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Cloning, soluble expression, and purification of the RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae a

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a

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Gaurav Chhetri , Arabinda Ghosh , Ramesh Chinta , Md. Sohail Akhtar & Timir Tripathi a

Molecular Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong -793022, India b

Department of Biotechnology. Indian Institute of Technology, Guwahati-781039, India

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Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Jankipuram Extension, Lucknow - 226031, India Accepted author version posted online: 31 Dec 2014.

Click for updates To cite this article: Gaurav Chhetri, Arabinda Ghosh, Ramesh Chinta, Md. Sohail Akhtar & Timir Tripathi (2014): Cloning, soluble expression, and purification of the RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae, Bioengineered To link to this article: http://dx.doi.org/10.1080/21655979.2014.1002301

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Cloning, soluble expression, and purification of the RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae Gaurav Chhetri1, Arabinda Ghosh2, Ramesh Chinta1, Md. Sohail Akhtar3 and Timir Tripathi1* Molecular Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong -793022, India

Department of Biotechnology. Indian Institute of Technology, Guwahati-781039, India Molecular and Structural Biology Division, CSIR-Central Drug Research Institute,

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Jankipuram Extension, Lucknow - 226031, India Short title: Recombinant expression & purification of Rpb5

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*Corresponding author

Dr. Timir Tripathi, Assistant Professor, Department of Biochemistry, North-Eastern Hill

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University, Shillong- 793022, India. Email: [email protected], [email protected]; Tel:

Abbreviations

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+91-364-2722141; Fax: +91-364-2550108.

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RNAP II, RNA polymerase II; Rpb, RNA polymerase II subunit; Ni-NTA, Nickil-nitrilotriacetic acid; TF, Transcription factor; IPTG, Isopropyl β-D-1-thiogalactopyranoside.

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ABSTRACT

We report the molecular cloning, expression, and single-step homogeneous purification of RNA

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polymerase II subunit RPB5 from Saccharomyces cerevisiae. RPB5 is a 210 amino acid nuclear protein that functions as the fifth largest subunit of polymerase II and plays a central role in transcription. The gene that codes for RPB5 was generated by amplification by polymerase chain reaction. It was then inserted in the expression vector pET28a(+) under the transcriptional control of the bacteriophage T7 promoter and lac operator. BL21(DE3) Escherichia coli strain

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transformed with the rpb5 expression vector pET28a(+)-rpb5 accumulates large amounts of a soluble protein of about 30 kDa (25 kDa plus 5 kDa double His6-Tag at N and C terminal). The protein was purified to homogeneity using immobilized metal affinity chromatography. RPB5 recombinant protein was further confirmed by immunoblotting with anti-His antibody. In this

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study, the expression and purification procedures have provided a simple and efficient method to

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cerevisiae RPB5 in gene expression and transcription regulation. Furthermore, it can provide

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additional knowledge of the interaction partners of RPB5 during various steps of transcription and gene expression.

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Keywords:

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RNA polymerase, subunit, gene expression, transcription, cloning, solubility, protein expression.

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obtain pure RPB5 in large quantities. This will provide an opportunity to study the role of S.

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INTRODUCTION In Saccharomyces cerevisiae, the RNA polymerase II (RNAP II) core enzyme is composed of 12 subunits called RNA Polymerase II subunits (RPBs) RPB1–RPB12. RPB1, RPB2, RPB3, RPB4, RPB7, RPB9, and RPB11 encode subunits unique to RNAP II, while RPB5, RPB6, RPB8,

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RPB10, and RPB12 subunits are the shared components of RNAP I, II, and III.1 The latter five

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they are present in all the three eukaryotic RNAPs. 2 These essential subunits are assembled along

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with a unique set of additional subunits to form either of the three classes of RNA polymerases I, II, or III.3 Studying the role of these subunits and their specific contribution to regulated gene

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expression can reveal many puzzles in biological context.

