Quest for the Clr2 protein

Quest for the Clr2 protein

In the yeast Schizosaccharomyces pombe, by expression and purification from Pichia pastoris

Gaikwad Sanket Gautam

Degree project in Applied Biotechnology (45 credits) Master of Science (2 years) 2013

Biology Education Centre and IMBIM -Department of Medical Biochemistry and Microbiology, Uppsala University

Supervisors – Daniel Steinhauf and Pernilla Bjerling

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Table of contents

Summary...4

1. Introduction...5

1.1 Schizosaccharomyces pombe as a model organism………....5

1.2 The SHREC complex………...6

1.3 Pichia pastoris as an expression system………6

1.4 pPICZαA and pGAPZαA an expression vectors………....6

1.5 Aim of the project...7

2. Materials and Methods...8

2.1 Media...8

2.2 Strains...8

2.3 Plasmid Mini Preparation...8

2.4 Restriction enzyme digestion of plasmid DNA and Shrimp Alkaline Phosphatase treatment...9

2.5 Gel Purification...10

2.6 Ligation...10

2.7 Competent cells preparations...10

2.8 Bacterial Transformation...11

2.9 Linearization of plasmid vector...11

2.9.1 Transformation by Electroporation...11

2.9.2 Transformation by Chemical method...12

2.10 Analysis of Protein by Sodium Dodecyl Sulfate-Poly- Acrylamide Gel Electrophoresis with coomassie staining...13

2.11 Detection of the protein by Immunoblotting ...13

2.12 Protein purification by Affinity chromatography ...14

3 Results...16

3.1 Restriction Enzyme Digestion...16

3.2 Gel Purification...16

3.3 Bacterial Transformation...17

3.4 Linearization of plasmid...18

3.5 Coomassie Stained SDS-PAGE...19

3.6 Immunoblot ………...19

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3.7 Purification of the protein...20

4 Discussion...22

4.1 Further Work...24

5 Acknowledgement...25

6 References...26

7 Appendix...28

7.1 Media recipe...28

7.2 Solutions used...28

7.3 P.pastoris expression vector’s Maps...30

7.4 Graphs showing concentration of the purified proteins by using

Bicinchoninic Acid Assay...31

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Summary

Epigenetics is the study of inheritable changes in gene expression without any change in the DNA sequence and it has been linked to cancer and disorders like mental retardation and chromosomal instability. DNA methylation and covalent histone modifications are two primary mechanisms related to epigenetics. To strengthen the knowledge in epigenetics, the fission yeast Schizosaccharomyces pombe is used as a model system. The SHREC complex that is involved in heterochromatin transcriptional gene silencing in S. pombe is composed of four proteins: Clr1 which is a zinc finger protein, Clr2 which has an unknown structure, Clr3 which acts like a histone deacetylase; the ‘Clr’ here stands for cryptic loci regulator and Mit1 which is a nucleosome remodeler. Previous studies have shown that Clr2 acts as silencing factor, which controls the transcription in the fission yeast S. pombe by formation of heterochromatin at various chromosomal locations. The Clr2 gene in the SHREC complex encodes a 62kDa protein with no known homology.

The aim of the study was to reveal the structure of the Clr2 protein by crystallization. The

post translational modifications in the P. pastoris as an expression system helps to obtain

properly folded protein which will lead to usable crystals. In addition, Clr2 and one of its

deletion constructs were purified from E. coli in order to determine the DNA binding ability

of the Clr2 protein. Protein-DNA binding interactions can be investigated using an

Electrophoretic Mobility Shift Assay (EMSA).

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1. Introduction

Epigenetics is defined as heritable changes in gene expression without any change in the DNA sequence. Epigenetic dysregulation might play a role in the earliest steps in cancer but it is firmly established that the overall epigenetical changes are a hallmark of cancer (Jones and Baylin, 2007). Epigenetic mechanisms are essential for normal development and maintenance of tissue specific gene expression patterns in mammals (Sharma et al., 2010).

DNA methylation and covalent histone modifications are two primary mechanisms related to epigenetics (Lund and Lohuizen, 2004). DNA is tightly packed around histone proteins to form chromatin in eukaryotic cells. The fundamental unit of chromatin is the nucleosome.

Each nucleosome contains an eight-histone tetramer core of H2A, H2B, H3 and H4 proteins wrapped by DNA. Post translational modifications are defined as the addition of chemical groups like acetyl groups, methyl groups, ubiquitin groups and phosphate groups to the core histones. The structure of chromatin plays an important role in gene regulation, genome propagation, differentiation, ageing, and angeogenesis of cells. Chromatin is divided into the two major types: euchromatin, which is lightly packed and easily accessible for active transcription, and heterochromatin is responsible for gene regulation, chromosomal integrity and it is more tightly packed compared with euchromatin (Knipe and Cliffe 2008).

1.1 Schizosaccharomyces pombe as a model organism

To increase the knowledge of epigenetics the fission yeast Schizosaccharomyces pombe is

used as a model system. S. pombe is a unicellular eukaryote with a medial division known as

fission rather than budding, which is the case for some other yeast like Saccharomyces

cerevisiae. The complete genome of S. pombe has been sequenced and was published in 2002

(Wood et al., 2002). S. pombe has many features similar to higher eukaryotes for example

centromere structure is similar to the centromere structure in humans in addition both histone

modifications and RNA interference (RNAi) are found in S. pombe (Goto and Nakayama,

2012; Wood et al.,2002).

