Cloning and recombinant protein
expression of the human Prmt5 gene
for the understanding of the histone arginine methyltransferase pathway of PRMT5
Bachelor thesis
L.M. van Maldegem
22-07-2011
Cloning and recombinant protein
expression of the human Prmt5 gene
for the understanding of the histone arginine methyltransferase pathway of PRMT5
Student: L.M. van Maldegem
E-mail: Lennart@van-maldegem.eu
Research Institute: Kyungpook National University, Daegu, South Korea
Department: School of Applied Biosciences
Research Coordinator: Prof. dr. E. di Luccio
University: Avans University of Applied Sciences, Breda, The Netherlands
Faculty: Academy of Technology, Health and Environment
Major: Forensic Laboratorial Investigation
Specialization: Analytical and Biochemistry
Supervisor Avans: Dr. ir. C.H. Verhees
Research Period: 07-03-2011 - 22-07-2011
The human protein arginine methyltransferases (PRMTs) family consists of 11 members. All of the PRMT members modify the characteristics of cellular proteins by transferring a methyl-group to the side chain of arginine amino acid in target proteins, which are important for both the normal and malign cellular processes. An increasing number of studies link PRMT5 no numerous pathologies that include cancers. Moreover, little is known about the PRMT5 pathway. Therefore, we focus on
understanding the structure, the exact cellular functions and the regulation of PRMT5.
PRMT5 is an arginine methyltransferase enzyme that methylates arginine on histones H3 and H4 at the loci R8 and R3 respectively, by transferring one or two methyl groups to the arginine side chains. Histones are the stage of diverse post-translational modifications that ultimately regulates the gene transcription. Arginine methylation is one prominent feature of the post-translational histone modifications in the regulation of chromatin structure and function. Arginine methylation, or any of the other histone modifications, can have both activating and repressive functions on the
transcription, which can lead in changing the chromatin status to an unregulated chromatin. Chromatins are a combination of DNA, histones and proteins. Transcription coregulators (TCs), bind to a nuclear receptor and play a key role in regulating of the compaction levels of the DNA in the chromatin. Tight packing will inhibit the transcription events. At the opposite, looser packing will permit the binding of the large complex of proteins which form the transcription factor and activate the gene expression.
PRMT5 is key in the regulation of the circadian cycle by modulating the regulation of expression of clock-genes. Recently, a trail of evidence link deregulated PRMT5 to the onset of carcinogenetic events. However, little is known about the exact biological functions and regulations of the PRMT5, as well as the PRMT family.
My research in the Di Luccio research group in South Korea was centered on understanding the structure of PRMT5. In this report, I describe the cloning of human Prmt5 and recombinant protein expression of a pure PRMT5 protein for functional and structural studies. The expected results will greatly add to our understanding of the human histone arginine methyltransferase pathways in general. My project is the first step of a multi-year team effort aiming at understanding the histone code, to unravel the regulatory cross-talks events within transcription co-regulators and the design of selective and specific drugs designed to modulate the activity of PRMT enzymes.
The primarily objective of this multi-year project is to solve the unknown protein structure of the human PRMT5 in order to better understand its function, mechanism and regulation. The first goal of my project was achieved by successfully cloning the human Prmt5 gene and by building a
recombinant expression vector for recombinant protein expression in E.coli. Next, I determined the optimum parameters for recombinant protein expression in E.coli BL21 strain. I successfully over-expressed human PRMT5 and purified it by affinity chromatography. I performed peptide mass fingerprinting by MALDI-TOF mass spectrometry to confirm PRMT5 on SDS gels. After introducing an ATP treatment to wash off HSP70, an E.coli chaperone protein, the PRMT5 showed no contamination anymore. With the acquired clean protein crystallization trays were set up. In one condition crystals were big enough for harvesting, but too small to determine the protein structure by X-ray
crystallography. This technique relies on growing protein crystals able to diffract at atomic resolution under a stream of X-ray radiations. The prerequisite for the growth of protein crystals is a pure protein sample.
Before you lies my graduation thesis “Cloning and recombinant protein expression of the human Prtm5 gene, for the understanding of the histone arginine methyltransferase pathway of PRMT5”. This scientific report has been written as part of my graduation for my bachelor title for the major Forensic Laboratorial investigation at the Avans University of Applied Sciences in Breda, the Netherlands.
This research has been accomplished between March 2011 and July 2011 at the structural biology laboratory of professor E. di Luccio at the school of Applied Biosciences at the Kyungpook National University in Daegu, South Korea.
First off all, I would like to thank my research supervisor, Prof. dr. Eric di Luccio, for supervising my research. Dr. Masayo Morishita I would like to thank for everything she taught me. I also like to thank Daan Mevius for making it very easy for me to adjust to the lab and the Korean life. Furthermore I like to thank my family for their insightful comments regarding the grammar and spelling which helped me to improve my thesis.
And last, for my Korean co-workers, 놀라운 시간을 내주셔서 감사합니다! 미래에 행운을 빕니다!
(Thank you for an amazing time, good luck in the future!) Daegu, July 2011,
SUMMARY ...3 ACKNOWLEDGEMENT ...4 1. INDEX ...5 2. INTRODUCTION ...6 3. THEORETICAL BACKGROUND ...7 3.1EPIGENETIC CHANGE ... 7 3.2TRANSCRIPTION COREGULATORS ... 8 3.3PRMT FAMILY ... 8 3.4PRMT5 ... 9 3.5LINKED DISEASES PRMT5 ... 10
4. MATERIALS AND METHODS ... 11
4.1CLONING ... 11
4.2TA-CLONING ... 11
4.3ALKALINE LYSIS ... 12
4.4RESTRICTION ENZYME DIGESTION ... 12
4.5SEQUENCING ... 12
4.6SWITCHING VECTOR, FROM CLONING TO PROTEIN-EXPRESSION VECTOR ... 12
4.7SMALL SCALE PROTEIN EXPRESSION ... 13
4.8LARGE SCALE PROTEIN EXPRESION ... .15
4.9BRADFORD ASSAY ... 15
4.10MALDI-TOF MASS SPECTROMETRY ... 16
4.11IONIC EXCHANGE CHROMATOGRAPHY ... 16
4.12PROTEIN CRYSTALLIZATION ... 17
5. RESULTS AND DISCUSSION ... 18
5.1CLONING OF HUMAN PRMT5 GENE ... 18
5.2SEQUENCING. ... 18
5.3SWITCHING VECTOR FROM CLONING VECTOR TO PROTEIN-EXPRESSION VECTOR ... 19
5.4SMALL SCALE PROTEIN EXPRESSION ... 19
5.5LARGE SCALE PRMT5 PROTEIN EXPRESSION ... 20
5.6MASS SPECTROMETRY ANALYSIS ... 21
5.7ATP TREATMENT TO REMOVE CHAPERONE PROTEINS ... 21
5.8CRYSTALLIZATION OF PRMT5 ... 22 6. CONCLUSION ... 24 7. REFERENCES ... 25 8. GLOSSARY ... 26 9. APPENDIX ... 27 9.1PLASMID MAPS ... 27 9.2BRADFORD ASSAY ... 28 9.3CRYSTALITZATION RESULTS ... 27
For the acquisition of my Bachelor of Applied Science (BAS) title, for the major forensic laboratorial investigation, I performed a graduation research at the biochemistry lab of Professor Eric di Luccio, Kyungpook National University in Daegu, South Korea. The biochemistry lab is performing research on new epigenetic therapies of cancer. This project will focus on a nuclear-receptor-binding protein PRMT5, a transcription coregulator, which is a histone arginine methyltransferase (HMTase) enzyme. The focus of the Di Luccio lab is to better understand the histone code pathways and the epigenetic regulators, which modulate the gene transcription. The primary goal is to understand the misreading of the histone code leading to physiopathological conditions that include cancers, neurodegenerative and cardiovascular diseases. The laboratory studies the functions, structures, and mechanisms of transcription factors for developing new drugs against cancers, neurodegenerative diseases and stroke.
