The pET-Rep recombinant plasmid construction and geminivirus Rep [C1] gene expression in Escherichia coli strain BL21

Geminivirus Rep [C1] gene produces a replication protein [Rep] that interacts with Retinoblastoma-related protein [RBR] when Geminivirus infect the plants. That interactions interfere with RBR and transcription factor function. So the mechanism of chili plant defense against symptoms of Pepper Yellow Leaf Curl Disease is blocked. The NPR1 gene is a transcriptional co-activator involved in the regulation defense mechanism of Systemic Acquired Resistance [SAR] in chili plants. In silico, Rep protein blocks the SAR mechanism that is regulated by the NPR1 gene. This study aimed to obtain a construction of pET-28a+ plasmid expression recombinant Rep [C1] gene and Rep protein that can be expressed in E.coli BL21. This research is useful to study the interaction of Geminivirus with transcription co-activator in chili plants. The construction was done by ligating plasmid and Rep genes which have been cut using restriction enzymes BamHI and SacI. The construction of a recombinant pET-Rep plasmid is confirmed by amplification and verification of the nucleotide sequence through sequencing techniques. Nucleotide sequence verification results proved that the Rep [C1] gene is successfully constructed into a pET-28a+ plasmid with proper position and orientation. The pET-Rep recombinant proceeds to the expression test using the E.coli BL21 host. Expression was performed using the induction method with IPTG. Proteins Rep is purified using the MagnehisTM Protein Purification System. Rep protein is visualized with SDS-PAGE. Visualization showed that Rep protein was successfully expressed in E.coli BL21 where protein size is estimated of 41,03 kDa in size.


Introduction
Currently, pepper plants still have problems with yellow leaf curl disease. The disease is an epidemic disease in pepper plants in West Sumatra, Indonesia. One of the major geminiviruses infecting many plants in Indonesia is Pepper Yellow Leaf Curl Disease [PepYLCV] [Begomovirus]. The virus has some components of functional protein-encoding genes designated as a coat of protein [CP], movement protein [MP], Replication [C1] and satellite beta [ß] [1].
One component of the functional protein-coding gene that plays a major role in the spread of virus particles is the Rep [C1] gene. The Rep [C1] gene is a replication and transcription regulatory element in geminivirus. The gene functions at the beginning of replication and initiates DNA replication [2]. The Rep [C1] gene has multifunctional characters including in the mechanism of rolling circle replication [RCR] and interacting with retinoblastoma proteins [3]. The Retinoblastoma-related protein [RBR] plays a significant role in cell regulation [4]. RBR suppresses rapid cell proliferation or development in adult tissues [5]. The Rep proteins that interact with RBR will interfere with some transcription factors. Interaction of Rep protein with RBR releases the transcription factor so that it activates the expression of Proliferating Cell Nuclear Antigen [PCNA] to produce DNA polymerase and the factors needed for viral replication [6].
Pepper plants have a Systemic Acquired Resistance [SAR] defense response. One of the transcription coactivators in pepper plants is the Non-expressor of Pathogenesis Related [NPR1]. The gene involved in defense mechanisms SAR in plants [7]. The NPR1 protein is the main regulator in the expression of Pathogen Related [PR] genes mediated by salicylic acid in SAR [8]. The NPR1 is also involved in basal resistance or when infected, Induced Systemic Resistance [ISR] and Pto mediated resistance [9]. The NPR1 gene expresses the PR1 gene in response to pathogenic infections. Structurally, the NPR1 consists of BTB/POZ, ankyrin and transactivation domains [10]. Molecular interaction between the NPR1 gene and Rep protein in response to Geminivirus attacks on pepper plants has not been proven so far. Binding of Rep protein to the NPR1 gene will cause suppression of NPR1 gene function. So the NPR1 gene could not stimulate expression of the Pathogen Related 1 [PR1] resistance gene. Therefore, it is necessary to elucidate the mechanism of NPR1 gene interaction with Rep protein for yellow leaf curl disease regulation in pepper plants [11].
Several steps to find out such the above mechanism has to involve the recombinant plasmid construction of the Rep [C1] gene and its downstream expression. Recombinant plasmid construction could be done to produce Rep proteins that could be used in the study of interactions with NPR1 gene transcriptional coactivators. The plasmid commonly used as the expression vector for such a goal is a pET-28a+ plasmid. This plasmid belongs to the expression vector that is facilitated with His-tag in the upstream and downstream of the inserted target gene. The presence of His-tag facilitates the phase of protein purification using E.coli strain BL21 as its host. E.coli BL21 has high expression capability and T7 RNA polymerase corresponding with the T7 promoter on the pET-28a+ plasmid.
This study was aimed to construct the pET-28a+ plasmid recombinant containing Rep [C1] gene sequence. So, the Rep protein can be expressed in E.coli BL21.