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1.2 The SHREC complex

The SHREC complex is composed of four proteins: Clr1 which is a zinc finger protein, Clr2 which has an unknown structure, Clr3 which acts like a histone deacetylase and Mit1 which is a nucleosome remodeler. SHREC is an effector complex that regulates heterochromatin transcriptional gene silencing (Sugiyama et al., 2007). Previous studies have shown that Clr2 acts as silencing factor, which controls the transcription in the fission yeast by formation of heterochromatin at various chromosomal locations (Bjerling et al., 2004). Several studies have been performed to understand the actual role of Clr2 but no solid conclusion has yet been reached. The Clr2

(wild type clr2 gene) encodes a 62kDa protein with no known homology, but recent bioinformatics studies on several fungal genome sequences revealed that there are genes with sequences similar to Clr2. Using these sequences three conserved motifs in the Clr2 protein were identified (Kristell et al., 2011). The atomic structure of Clr2 would give great insight into its function and it would also give insight into the interaction between Clr2 and other components of the SHREC complex.

1.3 Pichia pastoris as an expression system

The adoption of Pichia pastoris as an expression system had a slow start in the early 1980’s but two decades on the problems related to expression in yeast have been addressed and yeast is considered as an effective alternative system for recombinant protein production. Post translational modifications and glycosylation are also accomplished easier when using a yeast expression system. The most important benefit with yeast as an expression system is to get expression of the native proteins when compared to bacterial expression system which causes synthesis of improperly folded and inactive aggregated proteins or inclusion bodies (Cregg et al., 2009). P. pastoris has higher growth rate and is able to grow on a simple and inexpensive media than other yeast.

1.4 pPICZαA and pGAPZαA an expression vectors

pPICZαA is a 3.6kb size vector, which will express clr2

in the organism P. pastoris. The

vector has the AOX1 (Alcohol Oxidase 1) promoter, which allows a methanol induced high-

level expression. pGAPZαA is a 3.1kb vector with a GAP (Glyceraldehyde-3-phosphate)

promoter. The expression level of protein by the GAP promoter is slightly higher

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than that obtained from the AOX1 promoter. Alpha factor from the yeast S. cerevisiae helps to secret the recombinant protein into the growth media. Selection of both these vectors is done with Zeocin, an antibiotic that acts both on prokaryotic and eukaryotic systems. It belongs to the bleomycin or phleomycin antibiotics family, which is extracted from the bacteria Streptomyces verticillus. Antibiotics from this family act as strong anti-bacterial and anti-tumor drugs. Zeocin is toxic and it causes cell death by interacting with DNA and cleaving it (Chankova et al., 2007; Ehrenfeld et al., 1987). The polyhistidine tag present in vectors helps in purification of the proteins. The maps and details about these vectors can be seen in appendix 7.3.

1.5 Aim of the project

To test whether P. pastoris functions as a host system for the expression of the Clr2 protein

we used two different P. pastoris expression vectors. The post translational modifications in

the P. pastoris as an expression system helps to obtain properly folded protein which will

lead to usable crystals. Using a eukaryotic system like P. pastoris for the expression of the

Clr2 protein the chances of getting good Clr2 crystals are expected to increase. A complete

protein structure could help to resolve questions related to heterochromatin silencing in

fission yeast, and the role of the SHREC complex as a whole in S. pombe. An addition aim

was to purify Clr2 and a deletion construct from S. pombe in E. coli combined with Electro-

mobility shift assay technique (EMSA), which will give insight in order to find out more

about a possible DNA binding ability of the Clr2 protein.

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2. Materials and Methods

2.1 Media

To make chemically competent E. coli cells (DH5α), Luria Broth (LB) rich medium was used. The TOPO cloning vector which contained clr2

had an ampicillin resistance gene; LB medium with 150mg/ml ampicillin concentration was used. The media used for the P.

pastoris expression vectors pPICZαA and pGAPZαA containing Zeocin as a resistance gene was low salt LB media at p H 7.5, to prevent the inactivation of Zeocin (Invitrogen, R250- 01). The required concentration of Zeocin was 25-50mg/ml. P. pastoris cells were grown in two types of Yeast Extract Peptone Dextrose media with or without sorbitol, which is a sugar alcohol and acts as an osmotic stabilizer. The Zeocin concentration for yeast media was 100mg/ml. All media recipes can be found in the appendix.

2.2 Strains

The clr2

from S. pombe had previously been cloned into the TOPO cloning vector (Invitrogen, 1265035). The two expression vectors pPICZαA and pGAPZαA and the P.

pastoris yeast strain were obtained from Professor

Universitet (SLU). The clr2

and the deletion construct (Clr2-DC) had previously been cloned into the pTrcHis- TOPO prokaryotic expression vector (Invitrogen, 843974) and were available in the lab (Kristell et al., 2011).

2.3 Plasmid Mini Preparation

Topo cloning vector containing clr2

and P. pastoris expression vector strains (DH5α) were

streaked on their respective media. The Luria Agar (LA) media had an ampicillin

concentration of 150mg/ml and LA media with Zeocin with a concentration of 25mg/ml. The

samples were incubated overnight at 37°C. Positive colonies were picked and transferred into

10ml LB media containing ampicillin with a concentration of 150mg/ml and 10ml LB media

containing Zeocin with a concentration of 25mg/ml and the cultures were incubated overnight

on a shaker at 37°C. For the plasmid preparation the Gene JET Plasmid Mini Prep kit from

Thermo Scientific (K0503) was used. The samples were centrifuged at 4000rpm for 4 min at

4°C and the pelleted cells were resuspended in 250µl resuspension solution. 250µl lysis

solution was added to break the cell wall of the cells. 350µl neutralization solution was added

to the samples and the samples were mixed immediately by inverting the test tubes 4-6 times.

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The samples were centrifuged for 5 minutes at room temperature in a table top bench micro- centrifuge at 12000rpm. The supernatant was transferred without disturbing the precipitate to the supplied GeneJET spin column. Then column was centrifuged for 1 minute and the flow- through was discarded and the column was placed back into the same collection tube. Then 500µl of wash solution was added to the column. The GeneJET spin column was then centrifuged for 1 minute at 12000rpm and the flow-through was discarded. An additional 500µl wash solution was added and the columns were spun again at 12000rpm. To avoid residual ethanol in elute, the samples were centrifuged for 1 minute at 12000rpm. The GeneJET spin columns were transferred to 1.5ml test tubes and 35µl of elution buffer was added to the centre of the GeneJET spin column membrane to elute the plasmid DNA. The concentration of the plasmid DNA was measured using the NanoDrop.