The research group major focus is on understanding the human NSD family and PRMT5 pathway. In recent study a connection was made between the overexpression of the NSD-family and PRMT5 proteins in cancer, especially breast, prostate en lung cancer.
My research focused on the cloning and recombinant protein expression of a pure PRMT5 protein for functional and structural studies. This will greatly benefit the understanding of the human histone arginine methyltransferase of PRMT5. This research is only a small part of a multiple year project to understand the functional roles, unravel the regulatory cross-talks events and to design drugs which activate/silence the gene of the Prmt5 gene.
The first goal was to successfully clone the human Prmt5 gene and ligate the gene into an E.coli expression vector. The second step was to optimize the overexpression of the pure PRMT5 proteins. The third step would have been to purify the proteins even further using ionic exchange
chromatography (IEC), so a study could be performed on the 3D structure of the pure proteins using crystallography. The analysis of protein structures provides fundamental insight into most
biochemical functions and consequently into the cause and possible treatment of diseases.
Therefore, the main objective of this laboratory project is to solve the unknown 3D structure of the human PRMT5 protein. The gold standard in protein structure determination is protein X-ray crystallography. Unfortunately, we found E.coli chaperone proteins contaminant present in our sample. Therefore the third step of the project was to identify and remove the chaperone protein. After removing the E.coli chaperone protein the PRMT5 protein showed no impurities anymore. The final step preformed, was the crystallization of the PRMT5 protein. Crystal growth was clearly visible but the conditions for crystallization have to be optimized for the growth of big, cubical shaped protein crystals. Due to the limited time frame another researcher from the Di Luccio research group will continue the project.
The first chapter of this report will shed some light on the theoretical background of the project, followed by an extensive explanation of the materials and methods, results and discussion, and the conclusion. The last three chapters of the report contain the references, a glossary and an appendix with supplementary data.
3.1 Epigenetic changes
Until recently, the general idea was that carcinogenesis was initiated by damaged DNA which makes cells grow out-of-control. Recent studies have revealed that cancer can also be created by epigenetic events. The gene expression profile of cells will change overtime due to external forces like stress, diet, age, weather or pollution. These external factors affect the status of chromatin density in the cells, leading changes in genes
expression, which may trigger the cellular transformation. The cellular transformation is the early stage of carcinogenic events turning “normal” cells into
“primed” cells (Fig. 1). Primed cells are cells that are not cancerous cells yet, but they can change very easily into cancerous cells. Primed cells do not have a different DNA sequence but they will change the DNA methylation. Which will result in a change of protein production of a cell.
Histones are the stage of diverse post-translational modifications that ultimately regulates the gene transcription. Lysine methylation is one prominent feature of the post-translational histone
modifications in the regulation of chromatin structure and function. All the covalent histone modifications contribute to regulating the diverse activities associated with the chromatin and may be referred as a language of covalent histone modifications or histone code. Lysine or arginine methylation, or any of the other histone modifications, can have both activating and repressive functions on the transcription. The various covalent histone modifications define an array of signals whose integration determines the fate of the transcription. Like DNA methyltransferases, changes in the activity level of histone deacetylases, acetylase, methyltransferases, demethylases modulate the transcription of genes by changing the packing of the chromatin that ultimately control the
transcriptional events. In turn, it also controls the cell-cycle progression and developmental events. In cancer cells, methylation is frequently detected in the promoter regions of genes that control processes. The silencing of genes that regulates these processes can therefore promote tumor formation and growth. Little is known about how genes become silenced, or inactivated, via
methylation during transformation. An increasing number of studies link the altered cellular activities of DNA methyltransferases, which add methyl groups to DNA at cytosine residues, to tumor cells initiation along with being associated with several developmental abnormalities. [1,2]
3.2 Transcription coregulators
Transcription coregulators (TCs) are a category of proteins contributing to the transcription factor complex of proteins. TCs are responsible for biochemical activities such as recruiting the RNA polymerase and modifying the chromatin. Chromatins are a combination of DNA, histones and proteins. TCs bind to a nuclear receptor and they play a key role in regulating transcription events. The function of TCs is to regulate the compaction levels of the DNA in the chromatin. Tight packing will inhibit the transcription events. At the opposite, looser packing will permit the binding of the large complex of proteins forming the transcription factor and activate the gene expression. Aside from the transcription factors, transcription coregulators are highly regulated and can be the primary targets of hormonal control and signal transduction. This therefore raises new opportunities for the development of novels therapeutics. [2,3,4]
3.3 PRMT family
Eleven members of human protein arginine methyltransferases (PRMTs) family have been identified so far. The last couple of years the interest in the PRMT-family has rapidly growing, because of the emerging role of arginine methylation in cellular processes like signaling, RNA processing, gene transcription and cellular function. Almost all PRMTs have shown to have enzymatic activity and to catalyze arginine methylation. PRMT proteins modify the characteristics of cellular proteins by transferring a methyl-group to the arginine amino acid in target proteins, which are important for many benign and malign cellular processes. [5,6]
The eleven human PRMTs differ in both length and sequence, the length of the proteins varies between 316 and 956 amino acids. PRMT proteins are localized in the nucleus, in the cytoplasm or even attached to the plasma membrane. All the members have a common catalytic
methyltransferase domain, which consist of a region of 310 amino acids and subdomains, which are important for binding to the methyl donor to a substrate. The transfer of methyl groups to the arginine substrate in target proteins is a process, which involves posttranslation modification and has been recognized as a very important step for the modification of protein and their subsequent function. [7]
The posttranslation of protein arginine methylation and demethylation is playing a vital role in cellular functions, like the chromatin regulation of histones, which plays a big role in the regulation of the gene transcription. In figure 2 is it shown how the PRMT enzymes remove one methyl group, from S-Adonosyl-L-methionine (AdoMet) to create S-adonosyl-L-homosystein (AdoHcy), the methyl group will be transferred to an acceptor molecule, which is the terminal nitrogen atom of the guanidinium side chain of an individual arginine in the target protein.
