Prokaryotic Expression and Purification of DsMAPK from Dunaliella salina

Wenjing YUE, Xiaojie CHAI, Liying LIU, Tianxiang WU, Yiqiong LIU

Ministry of Agriculture’s Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea/Liaoning Provincial Key Laboratory of Marine Bio-resource Restoration and Habitat Reparation, Dalian Ocean University, Dalian 116023, China

Dunaliella salina is a kind of eukaryote that is known most tolerant to slat. It can withstand dramatic changes in salinity and has a wide range of salt tolerance. Dunaliella salina can grow in salt solutions with concentration ranging from 0.05 mol/L to the saturated concentration[1].Dunaliella salina has no cell wall and is natural protoplasts[2]. The strong salt tolerance,simple cell structure and facilitated culture conditions make the Dunaliella salina an important model organism to investigate the molecular salt tolerance mechanism of plants[3].Mitogen activated protein kinase family(MAPKs) are a class of serine/threonine protein kinases and they exist in all types of eukaryotes. The molecular weight of the MAPK family ranges from 38 to 55 kDa[4].MAPK,along with other signal molecules forms the MAPK cascade signaling pathway to accept the external stimulus and transduce the signals into cells, influencing the expression of specific genes.MAPK plays an important role in the growth, development, differentiation and apoptosis of eukaryotes[5]. Therefore, the separation of MAPK gene from the genome of Dunaliella salina and the in-depth study on its molecular mechanism will have an important scientific significance in the determination of molecular pathways that respond to salt stress in Dunaliella salina[6].

In this experiment, the gene of DsMAPK (GenBank Accession JQ782412) was successfully cloned.Its expression form under salt stress was analyzed with QRT-PCR.The results showed the expression of DsMAPK could be induced by salt stress. It participated in the salt tolerance of Dunaliella salina. The expression level of DsMAPK was up-regulated under salt stress. The recombinant expression vector pGS-21a-DsMAPK was constructed successfully in our study.The DsMAPK supernatant protein was collected through enlarged culture and sonication and purifiedwith GST-SefinoseTMKit. Then the soluble fusion protein with high purity was obtained. This study would lay a foundation for the preparation of antibodies and the further research on the role of DsMAPK protein in the molecular mechanism of Dunaliella salina’s salt tolerance at the protein level.

Material and Methods

Material

Strains and plasmids The strains of DH5α and E.coli BL21 (DE3)and the plasmids of pGS-21a and pMDCMGNCat were preserved by our laboratory.The pMD18-T Simple Vector was purchased from the TaKaRa Biotechnology(Dalian)Co.,Ltd.

Reagents The restriction endonucleases, Taq polymerase, IPTG, X-gal and T4 ligase were purchased from the TaKaRa Biotechnology (Dalian)Co., Ltd. The Nitrocellulose membrane, Anti-GST Antibody, HRP-IgG and Precipitation type single-component TMB substrate solution were purchased from the Tiangen Biotech(Beijing)Co.,Ltd.The GST-SefinoseTMKit(BSP032-7)and Prestained Protein Molecular Weight Marker were purchased from the Sangon Biotech(Shanghai) Co., Ltd. The Gel Extraction Kit was purchased from the Axygen Biotechnology Co., Ltd. Other reagents were all of analytical grade and made in China.

Methods

Construction of prokaryotic expression vector The primers were designed according to the open reading frame of DsMAPK gene. The restriction sites were added. The sequence of the upstream primer (P1,containing EcoR I restriction site) was as follows:5’-CGGAATTCATGGCAGACGAAGCCAAG-3’. The sequence of downstream primer (P2,containing Sal I restriction site) was as follows:5’-GTCGACTTAACAGGCAGACCCAGG-3’. The sequence of pMDCMGN-Cat was used as the template,and then the PCR was conducted with P1 and P2. The amplification conditions were as follows:pre-denaturation at 94 ℃for 5 min; denaturation at 94℃for 30 s,annealing at 60 ℃for 30 s,extension at 72 ℃for 1 min,35 cycles;extension at 72 ℃for 9 min. The PCR products were analyzed with 1.0%agarose gel electrophoresis. The target fragment was recovered and connected to the pMD18-T Simple Vector(T-A cloning). The recombinant plasmid (pMD18-MAPK) was constructed.Then the recombinant plasmid was transformed into E. coli DH5α. The positive monoclones were screened out on the anti-Amp plate. The pMD18-MAPK and pGS-21a plasmids were digested with EcoR I and Sal I and then connected with T4 ligase.The expression vector (pGS-21a-DsMAPK) was constructed and transformed into E. coli DH5α. The positive monoclones were screened out and sequenced by the TaKaRa Biotechnology(Dalian)Co.,Ltd.to verify the correctness of the reading frame.