Four different GeneJET spin columns with the same samples were prepared the same way as above. 70µl of elution buffer was added to the first column and the flow-through was centrifuged serially through the remaining columns to increase the final concentration of the plasmid DNA.

2.4 Restriction enzyme digestion of plasmid DNA and Shrimp Alkaline Phosphatase treatment

Suitable restriction enzymes for both pPICZαA and pGAPZαA vectors and clr2

were verified on the CLC workbench, sequence analysis software. The plasmids were opened with the restriction enzymes XhoI (Thermo Scientific FD0694) and KpnI (Thermo Scientific FD0524). The pPICZαA and pGAPZαA vectors were treated with Shrimp Alkaline Phosphates (SAP) in order to prevent self ligation of the cut plasmids by removing the 5´

phosphate group from the plasmids.

Each restriction enzyme digestion reaction mixture contained 5µl of plasmid DNA, 5µl of 10X Fast Digest Green Buffer, 2µl of each of the restriction enzymes and ddH

O to a final volume of 50µl. The samples were incubated at 37°C for 30 minutes followed by the addition of 1µl fast alkaline phosphatase (Thermo Scientific EF0654) only to pPICZαA and pGAPZαA samples, which were incubated for an additional 30 minutes at 37°C.

2.5 Gel purification

After the restriction enzyme digestion, clr2

and the opened, SAP treated, pPICZαA and

pGAPZαA vectors were gel purified. Gel purification was performed using a GeneJET gel

extraction kit from Thermo Scientific (K0692).

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The cut gel slices containing the DNA fragment were weighed and put into 1.5ml test tubes and 1:1 (volume: weight) volume of binding buffer was added to the cut gel slices. The gel mixture was incubated at 60°C until the gel slices were completely dissolved. The 800µl solubilised gel solution was transferred to the GeneJET purification column. The columns were centrifuged for 1 minute at 12000rpm at room temperature. The flow-through was discarded and the column placed back into the same collection tube. 700µl wash buffer was added to the column and centrifuged for 1 min, the flow through was discarded and the wash step was repeated one more time. The GeneJET purification column was transferred to the new 1.5ml test tube and 30µl of elution buffer was applied to the centre of the column membrane and centrifuged for 1 minute. After gel purification concentration of the samples were measured using the NanoDrop. The purified plasmids were stored at -20°C.

2.6 Ligation

Gel purified clr2

was ligated into the gel purified pPICZαA and pGAPZαA vectors with the ratio between insert and vector had to be 5:1. 8µl of pPICZαA and 9µl of pGAPZαA were mixed with 2µl 10X T4 DNA Ligase Buffer, 0.5µl T4 DNA Ligase Enzyme from Thermo Scientific (EL0011) and 0.75µl of 10 mM ATP (Adenosine tri-phosphate). To make the final volume of 20µl, clr2

was added. The samples were incubated at room temperature for 2 hours.

2.7 Competent cells preparation

DH5α cells from the -80°C stock were used for bacterial transformation. 50ml LB media was inoculated with DH5α cells and incubated at 37°C on the shaker. Cells were grown until the culture reached an OD

of 0.4. The cells were harvested by centrifugation at 3000rpm at 4°C for 10 minutes. The supernatant was discarded and the pellet was resuspended in 20ml TFBI (Transfer Buffer Solution I) solution and left on ice for 10 minutes

The tube was spun down again with the same conditions as above. Then the pellet was resuspended in 2ml TFBII (Transfer Buffer Solution II) solution followed by a 15 minutes of incubation on ice.

The recipes for TFBI and TFBII buffer can be found in the appendix. Cells were stored in 100µl at -80°C.

2.8 Bacterial Transformation

One aliquot described in 2.7 was thawed on ice. 4µl of ligated sample was mixed with 100µl

of DH5α competent cells and incubated for 20 minutes on ice, followed by a heat shock at

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42°C in water bath for 1 minute and then incubated immediately on ice for 5 minutes. The samples were streaked on LA plates with a Zeocin concentration of 25mg/ml and incubated overnight at 37°C.

2.9 Linearization of plasmid vector

The pPICZαA and pGAPZαA vectors containing clr2

were linearized and purified before transforming into P. pastoris as the linearized DNA helps to stimulate the integration between vector loci and the host genome locus by recombination. pPICZαA was linearized by SacI (Thermo Scientific ER1131) and incubated at 37°C for 2 hours and pGAPZαA with BspHI (Thermo Scientific FD1284) incubated at 37°C for 25 minutes as the latter shows star activity when incubated over longer periods. The linearized vectors were purified by ethanol precipitation.

2.9.1 Transformation by Electroporation

The goal with transformation is to insert the linearized recombinant vectors pPICZαA and pGAPZαA by electrical pulses into P. pastoris to express Clr2 protein. Electroporation was carried out with an electroporator from Bio-Rad. All steps were performed on ice. The P.

pastoris cells were grown to an OD

of around 0.5 at 30°C on a shaker. The cells were harvested by centrifugation at 5000rpm for 10 minutes at room temperature. The supernatant was discarded and the pellet was resuspended in 20ml of ice cold 1.2M sorbitol, which acts as an osmotic stabilizer. The cells were harvested at 2500rpm for 5 minutes at 4°C. The pellet was resuspended in 10ml of ice cold sorbitol and spun as above. The above two steps were repeated twice. After centrifugation the pellet was resuspended in 600µl of ice cold 1.2M sorbitol in order to obtain a cell density of 1x10

cells ml

. The cells were then divided into 200µl aliquots and mixed with between 500ng and 1µg of linearized DNA. Immediately after mixing, samples were transferred to ice-cold 2 mm cuvettes (Bio-Rad) and the cells were chocked using the electroporator at 2.25kv, 200Ω and 25mF. Then 1 ml of ice cold 1.2M sorbitol was added. The electroporated cells were spread on YPD plates containing Zeocin with a concentration of 100mg/ml. The plates were incubated at 30°C for 4 days.