Figure 2: Chemical overview of lysine/arginine methylation by histone methyltransferase enzymes
As there are three nitrogen atoms in guanidinium, the transfer of one to three methyl groups can be catalyzed from the cofactor AdoMet. Most commonly creating mono and demethylation reactions of arginine like omega-N-monomethylarginine (MMA), symmetrical dimethylarginine (sDMA) or
asymmetrical dimethylarginine (dDMA). Trimethylation has so far only been documented in yeast protein. [6,7,8]
According to their methylation status the PRMT enzymes are classified into different groups. Type-I (PRMT1-3-4-6 and 8) PRMT enzymes catalyze the formation of MMA and aDMA, type-II (PRMT5-7 and 9)enzymes form MMA and sDMA, the remaining three proteins (PRMT2-10 and 11) the enzymatic activity as well as the methylation function have not been determined. [7,9]
Interestingly, some of them have implications for disease processes and are involved in certain cancer types, cardiovascular disease, multiple sclerosis and spinal atrophy. Arginine methylation is also linked to signal transduction, proliferation, transcription regulation and RNA splicing. [10,11]
Figure 3: the human PRMT family. A) The different members of the PRMT family are indicated. Only the longest isoforms are shown. All PRMTs have at least one conserved Catalytic Domain (gray). Some members have an additional domain (highlighted green). B) Phylogenetic analysis of the protein sequences of all PRMTs using guide-tree calculations. The length of the lines indicates the relationship and distance between the PRMT proteins. [5]
B
Figure 4: the PRMT5 gene can be found at chromosome 14q11.2
3.4 PRMT5
This research was focused on the human PRMT5 protein. The human Prmt5 gene can be found on chromosome 14q11.2. The enzyme it forms, PRMT5, EC2.1.1.125, is localized in the cytoplasm and is able to form homo-oligomers. Two isoforms are known with a length of 637 and 620 amino acids, respectively. PRMT5 plays a role in cytokine-activated transduction pathways. Cytokine is important for the regulation of the immune system. [13,14]
PRMT5 methylates histones H3 and H4 at the loci Arg8 and Arg3 respectively, which modify the DNA packing in the nucleosome and ultimately affect the gene transcription. This leads to changing the chromatin status. PRMT5 is an enzyme that belongs to the super-family of histone methyl
transferase, which can catalyze MMA or sDMA, with a preference for MMA to the arginine residue of the histone protein, generally on the histone tail. The opposite reaction is the histone demethylation that removes methyl groups on lysine and/or arginine on histone tails. Similarly, the histone
acetylation and deacetylation are the transfer and the removal of acetyl groups on lysine and/or arginine. Both the histone methylation and acetylation participate in the chromatin regulation and both play a key role in regulating the gene transcription. Lysine or arginine methylation, or any of the other histone modifications, can have both activating and repressive functions on transcription. All the covalent histone modifications contribute to regulating the diverse activities associated with the chromatin and may be referred as a language of covalent histone modifications or histone code [2,15,16,17]
A recent study has proven that the PRMT5 expression profile significantly contributes to the regulation of the circadian oscillation by modulating the regulation of expression and alternative splicing of a subset of clock-genes. The interplay between the circadian clock and the regulation of the alternative splicing by PRMT5 helps organisms to synchronize physiological processes with daily changes in environmental conditions. [18,19]
3.5 Linked diseases PRMT5
A recent trail of evidence link alterations or amplifications of PRMT5 to several cancerous events that include osteosarcoma, breast-, colon-, lung-, cervix-, salivary gland-, kidney-,prostate-, pancreas- and ovary cancers. In addition that protein arginine methylation affects numerous pathways,PRMT5 has been cited to potentially alter the effect of p53, a tumor suppressor protein crucial in multi cellular organisms. PRMT5 may become a suitable target for the manipulation of the p53 pathway.[20,21,22] Taken together, the recently proven role of PRMT5, as an epigenetic regulator of gene expression, along with the linked diseases render an enzyme which is very interesting for us to study. [2,21]
4.1 Cloning
The cloning of the human Prmt5 gene was performed by specific designed primers (Table 1), with an Xho1 and a Not1 restriction site, which the specific gene out of a human cDNA library amplified by poly chain reaction (PCR). For the amplification Diastar™ EF-TAQ high-fidelity DNA polymerase from the bioscience company Solgent co. ltd and Vent® high-fidelity DNA polymerase from New England BioLabs Inc. were used, the PCR program is visible in table 2.
After amplification, 5 µL of amplified DNA was visualized on a 1% agarose gel. The remaining DNA was purified using a QIAprep® Spin Miniprep Kit from QIAGEN. The DNA was eluted in 50 µL. 3 µL was loaded on a 1% agarose gel to visualize results.
Table 1: Designed primers used for cloning and sequencing of Prmt5. The forward and reverse primer were
designed with restriction enzymes site (underlined), and used for cloning and sequencing. PRMT5_652F, a forward primer, which starts amplification at 652 base pairs, was only used to verify the correct sequence.
Primer Restriction
enzyme Primer sequence
Forward Not1 5’ AGC GGC CGC ATG GCG GCG ATG GCG GTC GGG GG 3’
PRMT5_652F ---- 5’ TCT AAT CAT GTC ATT GAT CGC TGG 3’
Reverse Xho1 5’ CA GGC CGC TCA TAT ACC ATT GGC CTC GAG CCC TGC 3’
Table 2: PCR Program used for the amplification of the Prmt5 gene. The denaturation, annealing and
elongation cycle was repeated 35 times.
PCR process Time in minutes Temperature in Celsius Repeat Initialization 1.00 95 Denaturation 0.30 95 Annealing 0.30 55 35X Elongation 4.00 72 Final Elongation 10.00 72 Final Hold 999.00 4
4.2 TA-Cloning
Using the purified DNA amplified by EF-TAQ, an overnight 10 µL ligation at 4°C was performed with 10X T4 DNA ligation buffer, TA-Vector, and T4 DNA ligase. The next day the ligation reaction was added to 200 µL DH5α chemical competent cells, after 30 minutes incubation on ice the sample was heat shocked at 42°C for 1 minute, followed by 5 minutes incubation on ice. 800 µL SOC medium was added and the sample was incubated at 37°C in a shaking incubator. After an hour, the sample was spun down, at 2000 rpm for 5 minutes, after the spinning 100 µL supernatant was left on top of the pellet. The pellet was resuspended in the remaining 100 µL supernatant. The sample was plated onto a LB/Amp/IPTG/X-gal plate, for a blue and white selection. The plate was incubated overnight at 37°C. The following day, four white colonies were randomly picked and cultured in 5 mL LB +Amp overnight at 37°C in a shaking incubator.
B
4.3 Alkaline lysis
Plasmidic extraction, using the alkaline lysis method was performed on the randomly picked four colonies. 1.5 mL of the culture was spun down, 3 times at 8,000rpm, for 1 minute, in 1.7 mL
Eppendorf tubes after each spin the supernatant was aspirated and a new 1.5 mL culture was loaded. After the third spin the cells, 200 µL LB liquid was kept on top of the pellets. The cells were
resuspended in the liquid by vortexing. 200 µL fresh 0.2 M NaOH, 1% SDS alkaline lysis solution was added, and the samples were inverted 10 times and left at room temperature to incubate for 2 minutes. 400 µL 5M CH3CO2K (pH 4.8) was added to the tubes, and again the tubes were inverted 10
times. The cell debris was spun down at 13,000rpm for 20 minutes. After the spin, the supernatant was transferred to new 1.7 mL Eppendorf tubes, which already contained 800 µL cold 100% EtOH and 50 µL 3 M CH3COONa (pH 5.3). After vortexing, the 4 candidates were spun down at 13,000 rpm
for 15 minutes at 4°C. The supernatant was aspirated and 500 µL cold 70% EtOH was slowly added to the pellet. The tubes were gently inverted 3 times before all of the liquid was aspirated. The tubes were kept open for the pellet to dry. After all of the EtOH was evaporated, 50 µL 10 mM Tris-HCl (pH 7.5), 1mM EDTA (pH 8.0)+ RNaseA (1 µg/mL), TE+RNaseA solution was added to elute the plasmids.