Progress has been made to identify and characterize various transcription factors. In

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contrast, there is less knowledge related to RPBs.3 RPB5 of S. cerevisiae has a molecular weight of approximately 25 kDa and consists of two domains, an eukaryote-specific N-terminal domain

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(16 kDa) and a smaller C-terminal domain (9 kDa).4 RPB5 is known to be present as two copies

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in RNAP II, and has been shown to homodimerize under in vitro conditions.5 RPB5 has been reported to interact with several general transcription factors or specific gene regulators.6

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Various studies on the human RNAP have suggested that RPB5 binds specific partners of RNAP II. Some previously reported specific partners are TFIIF and Taf15 (TAFII68).7 Few studies also

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small subunits are required for yeast cell viability and play a central role in RNA transcription as

showed that transcriptional transactivator protein X encoded by the human hepatitis virus may specifically interact with RPB5 to stimulate transcription in virally infected cells.5, 8, 9 However, interactions with these specific partners do not account for the fact that RPB5 is shared by all three yeast RNAPs.2,

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Earlier studies also proved the close relation of human RPB5 to the

archaeal subunit H.11and to viral RPB5-like subunits.12 These studies on human RPB5 promote calls for general and specific role of S. cerevisiae RPB5 in gene expression. 3

We have chosen Escherichia coli for producing large quantities of recombinant RPB5 protein because of its ability to grow rapidly and at high density on inexpensive substrates, combined with its well-characterized genetics.13-15 The expression of this gene was increased using 3% ethanol (v/v).16 Purified RPB5 can uncover concrete knowledge of the structure,

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interaction partners, and function of RPB5 in the mechanisms of gene expression operating at the

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PCR amplification and cloning of full-length rpb5 gene:

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RESULTS AND DISCUSSION

S. cerevisiae rpb5 gene was generated by PCR amplification using gene-specific primers. A

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single PCR product of the expected size (648 bp) was obtained (Figure 1, lane 2) and cloned to pSK+ vector. In order to verify the identity of the isolated fragment, the construct was digested

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with BamHI and HindIII. Two bands of expected sizes 3 kb and 648 bp were obtained (Figure 1,

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lane 3) confirming the identity of the rpb5 gene. Positive clones were sequenced and compared with the rpb5 sequences from Saccharomyces genome database. The rpb5 gene was subcloned

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into pET28a(+) expression vector (Figure 1, lane 4). The final construct was named as pET28a(+)-rpb5.

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RPB5 protein expression and solubility optimization: In the present study, both IPTG concentrations from low (0.5 mM) to high (1 mM) have shown

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molecular level.

almost same level of RPB5 protein expression (data not shown). Whereas, in the presence of 3% alcohol, increased fold of overexpression of RPB5 was found. In this study, we demonstrate the influence of temperature on RPB5 protein expression and solubility at 25 °C and 37°C. We found that soluble expression at 25 °C was higher than 37 °C (data not shown). SDS-PAGE

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results for overexpressed RPB5 protein is shown in Figure 2, lane 3. The overexpressed protein was not detected in the un-induced control sample (Figure 2, lane 2). Recombinant bacterial cells were collected by centrifugation and lysed by sonication. The supernatant and the pellet were collected and subjected to 12% SDS-PAGE analysis. The results

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show that the molecular weight of the recombinant product is 30 kDa, which corresponds to the

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Purification of RPB5 protein by affinity chromatography

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(Figure 2, lane 5) in comparison with the pellet fraction (Figure 2, lane 4).

Purification of recombinant S. cerevisiae RPB5 protein was accomplished by immobilized metal

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affinity chromatography (IMAC) on a Ni–NTA resin column. The SDS-PAGE analysis (Figure 2, lane 5) showed a single protein band with the expected molecular mass of the recombinant

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protein (30 kDa), based on the molecular mass of the S. cerevisiae RPB5 (25 kDa plus 5 kDa

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double His6-Tag at N and C terminal).17 Following IMAC, recombinant RPB5 protein was purified to homogeneity. The yield of purified recombinant RPB5 per liter of culture was

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approximately 45 mg/L. Table 1 shows the amount of cells and purification fold after each step. The presence of recombinant protein was confirmed with immunoblotting using anti-His

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antibody (Figure 3, lane 2).

For structural, functional, and biochemical studies pure recombinant protein in active

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predicted size of pET28a(+)-rpb5. RPB5 protein was predominantly present in soluble fraction

form is always a prerequisite. RPB5 is a 210 amino acid nuclear protein that functions as the fifth largest subunit of polymerase II and plays a central role in transcription.18, 19 RPB5 protein can be used for various protein–protein interaction studies. Detailed interaction studies of the RPB5 with other interacting partner can also help in complete elucidation of molecular mechanisms of transcription in S. cerevisiae. In RPB5 protein expression experiment, we noticed an increased

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expression rate under ethanol treatment.16 In addition, presence of ethanol to the growth media can mimic the heat shock response in E. coli and can enhance the solubility of some recombinant proteins.20, 21 In our work, 3% ethanol (v/v) was used to get the high-fold soluble expression of recombinant RPB5 protein.

