) were obtained after E. coli JM109 (pMAL-c2x-gapA-ChSase AC (cut MBP)), E. coli JM109 (pMAL-c2x-gapA-ChSase B (cut MBP)) and E. coli JM109 (pMAL-c2x-gapA-Hep A (cut MBP)) fermentation and treatment of ultrasonication and centrifugation (Fig. 1C). GAPDH increased the expression levels of ChSase AC, ChSase B and Hep A by 2.17, 1.43 and 1.64 times, respectively after Image J2X software calculation and normalization (data not shown). Enzymatic activity of the crude enzyme with GAPDH-ChSase AC improved 4.16 times as agreed with the increase of expression level. Moreover, enzymatic activity of the crude enzyme with GAPDH-ChSase B was 2.69 times higher than another crude enzyme with ChSase B. The impact of GAPDH on the enzymatic activity of Hep A presented the same trend and enzymatic activity raised 2.57 times (Table S2). All these results confirmed our hypothesis and indicate GAPDH has the potential to increase the expression levels and enzymatic activities of other proteins with various degree.
Arezi, B., Hogrefe, H.H., 2007. Escherichia coli DNA polymerase III epsilon subunit increases Moloney murine leukemia virus reverse transcriptase fidelity and accuracy of RT-PCR procedures. Anal. Biochem. 360, 84–91. Branlant, G., Flesch, G., Branlant, C., 1983. Molecular cloning of the glyceraldehyde-3-phosphate dehydrogenase genes of Bacillus stearothermophilus and Escherichia coli, and their expression in Escherichia coli. Gene 25, 1–7. Bustin, S.A., 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–193. Chen, S., Ye, F., Chen, Y., Zhao, H., Yatsunami, R., Nakamura, S., Arisaka, F., Xing, X.H., 2011. Biochemical analysis and kinetic modeling of the thermal inactivation of MBP-fused heparinase I: implications for a comprehensive thermostabilization strategy. Biotechnol. Bioeng. 108, 1841–1851. Chen, S., Huang, Z., Wu, J., Chen, Y., Ye, F., Zhang, C., Yatsunami, R., Nakamura, S., Xing, X.H., 2013. Combination of site-directed mutagenesis and calcium ion addition for enhanced production of thermostable MBP-fused heparinase I in recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 97, 2907–2916. Chen, Z., Li, Y., Feng, Y., Chen, L., Yuan, Q., 2015a. Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis. RSC Adv. 5, 76040–76047. Chen, Z., Li, Y., Yuan, Q., 2015b. Expression, purification and thermostability of MBP-chondroitinase ABC I from Proteus vulgaris. Int. J. Biol. Macromol. 72, 6–10. Chen, Z., Li, Y., Yuan, Q., 2015c. Study the effect of His-tag on chondroitinase ABC I based on characterization of enzyme. Int. J. Biol. Macromol. 78, 96–101. Duarte, M.D., Carvalho, C.L., Barros, S.C., Henriques, A.M., Ramos, F., Fagulha, T., Luis, T., Duarte, E.L., Fevereiro, M., 2015. A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2). J. Virol. Methods 219, 90–95. Gschaedler, A., Robas, N., Boudrant, J., Branlant, C., 1999. Effects of pulse addition of carbon sources on continuous cultivation of Escherichia coli containing a recombinant E coli gapA gene. Biotechnol. Bioeng. 63, 712–720. Hamai, A., Hashimoto, N., Mochizuki, H., Kato, F., Makiguchi, Y., Horie, K., Suzuki, S., 1997. Two distinct chondroitin sulfate ABC lyases. An endoeliminase yielding tetrasaccharides and an exoeliminase preferentially acting on oligosaccharides. J. Biol. Chem. 272, 9123–9130. Hara, M.R., Agrawal, N., Kim, S.F., Cascio, M.B., Fujimuro, M., Ozeki, Y., Takahashi, M., Cheah, J.H., Tankou, S.K., Hester, L.D., Ferris, C.D., Hayward, S.D., Snyder, S.H., Sawa, A., 2005. S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat. Cell Biol. 7, 665–674. Lee, M.C., Sung, K.L.P., Kurtis, M.S., Akeson, W.H., Sah, R.L., 2000. Adhesive force of chondrocytes to cartilage effects of chondroitinase ABC. Clin. Orthop. Relat. Res. 370, 286–294. Li, L., Khan, M.N., Li, Q., Chen, X., Wei, J., Wang, B., Cheng, J.-W., Gordon, J.R., Li, F., 2015. G31P, CXCR1/2 inhibitor, with cisplatin inhibits the growth of mice hepatocellular carcinoma and mitigates high-dose cisplatin-induced nephrotoxicity. Oncol. Rep. 33, 751–757. Liu, K., Zhang, G., Wang, Z., Liu, Y., Dong, J., Dong, X., Liu, J., Cao, J., Ao, L., Zhang, S., 2014. The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800 MHz radiofrequency electromagnetic radiation. Toxicol. Lett. 228, 216–224. Makris, E.A., Macbarb, R.F., Paschos, N.K., Hu, J.C., Athanasiou, K.A., 2014. Combined use of chondroitinase-ABC, TGF-1, and collagen crosslinking agent lysyl oxidase to engineer functional neotissues for fibrocartilage repair. Biomaterials 35, 6787–6796. Nazari-Robati, M., Khajeh, K., Aminian, M., Mollania, N., Golestani, A., 2013. Enhancement of thermal stability of chondroitinase ABC I by site-directed mutagenesis: an insight from Ramachandran plot. BBA Proteins Proteom. 