Research Article

Purification and Optimization of Prokaryotic Expression of CSN5B Protein of Arabidopsis thaliana  

Xu Niu1,2 , Lili Wang1,2 , Shenkui Liu3 , Yuanyuan Bu1,2
1 Key Laboratory of Saline-Alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin 150040, China
2 College of Life Science, Northeast Forestry University, Harbin 150040, China
3 The State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Lin’An, Zhejiang 311300, China
Author    Correspondence author
Molecular Soil Biology, 2020, Vol. 11, No. 2   doi: 10.5376/msb.2020.11.0002
Received: 22 Jan., 2020    Accepted: 12 Feb., 2020    Published: 26 Feb., 2020
© 2020 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Niu X., Wang L.L., Liu S.K. and Bu Y.Y., 2020, Purification and optimization of prokaryotic expression of CSN5B protein of Arabidopsis thaliana, Molecular Soil Biology, 11(2): 1-8 (10.5376/msb.2020.11.0002)

Abstract

CSN5B is a subunit of the COP9 signalosome (CSN) and plays physiological functions in the form of monomer or complex in plants. The CSN5B of Arabidopsis thaliana was cloned by RT-PCR technology and constructed into the prokaryotic expression vector pET-32a (+), which was introduced into E.coli BL21 host bacteria. Isopropylβ-D-1-thiogalactopyranoside (IPTG) was used to induce expression, and the cultural temperature and time was optimized. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to detect the induced recombination protein, and the protein purification was conducted. It was shown that the recombinant CSN5B introduced into E. coli could be induced to express, and the induction was relatively effective in the presence of 1mmol/L of IPTG at 30℃, 4h induction effect is better. The molecular weight of the induced recombinant protein was basically consistent with the theoretical molecular weight. This study provided a theoretical basis for in-depth further exploration of the functions of this gene in the future.

Keywords
CSN5B protein; Prokaryotic expression; Protein purification

The COP9 signalosome (CSN) is a multi-protein complex which was originally defined as a suppressor of photomorphogenesis in plants. Its plays an important role in plant growth and development (Serino et al., 2003). The CSN is acidic, binds to heparin and localizes in the nucleus (Chamovitz et al., 1996). It is a highly conserved multi-protein complex composed of 8 subunits and affects the key developmental pathways by regulating protein stability (Tuller et al., 2019). The pleiotropic and eukaryotic conserved CSN is the catalytic reaction center in the NEDD pathway, and its mainly exists in the form of complexes and monomers when it functions (Giovanna et al., 2003). When CSN is present as holocomplex, it can regulate the transcription of downstream genes, while the monomeric forms or small subcomplexes can increase protein stability(Wei et al., 2008). The CSN is an evolutionarily conserved multiprotein complex which controls many developmental processes of plant by regulating the activity of CULLIN-RING E3 ubiquitin ligases (CRLs) (Pacurar et al., 2017).

 

The pivot protein CSN5 is the fifth subunit of the CSN, and it is a regulatory component of the ubiquitin/proteasome system (Caroline et al., 2018). It is known that the CSN5 has two homologous, which are called CSN5A gene and CSN5B gene respectively in Arabidopsis thaliana. Previous studies had shown that VTC1 and CSN5B can interact with each other to influence the content of AsA in plants. Under the dark conditions, CSN5B can degrade VTC1 through the 26S proteasome pathway regulating the AsA content negatively (Wang et al., 2013). These studies indicate that CSN5B plays an important role in the growth and development of plants. Therefore, in this study, based on the cloned Arabidopsis thaliana CSN5B gene, the recombinant CSN5B protein was further expressed and purified prokaryotically, laying a foundation for indepth researches on the functions of CSN5B.

 

1 Materials and methods

1.1 Strains, vectors and main reagents

pET-32a (+) vector, Esc-herichia coli DH5α, BL21 (DE3) competent cells were prepared and stored in this laboratory. pEASYTM-T5 Zero was purchased from TransGen Biotech. The high-fidelity ExTaq enzyme series, pMD18-T Vector, dNTP Mix, T4 DNA ligase, and the reverse transcription kit were obtained from TaKaRa. The DNA restriction enzymes was purchased from Thermo scientific. The plasmid mini-extraction kit was received from CWBIO. The gel recovery kit was product of MBI(Fermentas). The sheep anti-mouse IgG (peroxidase covalent binding), eECL Western Blot and primers was purchased from CWBIO. Low molecular weight protein Marker (TransGen Biotech).