2.9.2 Transformation by Chemical method

10ml YPD media was inoculated with P. pastoris and incubated at 30°C overnight on a

shaker. On next day 140ml of fresh YPD was inoculated with the overnight culture and then

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incubated at 30°C overnight on a shaker. 1ml cell culture was taken out from the overnight culture and centrifuged at 2500rpm for 5 minutes. The pellet was resuspended in 1ml ddH

O.

The OD

(Optical Density) measured of the sample was around 2.5 units.

After OD measurement the amount of fresh culture needed to obtain 22.5 units at OD

was calculated. 70ml of fresh YPD media added to the overnight culture. The sample was spun down at 700rpm for 5 minutes. The pellet was resuspended with 150ml pre-warmed YPD at 30°C. 1ml was taken from the 150ml cell solution and spun down at 2500rpm for 5 minutes.

The cell culture was incubated at 30°C with vigorous shaking for around 5 hours to obtain an OD

of 0.6-0.7 (around two cell division). Single stranded carrier Salmon sperm DNA (Invitrogen, 1189883) was boiled for 5 minutes at 99°C to get maximum transformation efficiency. Two transformation mixtures were prepared. Mixture ‘A’ contained 0.88ml 1M Lithium Acetate mixed with 0.88ml 10x TE (Tris EDTA) at pH 7.5 and 6.24ml of ddH

O.

Mixture ‘B’ containing 1.2ml 1M LiOAc, 1.2ml 10x TE at pH 7.5 and 9.6ml PEG (Polyethylene glycol) was added to made final volume 12ml PEG/LiOAc (see appendix).

The 150ml culture with OD 0.62 was split into three 50ml Falcon tubes and the cells were harvested by centrifugation at 700rpm for 5 minutes. Then each pellet was resuspended in 30ml of ddH

O. Cells were pelleted again at 700rpm for 5 minutes. The supernatant was removed and each pellet was resuspended in 1ml of mixture ‘A’ and centrifuged as described above. The supernatant was removed and pellet was resuspended in 600µl of mixture ‘A’.

5µg of linearized pPICZαA and pGAPZαA plasmids were added to two out of three Falcon

tubes and to the third Falcon tube distilled water was added as a blank. 100µl single stranded

Salmon sperm DNA and the P. pastoris cells resuspended in 600µl of mixture ‘A’ were

added to all 3 tubes. 2.5ml of mixture ‘B’ was added to all the tubes followed by 1 minute of

vortexing. The tubes were incubated at 30°C for 45 minutes on a shaker. After incubation,

160µl dimethyl sulfoxide (DMSO) was added and the samples were mixed immediately. The

DMSO helped to permeabalise the yeast cell wall. The samples were incubated at 42°C for

20 minutes. The cells were harvested by centrifugation at 700rpm for 5 minutes. Each pellet

was resuspended in 3ml YPD media. The cells were incubated at 30°C for 90 minutes with

medium shaking (150rpm). The cells were harvested at 700rpm for 5 minutes and then

resuspended in 3.6ml 0.9% NaCl (Sodium Chloride) ddH

O. The YPD plates were streaked

with two different volumes 1ml and 500µl by utilizing the total 3.6ml resuspended sample

and incubated at 30°C for 5 days.

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2.10 Analysis of Protein by Sodium Dodecyl Sulfate - Poly Acrylamide Gel Electrophoresis with Coomassie staining

The recipe for acrylamid gels can be found in the Appendix. The available wild type clr2

and the 56 amino acids deleted from the clr2

construct, named clr2 deletion construct (clr2- DC), had previously been cloned into the E.coli expression vector, pTrcHis TOPO (Kristell et al., 2011). These two samples were inoculated in 10ml LB media with 150mg/ml ampicillin concentration at three different temperature conditions room temperature, 30°C and 37°C to select the best condition for high protein expression. The samples were grown overnight on a shaker. The 2ml cultures were harvested by centrifugation at 5000rpm for 5 minutes.

Concentrated blue loading dye is a pre-mixed loading buffer that was added to the samples, to act as reducing agent that helps to weigh down the DNA solution and the samples. The samples were then boiled for 10 minutes at 99°C to denature the proteins by reducing the disulfide linkages. The Page Ruler Plus Prestained Protein Ladder (Thermo Sceintific, 26619) and an equal amount of the cell lysate were loaded on the SDS-PAGE gel and the gel was run at 100V for 90 minutes. The SDS-PAGE gel was then stained with Coomassie blue dye for 2 hours on a shaker at room temperature followed by destaining with ddH

O overnight in cold room on a shaker.

2.11 Detection of the protein by Immunoblotting

The cell lysates of the empty vector, clr2

and the clr2-DC were grown at room temperature, 30°C and 37°C in LB media with an ampicillin concentration of 150mg/ml overnight on a shaker. An immunoblot was performed in order to analyse the expressed protein containing a 6xHis tag with anti-His-G antibody. The samples and a Page Ruler Plus Prestained protein ladder were loaded into the SDS-PAGE gel, and the gel was run for 90 minutes at 100V (Mini-Protean TGX from Bio Rad). The gel was removed carefully and placed into the transfer buffer (see 7.2 appendix) for 10 minutes to equilibrate the gel. The nitrocellulose membrane was soaked in 100% methanol for 15 seconds followed by transferring the nitrocellulose membrane into the transfer buffer to equilibrate for 5 minutes. The transfer cassette was placed with the black side down and a pre-soaked pad was placed on the black side. Three filter papers were placed on the pad followed by the gel and the nitrocellulose membrane. The membrane was covered with three filter papers and a pad on the top. By pressing the sandwich any bubbles present were removed, to avoid interference with transfer.