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Comprehensive studies of intermolecular protein interactions between polymerase II

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It is encouraging that many other unknown

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well conserved between yeast and human.7,

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interaction partners (proteins or protein complex) can be discovered using S. cerevisiae RPB5 protein which might be similar for human RPB5 as well because of the similarities between S.

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cerevisiae and human RPB5. Once more functions of the S. cerevisiae RPB5 subunit are discovered, several in vivo and in vitro assays should help illuminate the minor and major

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molecular details which underlie their critical but yet elusive functions. In conclusion, RPB5 is a

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common subunit among all the three RNA polymerases.2 Consequently, it is of interest to see the other interacting proteins or protein complexes that might affect the function of RNA

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polymerases I and III.

MATERIALS AND METHODS

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Materials

The molecular biology kits and Ni-NTA agarose were purchased from Qiagen, CA, USA. The

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subunit RPB5 can influence more functions that may be necessary for transcription. RPB5 is

dNTPs and enzymes were purchased from New England Biolabs, MA, USA. All other reagents and chemicals were purchased either from Sigma-Aldrich Chemical Company, St. Louis, MO, USA, or Sisco Research Laboratories, Mumbai, India and were of the highest purity available. Bacterial culture media was purchased from Himedia Laboratories, Mumbai, India. Genomic DNA isolation from S. cerevisiae:

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Genomic DNA of S. cerevisiae was manually isolated by method mentioned by Hoffman, et.al.26 Amplification of rpb5 gene: Gene-specific

primers,

5′-CGGGATCCATGGACCAAG-3′

(forward)

and

5′-

CCAAGCTTCTACATACAGATTC-3′ (reverse), were designed for PCR amplification of the S.

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cerevisiae rpb5 gene. Primers introduced BamHI and HindIII restriction sites on 5′ and 3′ ends,

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PCR reaction was performed using Phusion High-Fidelity DNA Polymerase (New England

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Biolabs, USA) with the following cycle parameters. Initial denaturation temperature of 98 °C for 120 s, 30 cycles of 98 °C for 40 s, 52 °C for 15 s, and 72 °C for 15 s, followed by a final

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extension of 72 °C for 5 min. PCR products were resolved using 1.0% agarose gel

Extraction Kit (Qiagen, USA).

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electrophoresis, and amplicon of the expected size (648 bp) was purified with the QIAquick Gel

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Cloning and subcloning of rpb5 gene:

The purified PCR product of rpb5 gene was ligated to pSK+ vector. The ligation product was

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used to transform in competent E. coli DH5α cells. The plasmid was isolated from the positive clones and sequenced to ensure sequence fidelity. Nucleotide sequencing was carried out by

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Xcleries lab (Gujarat, India). The plasmid was then digested with BamHI and HindIII, and the product was purified using agarose gel. It was then subcloned with BamHI and HindIII digested

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respectively. Synthetic oligonucleotides were purchased from Xcleris lab (Gujarat, India). The

pET28a(+) vector to generate the recombinant construct of pET28a(+)-rpb5. The recombinant plasmid was then transformed into chemically competent E. coli BL21(DE3) expression host cells by heat shock method and then spread onto agar plate containing kanamycin (50 μg/mL) to allow selection of colonies that successfully incorporated the plasmids. Plasmid DNA extraction was performed using the QIAprep Midiprep plasmid purification kit (Qiagen, USA).

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Optimization of recombinant RPB5 protein expression: Recombinant cells harboring pET28a(+)-rpb5 plasmid were screened on selective Luria Broth (LB) agar plates supplemented with kanamycin. A positive clone was picked up and grown overnight in 5 mL LB broth containing 50 µg/mL ampicillin at 37 °C with shaking (180 rpm).

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Frist, optimal expression conditions were standardized using 5 mL cultures. Two Isopropyl β-D-

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temperatures (25 °C and 37 °C) were tested at 18 h and 4 h incubation time course, respectively.