 

1.2 The extraction of total RNA and protein

The fresh leaves of Arabidopsis thaliana were collected, and the total RNA was extracted by the TRIzol method according to the reagent instructions. The total protein of the leaves was also extracted. The extracted RNA and protein was stored in a refrigerator at -80℃for later use.

 

1.3 Prokaryotic expression vector construction

The upstream primer CSN5B-F (5'-GGTACCATGGAGGGTTCGTCGTCGA-3, synthetic primer contains KpnI restriction site) and the downstream primer CSN5B-R (5'- GAATTCTCAATATGTAATCATAGGGTCTGGA-3, synthetic primer contains EcoRI cleavage site) was designed based on the CSN5B sequence (accession number: AT1G71230) in the NCBI database. Then, the total extracted RNA as a template and the oligo (dT) as a reverse transcription primer, the cDNA was obtained through the reverse transcription according to the TaKaRa's kit instructions. PCR reaction was conducted in total volume of 20μL including 1~50 ng cDNA with the final concentration of more than 10μmol/L as a template and 1μL each of the upstream and downstream primers. The reaction was performed under the condition of 94℃/ 30 s, 55℃/ 40s, and 72℃/90s for 30 cycles. The amplified product was identified on a 1.5% agarose gel. After the electrophoresis, it was purified using a Thermo Scientific kit, ligated to the pMD18-T vector and transformed into E. coli DH5α strain. The pMD18-T vector containing the target gene was identified by colony PCR and other methods. The target gene was double-digested with EcoRⅠand KpnⅠand purified. Then, it was ligated into prokaryotic expression vector pET-32a (+), which was double-digested with same restriction enzymes, and then transformed into E. coli DH5α. After that, throughout the identification by colony PCR and sequencing, the expression vector pET-32a (+)-CSN5B vector containing the target gene was successfully constructed.

 

1.4 Induced expression and detection of the foreign gene

The recombinant expression vector pET-32a (+)-CSN5B was transformed into E. coli BL21(DE3) plysS strain, and a single colony was picked and inoculated into LB medium with ampicillin (Amp). The plasmid was extracted and identified through the double enzyme digestion, the strains were preserved for the subsequent experiments. Then the correctly transfected strains was cultured at 37℃overnight, diluted in the fresh LB medium containing Amp on the second day and continued to grow until the OD600 reached from 0.6 to 0.8. At this moment, IPTG was added, and the shaking culture was continued at 30℃for 4h. Next, the cells were collected by centrifugation at 13000 r/min for 1min, and the total protein was extracted.

 

1.5 Refolding of recombinant protein inclusion bodies

5ml inclusion body lysis solution was added to dissolve the His-CSN5B protein inclusion bodies at room temperature for 4h. Then the sonication was employed for 15min at 50HZ (sonicate for 3S and stop for 5S in a cycle) followed by centrifugation at 13000rpm for 30min at 25℃, and the supernatant was collected. This supernatant was transferred out to obtain the renatured His-CSN5B fusion protein. Phosphate buffer: 0.2M NaH2PO4, 0.2M Na2HPO4; Inclusion body solution: 50ml phosphate buffer, 0.5M NaCl, 10mM imidazole, 8M urea.

 

1.6 Purification of the recombinant protein

First, 200µl Ni-NTA purification resin was loaded on the protein purification column and 400µl equilibration solution for the Ni-NTA resin equilibration was added.Otherwise,the refoldd His-CSN5B protein was added, and 2ml His protein washing buffer was added to wash the miscellaneous proteins for three times. Finally, 200µl His-protein elution buffer was added. After mixing together thoroughly and placing on ice for 15min, the purified pET-32a(+)-CSN5B protein which crossed the column was collected. Protein washing buffer: 50mmol / L Na2HPO4, 300 mmol / L NaCl, 20 mmol / L NaCl imidazole; Protein elution buffer: 50mmol / L Na2HPO4, 300 mmol / L NaCl, 200mmol / L NaCl imidazole.