4.4 Restriction enzyme digestion
Following the plasmid extraction step, a 10 µL enzymatic digestion was performed to confirm insertion of the Prmt5 gene in the TA plasmid. 0.1 µL of the enzymes Xho1 and Not1 were added, which cut the Prmt5 gene out of the TA-plasmid, to 1 µL 10X EzBuffer III, 0.1 µL 100X BSA( 10 mg/ml) 0.2 µL RNaseA (10 mg/ml) and 2 µL DNA. The samples were incubated at 37°C, after 2.5 hours of digestion they were taken out of the incubator and 1 µL digested plasmid, together with 10X loading buffer, was loaded on a 1% agarose gel.
4.5 Sequencing
4 positive candidates were cultured overnight in 5 mL LB+Amp at 37°C, the plasmids were extracted the next day, using HiYield™ Plasmid Mini Kit from the company RBC and eluted in 80 µL sterilized H₂O. 20 µL DNA of each candidate was sent away for sequencing, together with 3 primers. A new primer (Table 1, page 11) was designed to cover the complete DNA strain.
4.6 Switching vector, from cloning to protein-expression vector
20 µL DNA, of a perfect match candidate, 10X EzBuffer III, 100X BSA (10 mg/mL) and restriction enzymes Not1 and Xho1 were used for a 50 µL restriction enzyme digestion. In addition, 2 other digestions were setup one with 30 µL pTYB2 instead of DNA, the other with 30µL pTYB12 plasmid. The pTYB2 and pTYB12 are industrial designed plasmids for the benefit of protein expression, they have specific group, Intein, which will add a tag to the recombinant protein. The plasmids pTYB2 and pTYB12 are part of the protein affinity system IMPACT from New England BioLabs® Inc (Appendix 9.1, Page 27, Fig. 11 and 12)[23]. All the samples were incubated at 37°C. After 2 hours, 5 µL loading buffer was added and everything was loaded into 1% agarose gel. The Prmt5, pTYB2 and pTYB12 bands were cut out of the gel. A gel extraction was performed on the samples using the HiYield™ Gel/PCR DNA Mini Kit from the company RBC.
The acquired Prmt5 DNA was ligated using 10X T4 DNA ligation buffer, T4 DNA ligase and 1 µL DNA. For ligation into pTYB2 2 µL DNA was added, for the pTYB12 5 µL plasmid was added.
The samples were incubated overnight at 4°C, the next day the samples were taken out of the incubator and were incubate further on room temperature for 1 hour. In order to reduce the number of empty plasmid, the restriction enzyme EcoR1 was added to all the 10 µL ligation reactions, and the samples were incubated at 37°C for 2.5 hours. EcoR1 cuts between Not1 and Xho1 in the multiple cloning site of pTYB2, but is not capable to cut pTYB2 plasmids inserted with Prmt5.
The samples were transformed into DH5α chemical competent cells (see page 11). The cells were plated onto LB+Amp plates and incubated overnight at 37°C. From the plate several single colonies were randomly picked and cultured overnight in 5 mL LB+Amp at 37°C. A plasmidic extraction using alkaline lysis method was performed to get the plasmids out of the E.coli cells. On the acquired plasmids an enzymatic digestion was performed using 10 µL DNA and 5 µL master mix. The digestions were incubated for 2 hours at 37°C. Next, 1.5 µL loading-buffer was added in order to increase the density of the samples and allow them to sink into the wells. The loading-buffer contains a dye, making it possible to visualize migration of the samples. The samples containing the loading-buffer were loaded in a 1% agarose gel.
E.coli strains ER2566 and BL21 chemical competent cells, suitable for transformation and protein expression, were transformed with recombinant plasmids and incubated overnight at 37°C on LB+Amp agarose plates.
4.7 Small scale protein expression
For the protein expression we used the protein affinity system, Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) from the New England BioLabs® Inc [23]. From each of the 4 different plates (pTYB2-ER2566, pTYB2-BL21, pTYB12-ER2566 and pTYB12-BL21) 3 colonies were cultured overnight at 37°C in 5 mL LB+Amp. 1 mL of the overnight culture was inoculated in 75 mL LB+Amp. The cells were grown at 37°C until the optical density of the cells, at a wavelength of 600 nm (OD600), reached 0.6. The temperature of the incubator was cooled down to 15°C. As soon as the incubator and flasks were equilibrated on the new temperature, IPTG was added to create a 250 µM final concentration. IPTG is a highly stable synthetic analog of lactose. Recombinant protein
expression in pTYB plasmids is under the control of a lactose operon. The cells were incubated for 4 hours before spinning them down in conical tubes at 3,400 rpm for 10 minutes at 4°C. In order to help breaking open E.coli to extract recombinant proteins, a freeze-thaw method was used, thus, the pellets were stored overnight at -80°C
The cells were thawed and resuspended in 1 mL, 20 mM TRIS-HCL, 500 mM NaCl, 1 mM EDTA, IMPACT buffer +0.1% Triton X-100 (pH 8.5) + 1 mM PMSF, before transferring them to 1.7 mL Eppendorf tubes. Surrounded in ice, each sample was sonicated at 17-18 AMPS, 4 PULSER, for two times 1 minute. The samples were spun down at 13,000 rpm, for 10 minutes, at 4°C. The supernatant was transferred to new 1.7 mL Eppendorf tubes, 50 µL 50% chitin beads slurry was added to the soluble proteins. Chitin beads are part of the IMPACT system, and it will bind to the tag-protein Intein. The samples were incubated on ice for 10 minutes, before they were spun down at 10,500 rpm for 24 seconds.
After the spin the supernatant was aspirated and the chitin beads were washed 10 times with 1mL IMPACT buffer +0.1% Triton X-100 (pH 8.5). After the last wash all of the remaining supernatant was aspirated. 30 µL 1X SDS sample buffer containing 2% β-mercaptoethanol.
Using pTYB plasmids, a cysteine residue in the protein tag is adjacent to the first residue of the recombinant protein. Therefore, a thiol-containing agent such as 2% β-mercaptoethanol is added, to cleave the Intein-tag off the recombinant protein and left for a 10 minute incubation at room temperature, before applying a 5 minute heat shock at 95°C. The 95°C heat shock in SDS sample buffer denatures native proteins to individual polypeptides. The SDS detergent wraps around the polypeptide backbone, and in this process all the polypeptides will denature in a rod like structure with a uniform negative charge density, which makes proteins run linear on a SDS-PAGE gel to their molecular weight. In combination with β-mercaptoethanol the heat shock also denatures the proteins by reducing disulfide linkages, thus overcoming tertiary folding.
30µL from each sample was loaded on a 10% SDS-PAGE gel. The gel was stained for 1.5 hours using Coomassie™ blue R-250 Staining solution from Imperials Chemical Industries and destained for 2-3 hours with Coomassie blue R-250 Destaining solution.