In silico plasmid construction
The plasmid construction was carried out using the plasmid ApE editor win 2.0.36 application. The in silico construction was carried out according to the manual provided in the website.

Rep [C1] gene preparation
The Rep gene which is available in the pGEM-T Easy plasmid was verified using T7SP6 primer. Besides, verification was also performed using the C1-TD21-SmaI/BamHINT. The verified pGEM-Rep stock is then transformed into E. coli DH5α.

pET-28a+ plasmid preparation
Plasmid pET-28a+ was obtained from laboratory stock which has been propagated in E. coli DH5α. The isolation process is carried out using ATP TM Plasmid Mini Kit [Biotech, USA].

Rep gene [C1] and pET-28a+ plasmid restriction
The restriction of the Rep [C1] gene from the pGEM-Rep and pET-28a+ plasmid was done to cut out the insert. The restriction step was done twice using different restriction enzymes. The first restriction was carried out using the BamHI Fast Digest enzyme, while the second restriction was carried out using SacI. The 30 µL of cocktail composed of 17 µL nuclease-free water, 10 µL pGEM-Rep PCR and pET-28a+ plasmid, 2 µL FD10x buffer and 1 µL BamHI Fast Digest restriction enzyme was prepared. Amplification was run under the condition as presented in Table 2. After the first restriction, the restricted sequence was purified using isopropanol. Isopropanol was added to the restriction reaction using a 1:1 ratio. Incubated was done at -20˚C for 1 hour and subsequently centrifuged for 10 minutes twice at 10,000 rpm. The supernatant was removed while the pellets were washed with 70% ethanol and finally was dried. The pellet is dissolved with nuclease-free water before restricted with the SacI enzyme. The restriction was run using a PCR machine with the program presented in Table 3.

Ligation of Rep [C1] gene with plasmid pET-28a+
The ligation activity was carried out based on the protocol provided by Novagen [USA] using a 10x ligation buffer kit.

Transformation of pET-Rep into E. coli BL21
Transformation of pET-Rep into E. coli BL21 cell was done using the procedure described by Promega [Promega-USA] which was based using the heat shock technique.

Induction of pET-Rep plasmid
A single colony of E. coli BL21 was inoculated into 50 mL liquid LB media containing kanamycin 30 µg/mL antibiotics. The bacteria were propagated under agitation at a speed of 160 rpm at 37˚C until its OD [600] reached 0.6-1.0. The culture was stored at 4˚C for overnight. Expression induction was used by adding isopropyl β-D-1-thiogalactoside [IPTG] to a final concentration of 0.4 mM and followed by incubation for 2-3 hours at a temperature of 25˚C -30˚C.

Purification Rep protein
Purification of Rep protein was done using the MagneHis™ Protein Purification System [Promega, USA]. The purified protein was visualized using SDS-PAGE.    The plasmid DNA of pET-28a+ and the amplified Rep [C1] sequence were successfully digested. Figure 2 shows the plasmid conformation before circular-shaped restriction resulting in two bands. Restriction formed a single band indicating that the plasmid had been digested. Plasmid size before the restriction is 5369 bp and after the restriction is 5361 bp. The Rep [C1] gene size before the restriction was 1262 bp, while the size after being restricted was 1184 bp. Thus, ligation of both fragments should produce plasmid with 6545 bp in size.