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We tested and used 3% ethanol (v/v) to increase the expression fold of the recombinant gene according to Chhetri et.al.16 A 2 mL primary culture was inoculated in 400 mL of sterile LB

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broth supplemented with 50 μg/mL kanamycin and incubated at 37 °C with shaking (180 rpm) until the OD600 reached 0.5. Another 5 mL LB broth culture tube containing above-mentioned

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antibiotics was inoculated with the primary culture and was kept as un-induced control. After the

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OD600 reached, culture was induced with 0.5 mM IPTG and incubated for 18 h at 25 °C with continuous shaking. After 24 h, cells were harvested by centrifugation at 4 °C, and the pellet was

cocktail.

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resuspended in 20 mL of 50 mM Tris (pH 8.0) and 300 mM NaCl containing protease inhibitor

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Purification of recombinant RPB5 protein: Homogenized cells were lysed on ice using sonication at 50% amplitude for 30 cycles (30 s pulse

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1-thiogalactopyranoside (IPTG) concentrations (0.5 mM and 1 mM) and two different expression

on, off). The crude lysate was then clarified by centrifugation at 12,000 rpm for 20 min at 4 °C. The supernatant was filtered through a 0.45 μm pore PVDF membrane before affinity chromatography. Matrix was extensively washed with 10 bed volumes of equilibration buffer (50 mM Tris (pH 8.0), 300 mM NaCl). The supernatant was passed through the Ni–NTA agarose matrix (Qiagen, USA) and was washed with increasing concentration of imidazole. Elution of

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RPB5 recombinant protein was accomplished with elution buffer (50 mM Tris (pH 8.0), 300 mM NaCl, and 300 mM Imidazole). The purity was analyzed by 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) stained with 0.05% Coomassie brilliant blue R-250. The protein was dialyzed overnight against dialysis buffer (20 mM phosphate pH 8.0, 150 mM NaCl) at 4

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°C. Quantitation of recombinant proteins was carried out using Bradford method using BSA as a

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Western blotting

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His-tagged recombinant RPB5 purified protein was subjected to SDS-PAGE on 12% polyacrylamide gel and transferred to 0.45 μm polyvinylidene fluoride (PVDF) membrane

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(Whatman) for 90 minutes at 50 mV. Membrane was next blocked in 5 % skimmed milk and incubated with mouse anti-His antibody (1/3000 dilution). After three times washing with PBS,

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pH 7.4, membrane was then incubated with anti-mouse IgG alkaline phosphatase (1:1000). Both

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primary and secondary antibodies were diluted in PBS containing 3 % BSA. Membranes were then three times washed with PBS, pH 7.4. Signals were detected with the BCIP/NBT-Blue

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Liquid Substrate (Sigma-Aldrich Co, St. Louis, USA) and analyzed using gel doc system. Acknowledgement

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The study was supported by a research grant from Department of Biotechnology, New Delhi, India (BT/224/NE/TBP/2011 dated 13/04/2012). GC thanks DBT for providing fellowship.

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standard.

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Figure 1: PCR amplification and restriction digestion of S. cerevisiae rpb5 gene. Lane 1,

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DNA ladder. Lane 2: PCR product (648 bp). Lane 3: pSK+-rpb5 digested with Bam HI & Hind III; upper band (3 Kb) is pSK+ vector and lower band (648 bp) is the rpb5 gene. Lane 4:

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pET28a(+)-rpb5 was restricted with BamHI & Hind III; upper band (5.3 Kb) shows pET28a vector while the lower band (648 bp) shows rpb5 gene. 1 % agarose gel electrophoresis was used

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FIGURE LEGENDS

to visualize the bands.

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separated by 12 % SDS-PAGE, and stained with CBB. Lane 1: Molecular weight marker. Lane

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Figure 2: Rpb5 protein expression, solubility and purification analysis. Protein samples were

2: Rpb5 Un-induced control lysate. Lane 3: Rpb5 Induced lysate. Lane 4: Rpb5 protein pellet fraction after lysis. Lane 5: Soluble fraction after lysis. Lane 6: purified Rpb5 protein.

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2, Rpb5 recombinant protein detected by western blotting with a mouse anti-His antibody.

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Figure 3: Western blot analysis on PVDF membrane. Lane 1, Molecular weight marker. Lane

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Table 1. Amount of cells and purification fold after IMAC. Total protein/L culture (mg)

Protein yield after IMAC/L culture (mg)

~2.83

~240.8

~45

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Crude cell pellet/L culture (g)

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Cloning, soluble expression, and purification of the RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae.

We report the molecular cloning, expression, and single-step homogeneous purification of RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae...
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