4.8 Large scale protein expression
As a result of the small scale protein expression, in combination with the cleavage of the Intein-tag in the different vectors, a candidate was chosen for a large scale 12 liter expression. (Table 3, page 15) The E.coli transformant was cultured overnight at 37°C in 100 mL LB+Amp in a shaking incubator. The next morning 10 mL of overnight culture was inoculated in nine 2-L flasks each containing 1.33 L LB+Amp. The cells were grown at 37°C until the OD600 was 0.25, the temperature was cooled down to 15°C. An hour later when the temperature had reached 15°C, IPTG was added to acquire a 250 µM final concentration per flask. The cells were incubated for 5 hours before they were harvested using 250 mL Nalgene bottles, which were spun down at 6.000 rpm, for 8 min each time. All cells were scraped together and frozen at -80°C.
Next the cells were thawed and resuspended on ice for 1 hour in 150 mL IMPACT buffer + 0.1% Triton X-100 + 1 mM PMSF. PMSF is a serine protease inhibitor. Just before sonication 1.5 mL PMSF 100 mM was added to the suspension. The cells were sonicated on ice at 17-18 AMPS, 4 PULSER for fifteen times 2.5 minutes. The sonicated cells were spun down at 4,000 rpm for 30 minutes at 4°C. The lysate was transferred to new 250 mL Nalgene bottle and 1.5 mL 100 mM PMSF was added again before another sonication using the same settings as earlier, now only for five times. After the sonication the cell remaining debris was spun down at 6,000 rpm for 30 minutes at 4°C. The lysate was filtered to remove any remaining cell debris or other foreign particles, using a 0.45 µM filter disc attached to a 30 mL syringe. The filtered lysate was slowly loaded onto a chitin beads column kept at 4°C. After all of the lysate was applied, the whole column was washed overnight with IMPACT buffer +0.1% Triton X-100 with 1.5 L [24].
The next morning the Triton was removed from the column by washing the column with 250 mL IMPACT buffer. To remove any possible bacterial chaperone protein, the column was washed with a total of 150 mL IMPACT buffer + 10 mM ATP + 2.5 mM MgCl₂ [25]. After slowly applying 75 mL the valve was closed to let it soak. After an hour the remaining 75 mL was washed over the column.
To remove the ATP, the column was washed with 250 mL IMPACT buffer. To start the cleavage of the proteins 7.5 mL IMPACT buffer + 45 mM β-mercaptoethanol was washed over the column. OD280 was checked to verify of the added amount was sufficient. Depending on the cleavage properties (Table 3) the samples were left 2 to 5 days on the column to maximize the amount of proteins which can be cleaved.
The cleavage of the recombinant proteins from the Intein-tag depends of the amino acid adjectant to the N-terminus cysteine residue of Intein. The bod between the Intein tag and the recombinant protein can be cleaved by a thiol-containing agent, the cleavage can be looked up in the IMPACT cleavage chart [23]. In the case of PRMT5 the amino acid leucine is attached to Intein tag at the C-terminus of the gene and methionine at the N-C-terminus. Leucine has a reported 50% in vivo cleavage at 15°C, which means, that 50% of the proteins expressed in the pTYB2 vector lost the attachment to their Intein-tag. In the pTYB12 vector, the methionine has no reported in vivo cleavage.
Table 3: PRMT5 cleavage chart.
Intein tag Plasmid Amino acid Cleavage after
40 hours, 4 °C PRMT5 C-terminus pTYB2 Leucine (L) 75-90 %
PRMT5 N-terminus pTYB12 Methionine (M) 60-90 %
The proteins were eluted by 45 mL IMPACT buffer and concentrated by 50 mL 10k Amicon® Ultra Centrifugal Filters from the company Millipore™. For concentrating the sample they were spun down at 3,400 rpm 4°C at 20 minutes, after each spin the flow through was discarded. The sample was washed with 70 mL IMPACT buffer to remove the β-mercaptoethanol. The samples were
concentrated down to 400 µL. 2 µL and 6 µL of proteins were diluted in 1X SDS sample buffer to a final volume of 30 µL after applying a 5 minute 95°C heat shock they were run on a 10% SDS-PAGE gel.
4.9. Bradford assay
To determine the exact amount of proteins, a Bradford protein assay, was performed using a Bradford kit from Bio-Rad Laboratories Inc. The bovine serum albumin (BSA) standards used for the assay were diluted with IMPACT buffer (table 4). 995 µL 1X Bradford Dye Reagent was added to twelve 1.7 mL Eppendorf tubes. To the first 6 tubes 5 µL standard A to F was added. From the remaining six tubes two were filled with 5 µL IMPACT buffer, as blank, two were filled with 5 µL 40% pTYB2 sample. And the last two were filled with 5 µL 20% pTYB12 sample. The samples were diluted with IMPACT Buffer. All the samples were incubated for 5 minutes at room temperature, before the OD was measured at 595 nm. The results were plotted against the standard and the protein
concentration was calculated.
Table 4: BSA standard used for Bradford assay Standard BSA concentration in µg/mL
A 0 B 125 C 250 D 500 E 1000 F 2000
4.10 MALDI-TOF Mass spectrometry
To lever ambiguity about the nature of proteins separated on SDS gels, the band-samples were analyzed by a MALDI-TOF mass spectrometer. The separate protein bands were cleanly cut out of the gel, in a sterilized, non-keratin working environment in order to downgrade the risk of keratin
contamination. The small cut fragments of the SDS-PAGE gel were put in a 1.7 mL Eppendorf tube and they were destained by washing the samples seven times with 100 µL 500 mM NH₃HCO₃:ACN (6:4 ratio) destaining solution with an incubation time of 10 minutes after each wash. After the last wash the samples were incubated overnight at room temperature. The next morning the destaining solution was aspirated and the tube with the sample was tightly covered with parafilm, the covered top was penetrated by a needle. The samples were placed in a -80° freezer. When the samples were frozen solid they were placed for 2 hours in a freeze dryer. After 2 hours 20 µL trypsin, a serine protease, was added to the samples, which cleaved the proteins into small peptide fragment by cutting the basic lysine and arginine residues.
The samples were incubated on ice for 30 minutes before they were incubated in a water bath at 37°C, for 18 hours. After the incubation the samples were spun down at 12,000 rpm for 1 minute and all of the trypsin was transferred to a new tube. 20 µL 3% TFA, 60% ACN extraction buffer was added and the samples were incubated on ice again for 30 minutes. The extraction buffer was transferred to the tube filled with trypsin. The gel sample was washed again by pipetting 20 µL new extraction buffer up and down. The extraction buffer was transferred to the trypsin tubes, the trypsin tube was vortexed before frozen at -80°C. The frozen sample was freeze-dried until the sample was
concentrated to 20 µL. On the MALDI-TOF sample plate, 1 µL α-Cyano-4-hydroxycinnamic-acid was added as a matrix, and 1 µL sample was added, the plates were left to dry.