 

2 Results and analysis

2.1 Construction of the prokaryotic expression vector pET-32a(+)-CSN5B

First, the total cDNA of the model plant in Arabidopsis thaliana as a template, CSN5B was cloned by using pair of primers (CSN5B-F/CSN5B-R) through the PCR amplification, and the agarose gel electrophoresis was employed for detection. The results showed that the amplified fragment size was approximately 1074 bp as expected. After the purification from the gel, it was ligated into the pMD18-T cloning vector and transformed into E. coli DH5α. After the colony PCR and double-enzyme digestion identification, pMD18-T-CSN5B, the recombinant cloning vector containing the target gene fragment, was successfully obtained. In order to construct a prokaryotic expression vector of CSN5B protein, the target fragment from pMD18-T-CSN5B was cut by double digestion, cloned into the expression vector pET-32a (+), which was double-digested in the same way, and then transformed into E. coli DH5α strain. The constructed pET-32a(+)-CSN5B was double-digested again to ensure the correct size of the vector and target gene (Figure 1).Through these experiments, pET-32a(+)-CSN5B, the recombinant prokaryotic expression vector containing the target gene, was successfully obtained (Figure 1).

 


Figure 1 Enzymatic digestion identification of pET-32a (+)-CSN5B

Note: M: DS2000 Marker; 1-2: pET-32a (+)-CSN5B digested product

 

2.2 Small amount of induced expression of fusion protein and the optimization of induction conditions

The recombinant expression vector pET-32a(+)-CSN5B was transformed into E. coli BL21(DE3) plysS, and a single clone was selected and grown on LB culture medium (Amp+). After the induced expression by IPTG, SDS-PAGE detection was performed. The high concentrations of protein band existed in the host bacteria sample containing the recombinant plasmid pET-32a (+)- CSN5B. The target protein size is about 40kDa and the tag size such as His-tag in the pET-32a (+) vector is approximately 15kDa. Therefore, the total relative molecular weight of the fusion protein is about 55kDa, which was basically consistent with SDS-PAGE results.For large amounts of induced expression of the target protein, the optimization of the induction conditions were conducted, and it was shown that the addition of IPTG with the final concentration of 1.0 mmol / L when the bacterial solution was grown to an OD600 of 0.6 was best, and the cultivation was most efficient at 30℃for 4h after induction. These optimal conditions are used in all the subsequent experiments. As shown in Figure 2, the induced protein band can be seen from the second lane between 45-55kDa, and the bands gradually thickens with time (the position of the arrow in the figure). The size of the His-CSN5B fusion protein was consistent with the band position of the map, indicating that the expression of His-CSN5B fusion protein was successfully induced.

 


Figure 2 SDS-PAGE analysis of the effect of 1mmol/L IPTG concentration on the expression of His-CSN5B fusion protein at different times

Note: M: Low molecular weight standard proteins; 1: Not induced; 2-5: His-CSN5B protein induced at 30 ° C, 1 mmol / l IPTG for 1 h, 2 h, 3 h, 4 h

 

2.3 Large amounts of induced expression of the fusion protein

Based on the optimized induction conditions, the BL21 bacterial solution introduced with the recombinant plasmid was cultured in 100ml LB medium. When the OD600 value reached 0.6, 0.5 mol/L IPTG 100μL with a final concentration of 1mmol/L was added at 30℃. After 4h of induction, the bacteria cells were sonicated and the supernatant and precipitates were collected respectively as shown in Figure 3. The results showed that a large amount of His-CSN5B fusion protein were successfully induced. However, as it can be seen from the figure, the His-CSN5B fusion protein between 50-70kDa was expressed largely in the form of precipitates, and therefore, there naturation process of inclusion bodies were needed for the preparation of protein purification.

 


Figure 3 SDS-PAGE analysis of a large number of induced His-CSN5B proteins

Note: M: low molecular weight standard proteins;1: Expression of His-CSN5B fusion protein after small amount induction; 2: Expression of His-CSN5B fusion protein after large amounts of induction;3: Supernatant of total lysate after a large amount of induction of His-CSN5B fusion protein;4: Precipitates of total lysate after a large amount of induction of His-CSN5B fusion protein

 

2.4 Comparison of the soluble expression with the expression of inclusion bodies of pET-32a (+)-CSN5B fusion protein

The soluble expression of the isolated pET-32a (+)-CSN5B and the reconstituted inclusion bodies were analyzed by SDS-PAGE electrophoresis (separation gel concentration 12%), as shown in Figure 4. It can be seen from the Figure 4, after the induction of the pET-32a (+)-CSN5B fusion protein by IPTG, the amount of inclusion bodies extracted from E. coli was significantly higher than that of soluble expression in the bacterial solution. The renatured protein of inclusion bodies was detected by Western Blot (Figure 5), proving that the inclusion bodies of His-CSN5B fusion protein was successfully renatured.