Prokaryotic expression of fusion protein The correctly-constructed expression plasmid (pGS-21a-DsMAPK)was transformed into E.coli BL21 (DE3). The positive monoclones were picked out, inoculated in 5 ml of LB liquid medium containing 50 mg/L of ampicillin and placed in a thermostatic shaker overnight (37 ℃, 190 r/min).The next day,the bacterial solution was inoculated in 5 ml of new LB liquid medium containing 50 mg/L of ampicillin (v/v, 1%) and incubated at 37 ℃. When OD600of the bacterial solution reached 0.6-0.8, the IPTG was added in the treatment group with final concentration of 0.4 mmol/L but not added in the control group. Then the bacterial solution was incubated at 37℃ for 6 h. After centrifugation, the bacterial cells were collected. Then PBS buffer (0.01 mol/L)was added in the collected cells. After sonication and centrifugation (4 ℃,12 000 r/min,10 min), the supernatant and precipitate were obtained. After adding with sample buffer, the supernatant and precipitate were analyzed with SDSPAGE.

Enlarged culture and purification of recombinant protein A certain volume (100 ml) of expression bacterial solution was centrifuged at 8 000 r/min for 5 min.The bacterial cells were collected. After adding PBS buffer (0.01 mol/L), the bacterial cells were disrupted by ultrasonic wave (power, 30 W;working time,5 s;interval,5 s;total time, 20 min) on ice. After disruption,the solution was centrifuged at 12 000 r/min for 20 min at 4 ℃. Then the supernatant was passed through the 0.45 μm filter membrane. The filtrate was then purified with the GST-SefinoseTM Kit and the flow rate was controlled within 0.5-1.0 ml/min. The effluent was purified again to increase the amount of bind protein.The Elution Buffer was used to elute the protein(1 ml/time, flow rate controlled within 0.5-1.0 ml/min). New centrifuge tubes were used to collect the effluent step by step with 1 ml for each time. An equal volume (100 μl) of solution was taken from each tuber and mixed.Then an equal-volume 2 × Loading Buffer was added.The purified protein was detected with SDS-PAGE.

Western blotting analysis The purified fusion protein was analyzed with SDS-PAGE and then transferred to NC membrane (200 mA,1.5 h).Then the protein was closed with 3%bovine serum albumin and placed at 4 ℃overnight. After adding the Anti-GST Antibody(1∶2 000),the membrane was incubated at 37 ℃for 1 h. Then the membrane was washed with 0.01 mmol/L of PBST 3 times (5 min/time).After adding the HRP-IgG (1∶200), the membrane was incubated at 37 ℃for 1 h. Then the membrane was washed with 0.01 mmol/L of PBST 3 times (5 min/time). Finally, the precipitation type single-component TMB substrate solution was added on the membrane for the chromogenic reaction.

Results

Construction of prokaryotic expression vector

A 170 bp band was obtained by PCR (Fig.1A).The size of the protein was as same as expected. The recombinant plasmid (pGS-21a-DsMAPK) was digested with EcoR I and Sal I. The electrophoresis results(Fig.1B) showed the target fragment was connected to pGS-21a successfully, and the recombinant plasmid could be used for sequencing.The sequencing results showed the sequence of amplified fragment was completely consistent with that of open reading frame of DsMAPK gene, and the reading frame was correct. Therefore, the prokaryotic expression vector(pGS-21a-DsMAPK) was successfully constructed.