The transfer cassette was placed into the transfer tray with the black side towards the back so

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that the protein samples could transfer into the nitrocellulose membrane and not out to the filter paper. The transfer was carried at 100V for 60 minutes at room temperature, with an icepack in the transfer chamber, with constant steering to prevent the gel from melting during the transfer. To test the transfer, the SDS-PAGE gel was Comassie stained by the same method as in 2.10. The membrane was blocked in blocking buffer (7.2, appendix) for 60 minutes at room temperature on a shaker. The blocking buffer was removed and the primary antibody, anti His-G was added (Invitrogen, 838003), diluted 1:2500 in blocking buffer. The membrane was incubated with the primary antibody over night in the cold room on a shaker.

On the next day the primary antibody was removed and the membrane was washed several times with fresh washing buffer at different time intervals, twice a quick wash, then 5 minutes on the shaker followed by 15 minutes on shaker and then again 5 minutes on shaker ending with two quick washes. The wash buffer was changed in between each wash to get rid of antibodies. Wash buffer was removed and the membrane was incubated at room temperature for 60minutes with the secondary antibody, anti mouse IgG, Horseradish Peroxide linked antibody (GE Health care UK, NA931V), diluted 1:10000. This was followed by additional wash steps: 2 quick, then 5 minutes followed by two times 15 minutes, then 5 minutes and at last two quick washes. The wash buffer was removed and the membrane was transferred to 1x PBS buffer without Tween20. The membrane was soaked gently for few seconds in the ECL solution from Amersham

ECL Prime Western Blotting Detection Reagent (GE Health care UK, RPN2232) and the membrane was analyzed on Bio- Rad Gel Dock.

2.12 Protein purification by Affinity chromatography

To purify the expressed protein by affinity chromatography, the poly-histidine 6xHis tag protein, in the prokaryotic expression vector pTrcHis was used to bind nickel- nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices in a fast start column. The principle behind affinity chromatography is the interaction of the 6xHis tag with Ni-NTA matrices which allows the high affinity and selective binding between the 6xHis tag protein and Ni- NTA resin matrix. The presence of imidazole in elution buffer allowed for the release of the histidine tag proteins, as the structure of imidazole and histidine are similar.

The histidine-tagged proteins were purified using the Ni-NTA fast start kit from Qiagen

(30600). 240ml pre-warmed fresh LB containing ampicillin with a concentration of

150mg/ml was inoculated with 10ml overnight grown culture of E. coli containing the clr2

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or clr2-DC in the pTrcHis TOPO vector at 30°C. The culture was then incubated for 5 hours

on vigorous shaking to get a final OD

around 0.6. The cells were harvested by

centrifugation at 4000rpm for 20 minutes. The cell pellet was thawed for 15 minutes on ice

and cells were resuspended in 10ml native lysis buffer. The samples were incubated on ice

for 30 minutes and mixed 3 times gently in between. The lysate was centrifuged at

14,000rpm for 30 minutes at 4°C, to pellet cellular debris. The pellet was discarded and

supernatant was placed on ice for further processing. The resin in the fast start column was

resuspended by inverting the column to settle down the resin. The seal of the column outlet

was broken and the screw cap was loosened to wash out storage buffer. The supernatant was

added to the column and flow through was collected in 1.5ml test tubes. The column was

washed two times with 4 ml of native wash buffer. The washed fractions were collected in

the test tube for further analysis on SDS-PAGE. The bound protein to 6x His-tagged nickel

resin was eluted with 2ml of elution buffer. All fractions were checked with SDS-PAGE to

analyze the purification.

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3. Results

3.1. Restriction Enzyme Digestion

To ligate clr2

into the expression vectors pPICZαA and pGAPZαA, clr2

was cut out from the TOPO cloning vector successfully by restriction enzyme digestion, with the enzymes XhoI and KpnI. The expression vectors pPICZαA and pGAPZαA were also cut with the enzymes XhoI and KpnI. Samples were analyzed on 1% agarose gel. Figure 1, shows the result from the restriction digestion. In lane 2, the lower band is around 1.6 kb, which confirms the presence of the clr2

in the TOPO cloning vector. The two successfully opened expression vectors are observed as linear bands in lane number 3, which contain pGAPZαA at around 4kb and in lane number 4, which contains pPICZαA at around 4kb.

Figure 1: Restriction enzyme digestion of TOPO cloning vector and

pPICZαA and pGAPZαA

3.2. Gel Purification

The lower band from lane 2 containing clr2

and the bands from lane 3 and 4 in Figure1 were

cut out with a scalpel and purified. The concentration of the purified DNA was measured

using NanoDrop; Table 1 shows the values of DNA concentrations before and after the gel

purification.

R e s u l t s

P a g e | 17 Table 1: Different concentration of the samples before and after purification.

3.3. Bacterial Transformation

The purified plasmids and clr2

were ligated into the two vectors and transformed into E. coli (DH5α). The plasmids were extracted by plasmid mini preparation cleaved by restriction enzymes XhoI and KpnI and separated on a 1% agarose gel to confirm the ligation. Three different colonies were analysed from each pPICZαA and pGAPZαA transformants, for verification of the presence of clr2

in the plasmids. The plasmids were digested with the restriction enzymes XhoI and KpnI. The uncut or undigested plasmids were used as controls shows nick, linear and super-coil bands from top to bottom. Lower bands in cut products confirm clr2

presence in the expression vectors (Figure 2).

Figure 2: Verification of the Clr2⁺ gene in expression vectors

pPICZαA and pGAPZαA.

and 14 is a DNA Page Ruler 1kb Plus Ladder. Lane 2, 4, 6 show uncut pPICZαA vector candidates. Lane 3, 5, 7 shows three different candidates of cut pPICZαA vector by XhoI and KpnI enzymes. Lane number 8, 10, 12 show uncut pGAPZαA vector whereas lane 9, 11, 13 shows three different candidates of cut pGAPZαA vector by XhoI and KpnI enzymes.