1834, 479–486. Nelson, K., Whittam, T.S., Selander, R.K., 1991. Nucleotide polymorphism and evolution in the glyceraldehyde-3-phosphate dehydrogenase gene (gapA) in natural populations of Salmonella and Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 88, 6667–6671. Pojasek, K., Shriver, Z., Kiley, P., Venkataraman, G., Sasisekharan, R., 2001. Recombinant expression, purification, and kinetic characterization of chondroitinase AC and chondroitinase B from Flavobacterium heparinum. Biochem. Biophys. Res. Commun. 286, 343–351. Prabhakar, V., Capila, I., Bosques, C.J., Sasisekharan, R., 2005. Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification. Biochem. J. 386, 103–112. Prabhakar, V., Capila, I., Soundararajan, V., Raman, R., Sasisekharan, R., 2009. Recombinant expression, purification, and biochemical characterization of chondroitinase ABC II from Proteus vulgaris. J. Biol. Chem. 284, 974–982. Raran-Kurussi, S., Waugh, D.S., 2014. Unrelated solubility-enhancing fusion partners MBP and NusA utilize a similar mode of action. Biotechnol. Bioeng. 111, 2407–2411. Samuelsson, E., Moks, T., Nilsson, B., Uhlen, M., 1994. Enhanced in vitro refolding of insulin-like growth factor I using a solubilizing fusion partner. Biochemistry 33, 4207–4211. Sato, N., Shimada, M., Nakajima, H., Oda, H., Kimura, S., 1994. Cloning and expression in E: coli of the gene encoding the Proteus vulgaris chondroitin ABC lyase. Appl. Microbiol. Biotechnol. 41, 39–46.
4. Conclusions In conclusion, the role of GAPDH was investigated on the expression level and enzymatic activity of ChSase ABC I. It is interesting to note that GAPDH enhanced the expression level and enzymatic activity of ChSase ABC I by 2.25 and 2.99 times, respectively, as compared to ChSase ABC I. These results were also supported by RT-PCR study which revealed that the transcription level of ChSase ABC I was improved in the presence of GAPDH. Furthermore, GAPDH was found more potent than MBP in enhancing the expression level and enzymatic activity of ChSase ABC I. The findings of this study clearly demonstrate that GAPDH could be an attractive fusion protein for the successful expression of other proteins and might play a leading role to increase the expression levels of the fused protein. Acknowledgements The research was funded by the National Natural Science Foundation of China (grant number 21306002 and 21176018), Academic Leader of Beijing Polytechnic (DTR201601), the Fundamental Research Funds for the Central Universities (buctrc201613), the Key Project of Beijing Polytechnic (YZK2016028), the Science-Technology Foundation of Beijing Municipal Commission of Education (grant number KM201510858001), and Scientific Research Team of Beijing Polytechnic (TD201602). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec.2016.08. 016.
Z. Chen et al. / Journal of Biotechnology 236 (2016) 159–165 Schindler, S., Missbichler, B., Walther, C., Sponring, M., Cserjan-Puschmann, M., Auer, B., Schneider, R., Durauer, A., 2016. N(pro) fusion technology: on-column complementation to improve efficiency in biopharmaceutical production. Protein Expr. Purif. 120, 42–50. Sorensen, H.P., Mortensen, K.K., 2005. Advanced genetic strategies for recombinant protein expression in Escherichia coli. J. Biotechnol. 115, 113–128. Susan, M., Jasmina, N.R., Martin, L.B., Hermann, H., Francisco, S., Heinz, D., O’Connor, K.E., 2013. Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches. Biotechnol. Bioeng. 110, 1849–1857. Tkalec, A.L., Fink, D., Blain, F., Zhang-Sun, G., Laliberte, M., Bennett, D.C., Gu, K., Zimmermann, J.J., Su, H., 2000. Isolation and expression in Escherichia coli of
165
cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum. Appl. Environ. Microbiol. 66, 29–35. Wu, J., Chong, Z., Xiang, M., Ye, L., Xing, X.H., 2014. Controllable production of low molecular weight heparins by combinations of heparinase I/II/III. Carbohydr. Polym. 101, 484–492. Yamagata, T., Saito, H., Habuchi, O., Suzuki, S., 1968. Purification and properties of bacterial chondroitinases and chondrosulfatases. J. Biol. Chem. 243, 1523–1535. Ye, F., Kuang, Y., Chen, S., 2009. Characteristics of low molecular weight heparin production by an ultrafiltration membrane bioreactor using maltose binding protein fused heparinase I. Biochem. Eng. J. 46, 193–198.