Prokaryotic expression of fusion protein

After the expression bacteria(containing empty pGS-21a) in the control group were induced, an obvious band of GST-tag with molecular weight of 26 kD was shown on the electrophoretogram. After the expression bacteria (containing recombinant plasmid pGS-21a-DsMAPK) in the treatment group were induced, an obvious band of fusion protein with molecular weight of 78 kD was obtained. However, this band was not shown in the control group (Fig.2 a),which was in consistent with the expected results(the fusion protein contained a 52 kD of DsMAPK and a 26 kD of GST-tag). Fig.2A also showed the target protein was shown in both of the supernatant and precipitate samples, indicating part of the fusion protein was expressed in the soluble form.

Enlarged culture and purification of recombinant protein

Under the same induction conditions (final concentration of IPTG was 0.4 mmol/L; induced at 37 ℃for 6 h),the enlarged culture of the expression bacteria was conducted. The results showed the expression levels of GSTtag and the soluble fusion protein were relatively high (Fig.2B). After purified with GST-SefinoseTMKit, the expression products were detected with SDSPAGE. As shown in Fig.2C, a 26 kDa protein and a 78 kDa protein were obtained.It was indicated the fusion protein was expressed correctly and the expression level was relatively high.

Western blotting analysis

The Western blotting analysis(Fig.3) showed the fusion protein can bind with the anti-GST monoclonal antibody specially, indicating the fusion protein had a good immunological activity.Thus the fusion protein was successfully expressed in E. coli BL21(DE3).

Discussion

In recent years, more and more studies show that MAPK plays an important role in the regulation of growth and development, anti-adversity and other physiological process of plants[7-8].Therefore,the further research on the MAPKs has an important significance in the study on the molecular response of plants to environmental stresses and the key aspects of signal transduction[9-10]. The resistance of crops and other plants can be improved through transgenic methods based on the identification of key MAPKs in signal transduction related to the resistance of plants[11].Although the researches on MAPKs in plants have made a lot of progresses,the specific mechanisms are still unclear compared to that of MAPKs in animal and yeasts[12]. Therefore, the new membranes of MAPK family are needed to identify further and the relations among membranes are also needed to investigate to improve the MAPK cascade pathway model[13]. In the evolution of plants,a very sophisticated mechanism that is adapted to the bad environments has been developed.In Dunaliella salina,a highly salttolerant model organism,the DsMAPK has been studied rarely[14]. In this study, the DsMAPK was successfully cloned.It was also primarily proved the DsMAPK is related to the salt tolerance of Dunaliella salina.This will provide a basis for the further research on the role of DsMAPK in the salt-tolerance response of Dunaliella salina.

In this study, the classical prokaryotic expression system of E. coli was adopted to express the fusion protein. The expressed fusion protein existed in the supernatant and precipitate of bacterial cell suspension, indicating the existence forms of fusion protein included soluble form and inclusion. In order to reduce the expression level of the inclusion-styled protein, the concentration of the inducing agents should be as low as possible.The optimum temperature for the growth of E. coli was selected. The purified soluble DsMAPK protein can be used to screen out the proteins that can interact with DsMAPK protein to further understand the role of factors upstream or downstream the DsMAPK gene.In order to further investigate the effects of DsMAPK protein in the salt tolerance mechanism,the protein may be overexpressed in transgenic plants and then the salt tolerance of transgenic plants will be determined.

Conclusions

In our study, the prokaryotic expression vector (pGS-21a-DsMAPK)of DsMAPK was successfully constructed and successfully expressed in E.coli BL21 (DE3).After the enlarged culture and purification,high-purity soluble fusion protein was obtained from the supernatant. The western blotting analysis also primarily proved the fusion protein was GST-tagged DsMAPK protein.All the results above will not only lay a foundation for the antibody preparation and the further research on the role of DsMAPK protein in the salt tolerance of Dunaliella salina,but also provide a reference for the application of the DsMAPK gene in the improvement of salt tolerance of plants.