The plate was inserted into a Voyager DE™ -STR Matrix-Assisted Laser Desorption/Ionization with a Time Of Flight mass spectrometer (MALDI-TOF) from the manufacturer Applied Biosystems. The Data Explorer software setting were set on: instrument mode – reflector/Bin size 0.5, grid - 64%,
shots/spectrum – 150, Delay – 100 nsec, laser intensity - 2200 and mass range – 800 to 3000. The system was calibrated on an angiotensin-I standard. The recorded data was analyzed using MoverZ software from the company Genomic Solutions®. The results were cross-referenced to the MS-FIT database from the University of California.
4.11 Ionic Exchange Chromatography
As PRMT5 needs to be highly pure for crystallography (>98% purity), an additional purification step using ionic exchange chromatography was needed. In order to maximize the binding of PRMT5 onto either a strong anionic or cationic exchange column, the buffer was switched to a buffer with a pH that maximized the negative charges of the protein. Also the buffer has a very low ionic strength to facilitate the binding of the protein on the column. For the switching of the buffer centrifugal filter units were used which are permeable to only small molecule and impermeable to proteins of 30 kDa and above. 2 mL, 30 k Amicon® Ultra Centrifugal Filters from the company Millipore™ were used by washing it ten times with 50 mM TRIS buffer (pH 8.5) at 7,000 g for 1.45 minute. An ÄKTA prime ionic exchange chromatograph, equipped with a Q strong anion exchange resin column and a fraction collector, from the company Amerham Biosciences, was used. Solvent A was 50 mM TRIS buffer (pH 8.5), solvent B was 50 mM TRIS, 0.5 M NaCl (pH 8.5).
The setting for the fraction collector was set up to collect every 1 mL in a new 1.7 mL Eppendorf tube. The others settings are visible in table 5. After examining the chromatograms, the different peaks were pulled together. The pulled fractions were concentrated and the buffer was switched again, by washing the fragment ten times with a 20 mM TRIS buffer (pH 5.5) in 2 mL 30k Amicon® Ultra Centrifugal Filters from the company Millipore™. The results of the pulled fractions were visualized on a SDS-PAGE gel.
Table 5: Ionic Exchange settings
Flow in mL % A % B Flow (ml/min)
0.00 100.0 0.0 4.000
20.00 0.0 100.0 4.000
37.00 0.0 100.0 4.000
38.00 100.0 0.0 4.000
4.12 Protein crystallization
After acquiring the pure PRMT5 fraction, the protein concentration was measured using a Bradford assay. If the proteins were pure enough after the primary purification with the IMPACT system the buffer still needs to be switched to a 20 mM Tris buffer (pH 5.5). Before setting up crystallization trays, the sample was concentrated to a final protein concentration between 7 and 10 mg/mL. For the crystallization of the proteins, by hanging drop method, 24 well crystallization VDX trays from Hampton research were used, each well was loaded with 500 µL with a different Crystal Screen™ buffer from the company Hampton research. On the rim of each well mineral oil was applied. On a 22 mm siliconized glass circle cover slides, from Hampton research, 1 µL of Crystal Screen™ buffer was added. The buffer added to the glass plate was identical to the buffer already present in the well. 1 µL of samples was added to the 1 µL buffer on the glass plate and the drop was mixed by pipetting the droplet several times. Next the glass was inverted using a tweezer and placed on top of the corresponding well. The mineral oil on the rim acts as an airtight seal between the glass plate and well, so the buffer and the droplet can have interaction in their own specific environment. The samples were incubated in a temperature controlled environment at 13°C, and checked daily with a Nikon Eclipse 80i microscope. Formed crystals were harvested and stored in liquid nitrogen at -196°C, until further use as either microseed, for the catalization of PRMT5 protein crystals growth in future samples or for determining the crystal structure of by protein X-ray crystallography.
5.1 Cloning of human Prmt5 gene
After amplification of the human Prtm5 Gene by EF-TAQ polymerase and Vent polymerase, the results were analyzed by a 1% agarose gel (Fig. 5A) and the amplified PCR-product was purified. These results were also put on a 1% agarose gel (Fig. 5B). The size of the human Prmt5 gene is 1911 base pairs. In both figures, bands are shown at the expected 1.9 kilobase pair using the 2-Log DNA Ladder. The concentration of DNA was calculated by absorbance, readings were performed at 260 nm. The concentration of DNA amplified by Vent polymerase was 120 ng/mL, the EF-TAQ has a concentration of 60 ng/mL.
5.2 Sequencing
The DNA amplified by EF-TAQ polymerase was ligated into a TA vector. After E.coli transformation and a blue and white selection on LB+Amp+IPTG+X-gal plates, 8 colonies were checked if they had the correct insert. The results show (Fig. 5C) that there was insertion in 7 out of the 8 samples. Samples 1 to 4 were sent away for sequencing. The results are shown in table 6.
Table 6: Sequencing results of the Prmt5 gene Candidate Sequencing results
1 1 Silent mutation TCA TCG
2 Perfect match
3 2 Mutations
4 1 Mutation
Figure 5: Cloning of human Prmt5 gene into TA-Vector
A) PCR amplification results of Prmt5(1.9 kb) using Vent polymerase and EF TAQ polymerase. B) PCR results of Prmt5 (1.9
kb)using Vent polymerase and EF TAQ polymerase after purifying DNA using QIAprep Spin Miniprep Kit C) Restriction enzyme digestion of Prmt5 (1.9 kb) in TA vector(2.7 kb). Insertion of Prmt5 visible in all samples except sample #7
5.3 Switching vector from cloning vector to protein-expression vector
On the candidate with the 100% correct nucleobases sequence, the plasmid was switched from TA- vector, used for cloning, to pTYB2 and pTYB12 plasmids which maximizes the protein expression. The Prtm5, pTYB2 and pTYB12 fragments were cut out of a 1% agarose gel after restriction enzyme digestion (Fig. 6A). The Prmt5 gene was ligated into the pTYB2 and pTYB12 vector (Fig. 6B and 6C).
5.4 Small scale protein expression
The Prmt5-pTYB2 and Prmt5-pTYB12 plasmids were transformed into chemical competent E.coli cells BL21 and ER2566 strains, suitable for protein expression. A 75 mL small test scale protein expression was performed on 3 samples from each condition samples (Fig. 7A and 7B). With the test expression it is able to show if a E.coli strain can express human proteins. The small scale expression can also be used to optimize the protein expression parameters, before scaling up to a 12 L expression. The expression of the pTYB2 samples is shown only in Prmt5-pTYB2-ER2566#1, a smear is visible from 90 kDa to 60 kDa, were a single band at 72 kDa was expected. For the pTYB12 expression is clearly shown in Prmt5-pTYB12-ER2566#3 and Prmt5-pTYB12-BL21#1. They show a single band at the expected protein weight of 72.7 kDa.
B
A
C
C
Figure 6: Switching Prmt5 gene from TA-vector into pTYB2 and pTYB12.
A) Gel extraction after restriction enzyme digestion of Prtm5 (1,9 kb) in TA-Vector (2,7kb). Also gel extraction was performed on pTYB2 and pTYB12 vector B)Restriction enzyme digestion of Prmt5 (1.9 kb) in pTYB2 plasmid(7.4 kb). Insertion of Prmt5 visible in sample #2 C) Restriction enzyme digestion of
Prmt5 (1,9 kb) in pTYB12(7,4 kb)
plasmid. Insertion of Prmt5 visible in samples 1,4, 5, 8 and 10 - 16
↑ Figure 7: Small scale expression of PRMT5(72.7 kDa) in pTYB2 and pTYB12 plasmid, in ER2566 and BL21. A)
Small scale pTYB2 shows expression in ER2566 #1, but no clear band visible. B) protein expression of small scale rmt5-pTYB12 is shown in sample ER2566 #3 and BL21#1 with nice sharp bands at 72 kDa.