Samples Concentration before Gel

purification

Concentration after Gel purification

Topo cloning vector with clr2

486.9ng/µl 10.3ng/µl

pPICZαA 172.4ng/µl 7.1ng/µl

pGAPZαA 173.0ng/µl 5.1ng/µl

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3.4. Linearization of plasmid

The vectors containing clr2

were obtained through bacterial transformation (Figure 2). In order to express Clr2 protein in the yeast P. pastoris, transformation by electroporation with linearized recombinant vectors was essential. The linearization aimed to open the vectors in the promoter regions. The linearized vectors contained DNA sequences at each end that stimulates integration between locus shared by vector and host genome. This intermolecular recombination by a single crossover recombination event is a low frequency or sometimes rare event. The pPICZαA plasmid was opened at the AOX1 promoter by SacI and pGAPZαA was opened at the GAP promoter by BspHI, both enzymes give sticky ends. In Figure3, both pPICZαA and pGAPZαA uncut vectors can be seen as a positive controls and in addition, cut pPICZαA and pGAPZαA vectors by the enzymes XhoI and KpnI, to show the presence of clr2

, also as a positive control. We had to assure the complete linearization of the recombinant vectors before transforming into P. pastoris. It was observed that the linearization of pPICZαA vector with SacI was not complete when compared with linearization of pGAPZαA vector by BspHI. A small band was observed in the case of pPICZαA linearization at around 1kb size in lane 4 which can be seen in Figure 3.

Figure 3: Linearization of the

pPICZαA and pGAPZαA vectors.

R e s u l t s

P a g e | 19

3.5. Coomassie Stained SDS-PAGE

clr2

and clr2-DC had been cloned and expressed in E. coli expression vector pTrcHis. The cell lysate of the pTrcHis empty vector was used as a negative control to compare presence of Clr2 and the deletion construct protein in the cell lysate. Samples were analysed on Coomassie stained SDS-PAGE. After destaining of the gel several protein bands were observed at different sizes on the gel as seen in Figure 4.

Figure 4: Coomassie stained SDS-PAGE gel. Lane 1 is a Page Ruler Plus Prestained Protein Ladder.

Lane 2 is the empty vector (EV) at Room Temperature (RT), Lane 3 is the Clr2 wild type (WT) at RT, Lane 4 is the deletion construct (DC) at RT, Lane 5 is the EV at 30°C, Lane 6 is the WT at 30°C, Lane 7 is the DC at 30°C, Lane 8 is the EV at 37°C, Lane 9 is the WT at 37°C and Lane 10 is the DC at 37°C.

3.6. Immunoblot

Coomassie staining was performed to confirm the presence of the proteins in the cell lysate

(Figure 4). The analysis by immunoblot was needed for the specific verification of wild type

Clr2 (WT) and deletion construct (DC) protein. Anti His-G antibody was used as primary

antibody to detect the expressed protein and anti mouse IgG as secondary antibody. The cell

lysate of empty vector (EV) pTrcHis was used as a negative control, at three different

temperature conditions to compare with cell lysates of the Clr2 and the DC. The three

different temperature conditions were used to grow the culture with the intention to

understand the improved protein expression as seen in Figure 5.

P a g e | 20 Figure 5: Immunoblot to detect the Clr2 and DC protein. The cell lysates of EV, WT and DC at different temperature conditions with 150mg/ml ampicillin concentration. Lane 1 shows the Page Ruler Plus Prestained Protein Ladder, lane 2, 3, 4 shows EV, WT and DC at room temperature. Lane 5 shows EV, lane 6 shows WT Clr2 at 62kDa marked with a square and lane 7 shows DC at around 56 kDa, marked with square at 30°C. Lane 8 shows EV, lane 9 shows WT Clr2 at 62 kDa marked with light square and lane 10 shows DC at 56 kDa marked with square at 37°C.

3.7 Purification of the protein

The supernatant, flow through, wash and elute samples were detected through immunoblot.

The supernatant, flow through and wash samples were used as a control to differentiate the

extent of purification with elute, which is final product. The purified Clr2 protein was

observed at a 62kDa size from lanes 6 to 9, spotted in box together with some unspecific

proteins below 30kDa, as seen in Figure 6. Whereas, the purified DC protein was observed at

56kDa size from lanes 6 to 9, spotted in box also with some unspecific proteins below 30kDa,

as seen in Figure 7. The concentration of purified Clr2 and DC protein was determined by

Bicinchoninic Acid Assay as seen in Table 2.

R e s u l t s

P a g e | 21 Figure 6: Immunoblot of purified WT Clr2. Lane 1 is the Page Ruler Plus Prestained Protein Ladder, lane 2is the supernatant, lane 3 is the flow through, lane 4 is wash 1 and lane 5 is wash 2. Lanes number 6 to 9 are the elute.

Figure 7: Immunoblot of purified DC. Lane 1 is the Page Ruler Plus Prestained Protein Ladder, lane 2 is the supernatant, lane 3 is the flow through, lane 4 is wash 1 and lane 5 is wash 2. Lanes 6 to 9 are elute.

P a g e | 22 Table2: Purified protein concentration by Bicinchoninic Acid Assay

4. Discussion

The aim of this study was to obtain crystals of the Clr2 protein to solve its 3D structure, which is important to get a further understanding of the function of the Clr2 protein. The work was divided into several steps and started with producing the recombinant expression vectors pPICZαA and pGAPZαA cloned with clr2

(Figure 2). The recombinant vectors were then transformed into P. pastoris for Clr2 protein expression. The fully expressed protein was planned to be purified, as the purity of protein is one of the prerequisite conditions to get good crystals.