[1]HE QH (贺庆华),ZHANG QL (张庆莲).Research on salt tolerance mechanism of Dunaliella salina (盐藻的耐盐机理的研究) [J].Journal of Southwest University for Nationalities (西南民族学院学报(自然科学版)),2009,35(3):487-490.

[2]HOU YJ(侯永杰), LI QH(李庆华), XUE LX (薛乐勋).Construction and expression of prokaryotic expression vector of FLA8 of Dunaliella salina(杜氏盐藻基因FLA8 原核表达载体的构建和表达) [J].Journal of Zhengzhou University(Medical Sciences),2013,48(1):31-34.

[3]CHEN JY (陈建勇), WANG C (王聪),WANG J (王娟), et al. Research advance on MAPK signal pathway(MAPK信号通路研究进展)[J].Journal of Chinese Medical Sciences(中国医药科学),2011,1(8):32-34.

[4]LI XJ (李秀娟),CHAI XJ (柴晓杰),TAO XY (陶晓迎),et al.Prokaryotic expression and purification of DsRab, a small G protein of Dunaliella salina (盐藻小G蛋白DsRab 的原核表达及纯化) [J].Biotechnology Bulletin (生物技术通报),2014,4:178-182.

[5]DING Y (丁洋), ZHAO RR (赵瑞瑞),SHEN L (申琳),et al.Role of MAPKs in the signal transduction of plants under stresses (MAPKs 在植物逆境信号转导中的作用)[J].Journal of Beijing University of Agriculture (北京农学院学报),2009,24(3):77-80.

[6]BAI XM(白雪梅),ZHANG Y(张岩),TAO BQ (陶柏秋).Research methods of mitogen activated protein kinase(MAPK)(促分裂原活化蛋白激酶(MAPK)的研究方法)[J]. Inner Mongolia Petrochemical Industry (内蒙古石油化工),2011,3:5-7.

[7]ZHANG JJ(张军杰),ZHOU H(周会),HU YF(胡育峰),et al.Cloning and prokaryotic expression of sbel cDNA of corn(玉米基因sbe1 cDNA 的克隆与原核表达)[J]. Acta Agriculturae Nucleatae Sinica(核农学报),2009,23(1):70-74.

[8]YANG YD (杨亚东),GUO HL (郭辉力),LIU WB (刘文彬), et al. Construction,expression and purification of fusion protein of 4CL of tobacco and STS of Japanese knotweed (烟 草4CL与虎杖STS 融合基因的构建、 表达及融合蛋白纯化)[J].Journal of Beijing University of Agriculture (北京农学院学报),2014,29(3):1-5.

[9]SUN BX (孙柏欣), LIU CY (刘长远),CHEN Y (陈彦), et al. Research advance on gene expression system(基因表达系统研究进展) [J]. Modern Agricultural Science and Technology (现代农业科技),2008,2:205-209.

[10]SUN QC (孙庆春),ZHENG CS (郑成淑),LIANG F(梁芳),et al.MAPKs and their signal transduction in vital movement of plant cells(MAPKs 及其在植物细胞生命活动中的信号转导) [J]. Advances in Horticulture (园艺学进展),2009,482-487.

[11]YU SW, TANG KX. MAP kinase cascades responding to environmental stress in plants [J]. Chinese Bulletin of Botany (植物学通报), 2004, 46(2):127-136.

[12]GUO L (郭丽), DUAN JX (段金秀),LIANG ZX(梁占训),et al.Construction of plant expression vector for MAPK gene of Dunaliella salina and its transformation into tobacco ( 杜氏盐藻MAPK 基因植物表达载体的构建及对烟草的转化) [J]. Grassland and Turf(Bimonthly),2006,4(117):32-36.

[13]LIU Y, REN D, PIKE S, et al. Chloroplast generated reactive oxygen species are involved in hypersensitive response-like cell death mediated by a mitogen-activated protein kinase cascade[J].Plant J,2007,5l:4l-54.

[14]GOYAL A. Osmoregulation in Dunaliella, Part II:Photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta [J]. Plant Physiol Biochem,2007,45(9):705-710.