5.5 Large scale PRMT5 protein expression
12 L large scale expressions were performed on both the pTYB2-ER2566#1 and the pTYB12-BL21#1 (Fig. 8). The PRMT5-pTYB2 sample shows a bright band at 60 kDa, and a fade band at 72 kDa. The Prmt5-pTYB12 samples show a similar profile except for an extra band at 100 kDa.
A Bradford assay was performed to measure the exact protein concentration (Appendix 9.2, Page 28, table 7 and 8, Fig. 12). The pTYB2 sample has a final volume of 410 µL with a protein
concentration of 3.3 µg/µL, the pTYB12 sample has a protein concentration of 3.9 µg/µL and a final volume of 400 µL. This is also on the SDS-PAGE gel (Fig. 8) where the bands of the Prmt5-pTYB12 are substantial brighter than the Prmt5-pTYB2 expression. This is most probably the result of the ~50% in vivo cleavage in the pTYB2 sample (Table 3, page 15). The protein production yield of the Prmt5-pTYB2 is 112.8 µg/L culture and the yield of Prmt5-pTYB12 is 131.3 µg/L culture (Table 8).
Although the expected molecular weight of the PRMT5 protein is 72.7 kDa. It is highly likely that the protein visualized at 60 kDa is the overexpressed PRMT5 despite being 12 kDa smaller than expected. It might be the result of cleavage by protease enzymes which can hydrolyze peptide bonds, which link the amino acids together. An
anti-protease treatment was applied but it is likely PRMT5 cleaved by residual E.coli proteases. Figure 8: SDS-PAGE gel of 12 L human PRMT5 (72.7 kDa) recombinant protein expression.
The pTYB2 (left) and the pTYB12 (right) show a similar protein profile. The pTYB12 sample shows, as expected brighter bands. The bright band at 60 kDa is most likely cleaved PRMT5 protein.
The band at 72 kDa is either the non-cleaved PRMT5 or an E.coli chaperone protein, most called HSP70, with a protein weight of 70 kDa. Expressing higher eukaryote proteins into E.coli is a challenge. E.coli might have issues with folding human protein properly. Therefore, bacterial chaperone gets stuck to the recombinant protein. Also if the recombinant protein is unstable, for instance a misfolded protein, the bacterial chaperones dock around. The 100 kDa contamination is a unknown contamination and it is expected to be removed by purifying the protein samples with ionic exchange chromatography.
5.6 Mass spectrometry analysis
In order to further identify the protein bands at both 60 and 72 kDa a peptide mass fingerprinting using MALDI-TOF mass spectrometry was performed after trypsin digestions of the samples (Fig. 8). The results, analyzed by computer software from MoverZ and cross-referenced against the MS-FIT database of the University of California, showed in both the samples at 60 and 72 kDa, a mixture of chaperone proteins from E.coli and human transcription factor protein, most-likely PRMT5. The E.coli chaperone protein detected at 72 kDa was identified as HSP70 protein, also a human protein was
detected. Unfortunately, due to the resolution, no reliable confirmation was possible for this protein. Similarly, in the 60 kDa sample two proteins were detected. One could be identified as an E.coli chaperone, the other one was identified as human protein. In both the 60 and 72 kDa samples, the human protein identified belong to the transcription family super-family of protein.
Therefore, it is very likely PRMT5. Unfortunately, quantification or further identification was not possible due to the resolution limits of our peptide mass fingerprinting data.
5.7 ATP treatment to remove chaperone proteins
To get rid of chaperones contaminations, which are ATPase enzymes, we introduced an extra wash step with ATP and MgCl₂ specifically designed to help release protein chaperones from the recombinant proteins. The ATP wash will remove the chaperone protein by providing the docked chaperone new energy to get itself detached. The results gathered from the chaperone treatment are very positive (Fig. 9). The HSP70, a chaperone protein, is clearly visible at 70 kDa, together with another unknown chaperone ATPase protein at 140 kDa, which could possibly be the result of protein-protein binding between two HSP70 proteins. Due to the heat shock procedure this is unexpected and it seem like it is another ATPase enzyme. The single band on the PRMT5 gel means it was pure enough for setting up crystallization trays (Fig. 9). Although no contamination was detected, it is recommended for the future to perform a purification by ionic exchange chromatography(IEC). It is expected that the IEC, a secondary purification step after the IMPACT system, purifies the PRMT5 protein even further. Figure 9: SDS-PAGE gel of human
PRMT5 (72.7 kDa) after ATP/MgCl₂ treatment. Left of the marker 10
mL of ATP/MgCl₂ wash buffer was collected after a 1 hour soak, and concentrated to 50 µL. Right of marker, the PRMT5 sample shows no other bands except the expected ~60 kDa human PRMT5. The reason the 5 µL shows a fainter band than the 2µL is because a trainee made a mistake loading that sample.
5.8 Crystallization of PRMT5
The analysis of protein structures provides fundamental insight into most biochemical functions and consequently into the cause and possible treatment of diseases. Therefore, the main objective of this laboratory project is to solve the unknown 3D structure of the human PRMT5 protein. The gold-standard in protein structure determination is protein X-ray crystallography. This technique relies on growing protein crystals able to diffract at atomic resolution under a stream of X-ray radiations. The prerequisite for the growth of protein crystals is a pure protein sample.
On the pure PRMT5 proteins, acquired after the ATP wash, another Bradford assay was preformed, distinguishing the protein concentration on 1.0 mg/mL in a volume of 450 µL. For crystallization the protein concentration has to be between 7 and 10 mg/mL in average. The sample was concentrated down to 54 µL with a new concentration of 8.6 mg/mL. 1 µL sample was mixed with 1 µL buffer on a siliconized glass plate, in total 54 times a 1 µL drop of sample was combined with a different
crystallization buffer.
After a week of daily checking the growth under the microscope, in 19 out of the 54 samples precipitation was visible, 7 other situation look promising of forming crystals. Three conditions gave so called interesting “hit”, one condition showed the formation of needle crystals, one condition provided small crystals in a combination with quasi crystals, and one condition changed overtime from clear drop to microcrystals to small needle crystals to a clear drop again.
The three conditions were a hit was detected used the following buffer, for the needle crystal (A) 0.2 M Ammonium sulfate, 0.1 M Sodium trihydrate pH 4.6, 25% w/v polyethylene glycol 4,000, for the small crystals and quasi crystals (B) 2.0 M Sodium chloride, 10% w/v polyethylene glycol 6,000 and for the changing crystals (C) 0.5 M Sodium chloride, 0.01 M magnesium hexahydrate, 0.01 M
hexadecyltrimethylammonium bromide. The three situations will be referred from this point onward as condition A, B and C.