 


Figure 4 SDS-PAGE analysis of a large number of induced and renatured His-CSN5B proteins

Note: M: low molecular weight standard proteins; 1: Not induced; 2: Expression of His-CSN5B fusion protein after large amounts of induction; 3: Supernatant of total lysate after a large amount of induction of His-CSN5B fusion protein; 4: Precipitates of total lysate after a large amount of induction of His-CSN5B fusion protein; 5: Renatured protein of His-CSN5B inclusion bodies;

 


Figure 5 Western Blot analysis of protein induction and renaturation

Note: 1: Expression of His-CSN5B fusion protein after large amounts of induction; 2: Renatured protein of His-CSN5B inclusion body;


2.5 Purification of the fusion protein

In order to purify the target protein, the 6 × His tag fused to the recombinant CSN5B protein was used, and the Ni-NTA His Bind Resins affinity chromatography was employed for the purification. The purified protein was detected as a single band in the SDS-PAGE and the size was consistent with the above results (Figure 6). The size of the purified His-CSN5B fusion protein was consistent with the protein expressed in small amount induction.

 


Figure 6 SDS-PAGE analysis of a large amount of induced and purified His-CSN5B protein;

Note: M: low molecular weight standard proteins; 1: Not induced; 2: Expression of His-CSN5B fusion protein after large amounts of induction; 3: Precipitates of total lysate after a large amount of induction of His-CSN5B fusion protein; 4: Renatured protein of His-CSN5B inclusion bodies; 5: Purified His-CSN5B protein;

 

3 Discussion

Ascorbic acid is an important plant-derived antioxidant, which constitutes the main source of human dietary vitamin C. AsA plays an important role in plant growth and development (Smirnoff et al.,2000; Hemavathi et al., 2010; Zhou et al., 2012). On the one hand, AsA is synthesized through a variety of biological pathways including D-glucose (Loewus et al.,1999), D-galacturonic acid (Davey et al., 1999), and D-man/L-Gal pathway(Wheeler et al., 1998) .Among them, the last pathway is particularly important in plants. On the other hand, the environmental signals such as light stress can also induce ASA synthesis (Smirnoff et al., 2000). Light affects the accumulation of AsA in Arabidopsis leaves, therefore, the content of AsA in leaves are influenced by light. AsA content in Arabidopsis leaves under the appropriate high light intensity is higher than that in low light conditions (Yabuta et al., 2007).

 

The CSN5B subunit in the COP9 signaling complex (CSN) can affect the AsA biosynthesis and regulate the plant responses to oxygen and salt stress. Furthermore, the CSN5B participates in the 26S protease system indicating the regulative function of CSN5B as a photomorphic factor in AsA synthesis (Wang et al., 2013).

 

Due to the low cost of the prokaryotic expression system, this study tried to achieve the expression and optimization of CSN5B recombinant protein in E. coli. In order to improve the stability of the target protein, through the fusion expression with tag protein, the optimal conditions including a suitable induction temperature were selected. As a result, the expression level of CSN5B in prokaryotic cells was increased and a large amount of purified CSN5B protein was obtained. It was found that the induction temperature, for example, 30℃ promoted the expression of CSN5B fusion protein enough, but the expression level of total protein decreased significantly. The fusion expression with soluble tag protein not only promoted the expression of CSN5B in inclusion bodies, but also it did not affect the expression level of total protein.

 

In summary, the optimal expression of CSN5B recombinant protein in the prokaryotic system is still a low-cost, fast and effective method. Through the indepth research and optimization of this study, it is expected that the production level of CSN5B recombinant protein can be improved and provide a foundation for further analysis of the functions of CSN5B.

 

Acknowledgements

This work was supported by the Fundamental Research Funds for the Central Universities (2572016CA14), Heilongjiang Province Government Postdoctoral Science Foundation (LBH-Q18008), and State Key Laboratory of Subtropcal Silviculture (KF201707) awarded to Yuanyuan Bu. Further supported by the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT17R99) awarded to Shenkui Liu. The funders had no role in study design.

 

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