Transformation of pET-Rep into E.coli BL21
The success of ligation between pET-28a+ and Rep gene sequence was verified using the PCR technique. Figure 3 indicates that the ligation between pET-28a+ and Rep [C1] gene did occur. Using primer T7_PFTR, PCR analysis could produce a single fragment with 1496 bp in size [B-C], while amplification using primer C1 SmaI/BamHINT produced a single fragment with 1120 bp in size.
The success ligation between plasmid and target genes could be influenced by several factors. The ends of the plasmid and target gene must be compatible, therefore the same restriction enzyme should be used in the restriction process. The concentration and composition of the ligase and buffer enzymes in the cocktail must be appropriate. Furthermore, the ratio of plasmids compared to insert genes must be carefully measured. This generally is determined by the concentration and length of the nucleotides of both plasmid and target genes. Some external factors such as the incubation temperature during the ligation process could influence the success rate of ligation [12].
Further verification was performed using the PCR colony technique. The result is presented in Figure 4.  Figure 4a shows that colony A produced some bands while colony C produced one single band. Colony B and colony D, unfortunately, produced no products. Based on that result colony A was reamplified with specific primer C1 and finally exhibited a single fragment of 1120 bp in size [ Figure  4b]. The positive colony was processed to further sequence analysis.

pET-Rep sequence analysis
Sequence analysis was performed in order to verify the orientation of the inserted gene target. Sequence data were further analyzed using Vecsreen software and the result is presented in Figure 5. Since reverse primer produced low-quality sequence data, the analysis was performed from the forward primer. Figure 6 shows that the codon start of the Rep [C1] gene is located on the downstream position of the T7 primer sequence. Meaning that its orientation is correct, theoretically enabling transcription initiation.   The GCC is a BamHI restriction site. ATG is a codon start.
Further analysis was carried out to verify the integrity of the sequence. The nucleotide sequence in Figure 6 proves that the orientation of the Rep [C1] gene is correct. This fact is based on that the nucleotide sequence of the T7 promoter [TCGCATGCTCCGAGCCGCCAT] followed by the restriction site of the BamHI enzyme is located between gene insert and start codon. Based on this it can be ascertained that the orientation of the Rep [C1] gene is correct. Moreover, to verify the identity of the sequence, BLAST and Clustal analysis were run. BLAST data using 692 nucleotides against nucleotide available in the NCBI database exhibited 99% identity with Rep [C1] nucleotide sequence isolated from PepYLCV strain Tanah Datar [TDWS]. Two base substitutions were found after comparing both nucleotide sequence data [see Figure 7].  The two nucleotides undergo substitution from base A to G in the position of 55 th and 273 rd . Despite we found two substitution events, the analysis was continued by comparing the amino acid sequence. The result is presented in Figure 8. Alignment of both amino acid sequences exhibited that only 1 nucleotide substitution [base A to G in the 55 th ] causes a change in amino acid composition from N [asparagine: asp] to D [aspartic acid: asn], while nucleotide substitution at position 273 rd does not change any amino acid variation. However, asparagine is an analog of aspartic acid [13] so that the structure and function of Rep proteins could be almost similar. Analysis of protein structure using ProFunc software [http://www.ebi.ac.uk/thornton-srv/database/ProFunc] can be seen in Figure 9-10. Analysis of the secondary structure of the amino acid sequences of the two Rep proteins shows similar shape and arrangement of their α-helices and β sheets. A similar result is shown also from tertiary structure analysis [Fig 10]. They have also similar conformation shapes at each rotational degree of their structure. Based on this data, we assume that both proteins are analog and should have similar activity [14].

Induction of expression
To test, whether the inserted gene could be expressed or not, expression analysis was performed. The Rep [C1] gene expression was induced with 0.4 mM IPTG for 3 hours. Induction was performed at temperatures ranging from 25 to 30 ˚C [12]. Since the Rep protein doesn't have a signal peptide domain, we assumed that Rep protein will be produced as intracellular protein. For that reason, the purification of Rep protein was performed using His-tagged approach [ Figure 11]. SDS-PAGE analysis shows that Rep protein bands were larger than 40 kDa based on the unstained protein ladder. The protein produced from the expression is corresponding with the expected size which is about 41.03 kDa [ Figure 11]. The expectation was performed using Promega Biomath Calculator software which is available online.

Conclusion
The Rep [C1] gene was successfully constructed into the plasmid expression pET-28a+ with the correct orientation. The gene was inserted between the SacI and BamHI cloning sites. Expression analysis showed that Rep protein was successfully expressed in the cell of E. coli BL21 conferring 41.03 kDa in size.