The recombinant vector pPICZαA was linearized with SacI according to the P. pastoris expression vector manual (Invitrogen V195-20), enzyme SacI was supposed to cut only once in the AOX1 promoter in order to linearize the vector. But in practice the complete linearization of the pPICZαA vector could not be obtained. After cross checking clr2

sequence it was observed that SacI has a recognition site in clr2

, which cuts pPICZαA both in AOX1 promoter and in clr2

. So it was cleared that the 1kb band (lane 4; Figure 3), was a small piece of each of half of the AOX promoter and clr2

. Therefore I tried the enzyme PmeI (MssI). Surprisingly, it was also unable to linearize pPICZαA in the AOX1 promoter as observed several bands instead of linear one (data not shown). One reason could be that the sequence analyzed through the CLC workbench software might be incorrect in case of the pPICZαA. The solution to this problem would be to sequence pPICZαA and thus find the exact enzyme that will linearize the vector at a unique site in the AOX1 promoter.

Samples Concentration of wild type Clr2 protein

Concentration of DBD protein

First Batch purification 0.2mg/ml 0.21mg/ml

Second Batch Purification

0.38mg/ml 0.38mg/ml

Third Batch Purification

0.35mg/ml 0.37mg/ml

D i s c u s s i o n

P a g e | 23

The pGAPZαA was linearized successfully with BspHI enzyme in the GAP promoter (lane 7;

Figure 3). The transformation of the linearized and purified vector in order to express the Clr2 protein did not work. There could be several possibilities for the failure; first the vector should enter the yeast cell and then the intermolecular recombination between linearized vector and host genome, by single crossover recombination has to take place, which is an unlikely and rare event (Shixuan and Geoffrey, 2004). The transformation efficiency in case of P. pastoris as a host organism is low compared to that of prokaryotes, like E. coli. The failure in getting colonies questioned the stringency of both the YPD and YPDS plates containing Zeocin. To check if the plates did not allow the growth of the transformed colonies, the P. pastoris expression vector containing a Pichia pastoris strain with Zeocin resistance from ICM, Uppsala University, were streaked on both YPD and YPDS plates containing Zeocin. Optimal Zeocin resistant colonies were observed on the YPD and YPDS with Zeocin plates after 3 days, which confirmed that the cells were able to grow on the plates. Activity of the Zeocin antibiotic was checked by streaking a P. pastoris strain without the Zeocin resistance as negative control. Equally, changing the P. pastoris competent strain to GS115 from ICM, Uppsala University did not result in successful transformation either.

The attempt of transformation through chemical method did not work either.

To overcome this, one could use the pPICHOLI shuttle vectors, which have an autonomous replication site (ARS), as the yeast origin of replication. The linearization of these vectors is not required since it has both a yeast origin and a bacterial origin of replication. It can be a good option because of its dual origin of replication in both prokaryotes and eukaryotes (Lueking et al., 2003).

clr2

and clr2-DC were expressed in pTrcHis prokaryotic expression vector and were

analysed on Coomassie stained SDS-PAGE gels. The presence of both proteins was

confirmed by immunoblot, by comparing their sizes with the page ruler plus pre-stained

protein ladder (Figure 5). Different temperature conditions like room temperature, 30°C and

37°C showed fluctuations in the protein expression. The protein expression at room

temperature was negligible, whereas at 30°C and 37°C they were almost similar (Figure 5).

P a g e | 24

The culture containing both expressed protein samples were purified by fast start Ni-NTA column. The purification of both proteins was good according to immunoblot (Figure 6 and 7). The results of the immunoblot helped to understand the extent of Clr2 protein and DC protein’s purification. But some unspecific bands at 30kDa size were observed that could also be seen in the empty vector at 30kDa (Figure 5). The obtained purified samples can be purified with size exclusion chromatography, in order to get rid of unspecific and unnecessary lower molecular weight proteins.

4.1 Further Work

To send pPICZαA and pGAPZαA vectors containing clr2

for sequencing. Then try out a

new protocol for transformation of vectors into P. pastoris. Further purification can be done

by size exclusion chromatography. After size exclusion chromatography the resulting sample

of the Clr2 protein could then be sent for screening and crystallization. In addition we would

like to investigate whether the Clr2 protein is binding to DNA by using an electrophoretic-

mobility shift assay (EMSA). If the Clr2 has DNA binding activity we want to investigate, if

this is compromised in the Clr2 DC.

A c k n o w l e d g e m e n t

P a g e | 25

5. Acknowledgement

Sincere thanks to Pernilla Bjerling for giving me an opportunity to work on this interesting project. I am thankful to Daniel Steinhauf, my supervisor for his unconditional tutelage. I also would like to thank all the group members and the lab corridor mates for great scientific atmosphere with loads of fun. I would like to thank Sanjeewani, Post Doc from ICM for her valuable suggestions during my project.

I want to take an opportunity to thank my friends and dear one’s for the support and encouragement. I would like to extend my sincere appreciation to my housemates and all those who knowingly and unknowingly helped me to complete this endeavour.

I would like to express my deepest gratitude to my teacher, S.N.Goenka, my siblings and all

family members. And last but not least, Mummy and Papa this one is for you thank you very

much!

P a g e | 26

6. References

Bjerling, P., Ekwall, K., Egel, R., and Thon, G. (2004). A novel type of silencing factor, Clr2, is necessary for transcriptional silencing at various chromosomal locations in the fission yeast Schizosaccharomyces pombe. Nucleic acids research 32, 4421-4428.

Chankova, S. G., Dimova, E., Dimitrova, M., and Bryant, P.E. (2007). Induction of DNA double-strand breaks by zeocin in Chlamydomonas reinhardtii and the role of increased DNA double-strand breaks rejoining in the formation of an adaptive response. Radiation and environmental biophysics 46, 409-416.

Cregg, J. M., Tolstorukov, I., Kusari, A., Sunga, J., Madden, K., and Chappell, T. (2009).

Expression in the yeast Pichia pastoris. Methods in enzymology 463, 169-189.

Ehrenfeld, G.M., Shipley, J.B., Heimbrook, D.C., Sugiyama, H., Long, E.C., van Boom, J.H., van der Marel, G.A.,Oppenheimer, N.J., and Hecht, S.M. (1987). Copper-dependent cleavage of DNA by bleomycin. Biochemistry 26, 931-942.

Goto, D.B., and Nakayama, J. (2012). RNA and epigenetic silencing: insight from fission yeast. Development, growth & differentiation 54, 129-141.

Jones, P.A., and Baylin, S.B. (2007). The epigenomics of cancer. Cell 01.029.

Knipe, D.M., and Cliffe, A. (2008) Chromatin control of herpes simplex virus lytic and latent infection. Nature reviews Microbiology 6, 211-221.

Kristell, C. (2011) Paper III in the thesis, Chromatin Dynamics in the Fission Yeast, Schizosaccharomyces pombe, Uppsala University.

Lueking, A., Horn, S., Lehrach, H., Cahill, D.J. (2003). A dual expression vector allowing expression in E.Coli and P.Pastoris. Methods in Molecular Biology 205, 31-42.

Lund, A.H., and van Lohuizen, M. (2004). Epigenetics and cancer. Genes & development 18,

2315-2335.

R e f e r e n c e s

P a g e | 27

Sharma, S., Kelly, T.K., and Jones, P.A. (2010). Epigenetics in cancer. Carcinogenesis 31, 27-36.

Sugiyama, T., Cam, H.P., Sugiyama, R., Noma, K., Zofall, M., Kobayashi, R., and Grewal, S.I. (2007). SHREC, an effector complex for heterochromatin transcriptional silencing. Cell 128, 491-504.

Wood, V., Gwilliam, R., Rajandream, M.A., Lyne, R., Stewart, A., Sgouros, J., Peat, N., Hayles, J., Baker, S. (2002). The genome sequence of Schizosaccharomyces pombe. Nature 415, 871-880.

Wu, S., and Letchworth, G.J. (2004). High efficiency transformation by electroporation of

Pichia pastoris pre-treated with lithium acetate and dithiothreitol. BioTechniques 36: 152-

154.

P a g e | 28

7. Appendix

7.1 Media Recipe Luria Broth (1 litre LB) 10g Trypton

10g Sodium Chloride (NaCl) 5 g Yeast Extract in 950ml ddH

O For plates 10g of Agar was added

Antibiotics used were ampicillin with 150mg/ml concentration and for Pichia vectors zeocin with 50 mg/ml concentration.

Yeast Peptone Dextrose (1litre YPD) 20g Peptone

10g Yeast Extract in 900 ml ddH

O

100ml 20% filter sterilized Dextrose (Add this after above liquid autoclaved) 20g Agar to make plates

Zeocin with 100mg/ml concentration for plates and media that requires antibiotic selection Yeast Peptone Dextrose with Sorbitol (1litre YPDS)

20g Peptone 10g Yeast Extract 182.9g Sorbitol

100ml 20% filter sterilized Dextrose (Add this after above liquid autoclaved) 20g Agar to make plates

Zeocin with 100mg/ml concentration for plates and media that requires antibiotic selection

7.2 Solutions used

Transformation Buffer I (TFBI) 1.18g, 30 mM KC

H

O

4.84g, 100 mM RbCl 0.59g, 10 mM CaCl

x 2H

O 3.2g, 50 mM MnCl

x 2H

O 69 ml 15% v/v Glycerol ddH

O up to 400ml

Adjust the pH5.8 with glacial acid (vinegar acid) and then filter sterilize.

Transformation Buffer II (TFBII) 0.42g 10 mM MOPS

0.24g 10 mM RbCl

2.21g 75 mM CaCl

x 2H

O 34.5ml 15% v/v Glycerol ddH

O up to 200ml

Adjust the pH6.6 with KOH and then filter sterilize

A p p e n d i x

P a g e | 29

10% SDS-PAGE gel

Separation gel Stacking gel ddH

O 7.9 ml 2.7ml

30% Acrylamide mix 6.7ml 670µl

1.5M Tris (pH 8.8) 5ml 500µl (pH 6.8) 10% SDS 200µl 40µl

10% Ammonium persulfate 200µl 40µl TEMED 8µl 4µl Running Buffer (1x)

28.8g Glycine 6.4g Tris 2g SDS

Dissolved in 1 litre of dd H

O Transfer Buffer (10x) 144g Glycine

30.2g Tris 10g SDS

In 1 litre of dd H

O Transfer Buffer (1x) 100ml 10x transfer buffer 100ml Methyl Alcohol 800ml dd H

O

Blocking Buffer 2g BSA

40ml dd H

O PBS (1x) 8g NaCl 0.2g KCl 1.44g Na

HPO

0.24g KH

PO

Dissolved in ddH

O, adjust pH to 7.4 and sterilized by filter sterilization Wash Buffer

5 ml 20% Tween-20

Diluted in 995ml 1x PBS

P a g e | 30

7.3 P.pastoris expression vector’s Maps

Figure 8: pPICZalphaA Vector map

Figure 9: pGAPZalphaA Vector map

A p p e n d i x

P a g e | 31

7.4 Graphs showing concentration of the purified proteins by using Bicinchoninic Acid Assay

Figure 10: Measurement of the purified Clr2 and DC protein concentration.

Concentration of Bicinchoninic Acid Assay (BSA) reagent versus Absorbance in order to get linear equation to measure the purified protein concentration of the first batch purified samples.

Figure 11: Measurement of the purified Clr2 and DC protein concentration.

Concentration of BCA reagent versus Absorbance in order to get linear equation to measure the purified concentration of the second and third batches purified samples.

y=1.6344 x - 0.1819

-0,5 0 0,5 1 1,5 2 2,5

0 0,5 1 1,5

Concentration On Y- Axis

Absorbance on X- Axis

Serie1 Linjär (Serie1)

y = 1.2318x - 0.1827

-0,5 0 0,5 1 1,5 2 2,5

0 0,5 1 1,5 2

Serie1 Linjär (Serie1)