The crystallization of condition A is the most promising, on day 1 small needle crystals were visible, between day 1 and 2 the growth was spectacular and big needle structures were formed. The growth of crystals stabilized after day 4. (Fig. 10 and Appendix 9.3, Page 29, Fig. 14)
Figure 10: Crystallization of human PRMT5 in condition A. A) Needle crystals visible, formed in a buffer(condition A) of 0.2 M Ammonium sulfate, 0.1 M Sodium trihydrate pH 4.6, 25% w/v polyethylene glycol 4,000. Image A is the 2 µL seen through a Nikon Eclipse 80i Nikon Plan Fluor 4X/0.13 WD 17.1 lens. B) Close up of the drop in A, seen through a Nikon Fluor Plan 10X/0.30 DIC L/N1 WD 16 lens.
In condition B crystals were visible from day 2 onward, close-ups of the crystals show a combination of small crystals and quasi crystals. After day 2, no significant growth was recorded anymore. (Appendix 9.3, Page 30, Fig. 15)
In condition C microcrystals were visible on day 1, on day 2 no crystals were visible anymore, from day 4 to day 5 small needle crystals were present in the sample drop. On day 6 the small needle crystals disappeared again. On day 7 the droplet remained completely clear of crystals. (Appendix 9.3, Page 31, Fig. 16)
From the three conditions, condition A was the only situation which provided big enough crystals to be harvested, although their length of 0.25 mm, they were too small, overlapped with each other and misshaped for determining the protein structure by X-ray crystallography analysis. Single and singular crystals are a prerequisite for protein X-ray crystallography. The current PRMT5 crystals are not usable for data collection as the crystallization conditions need to be further optimized in order to growth single crystals large enough. The 0.25 mm crystals can be harvested and be broken up, these crystal seeds can help to catalyze the growth of PRMT5 crystals in future crystallization to catalyze the growth of PRMT5 crystals. For the future crystallization, condition A will be optimized. The optimization of “hit” conditions is necessary to optimize the growth of crystal structures for the gain of a single crystal with a size of at least 0.5 mm in each dimension.
The cloning and insertion of the Prmt5 gene into the E.coli expression vector pTYB2 and pTYB12 was successfully achieved. The optimum PRMT5 protein expression conditions were achieved by using the pTYB12 plasmid with the BL21 E.coli strain as host cells.
The SDS-PAGE protein gel performed on PRMT5 purified by affinity chromatography following a 12 L culture showed several bands. In order to lever ambiguities and identify proteins on the SDS-PAGE gel, we extracted the proteins from the gel and sequenced them by peptide fingerprinting using MALDI-TOF mass spectrometry. Mass spectrometry analysis shown that the proteins samples contain a mixture of human and E.coli proteins. However the human protein could not clearly be identified. But the human sequenced Prmt5 gene was ligated into a pTYB12 plasmid and under the dependence of a strong inducible promoter region, which renders highly likely the presence of PRMT5 protein. The E.coli protein was identified as the HSP70 protein. HSP70 is an ATPase protein. Thus we introduced and optimized an ATP/MgCl₂ washing step to remove the chaperone protein from the sample.
Also it is shown that the PRMT5 protein most probably been cleaved by protease enzymes, which make it run at ~60 kDa instead of the 72.7 kDa.
After removing the chaperone protein the level of purity of the PRMT5 protein was sufficient for setting up crystallization trays. In three condition crystals started forming, in one condition the crystals were big enough to be harvested. However, due to their twinned and overlapping nature, our current PRMT5 needle shape crystals will not be used for determining the protein structure of PRMT5 by X-ray crystallography. The conditions have to be optimized for the growth of non-overlapping crystals of at least 0.5 mm in each dimension. Due to the limited time frame, another researcher from the Di Luccio research group will optimize the crystallization condition and further continue the project. Nevertheless, the obtention of initial crystals hits is an important step in the project.
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[2] Epigenitic therapy of cancers and histone
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[25]Removal of DnaK contamination during fusion protein
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aDMA Asymmetrical dimethylarginine.
Amp Ampicillin, an antibiotic.
BL21 E.coli strain, used for the expression of protein.
BLAST Basic local alignment search tool, bioinformatics program to check sequences.
Bp Base pare, unit to express the size of a DNA-strain.
Chitin Beads Part of the IMPACT system, the beads bind to the Intein tag.
DH5α E.coli strain, used for the cloning of genes.
E.coli Bacteria used as a host for plasmids.
EF TAQ A DNA polymerase used in PCR amplification, adding an A extra to the amplified
fragment.
ER2566 E.coli strain, used for the expression of protein
HMTase Histone Methyltransferase are enzymes that catalyze the transfer of one to
three methyl group.
IEC Ionic exchange chromatography, a system to separate protein by their ionic
composition.
IMPACT system Protein affinity system by NEB (New England Biolabs)
Intein A tag protein, part of the pTYB2/pTYB12 plasmid, which attaches to the
recombinant protein, the Intein tag binds to Chitin beads.
kDa Unit to express the molecular weight of protein
KNU Kyungpook National University, university in Daegu, South Korea. Place where
the research has been performed.
LB Broth Medium Luria-Bertani Broth medium, a rich medium to grow E.coli cells in.
MALDI-TOF Matrix-Assisted Laser Desorption/Ionization - Time Of Flight is a soft ionization mass spectrometry method.
MMA Omega-N-monomethylarginine
NSD-family Three nuclear receptor binding SET domain proteins
OD Optical density, a synonym for the absorbance in spectroscopy
PCR Polymerase chain reaction, methods used for the amplification of DNA
Prmt5 Human gene found on chromosome 14, PRMT5 refers to Protein aRginine
MethylTransferase event number 5.
pTYB12 Plasmid used for recombinant protein expression with an Intein tag at the
N-terminus of the multiple cloning site, part of the IMPACT system.
pTYB2 Plasmid used for recombinant protein expression with an Intein tag at the
C-terminus of the multiple cloning site, part of the IMPACT system.
sDMA Symmetrical dimethylarginine.
SDS-PAGE SDS-PAGE gel is technique widely used in genetic to separate proteins by molecular weight.
TA-vector A high copy plasmid used for cloning of genes. Has a sticky end with a T on both side on the open plasmid.
TCs Transcription coregulators interact with transcription factors to either activate
or repress transcription.
9.1 Plasmid maps
Figure 11: pTYB2 Plasmid
Figure 13: Bradford protein assay graph, the R² showed a result of 0.993
9.2 Bradford Assay
Table 7: OD results from the Bradford assay BSA standards. The results are plotted in figure 12.
µg/mL O.D. 0.0000 0.0000 0.1250 0.0350 0.2500 0.0540 0.5000 0.1640 1.0000 0.2800 2.0000 0.5230
Table 8: protein concentration of pTYB2 and pTYB12 from a 12 L large scale expression Sample name Sample
1 Sample 2 Concentration in mg/mL Concentration µg/mL Volume in µL Yield µg/ L Culture blank 0.000 -0.001 0.000 0.000 --- --- PRMT5 pTYB2 2 µL 0.366 0.340 3.30 3297.83 410 112.8 PRMT5 pTYB12 1 µL 0.207 0.214 3.94 3933.11 400 131.3 y = 0.2676x R² = 0.993 0,0000 0,1000 0,2000 0,3000 0,4000 0,5000 0,6000 0,0000 0,5000 1,0000 1,5000 2,0000 2,5000 O. D . 595 mg/mL
Bradford Assay
9.3 Crystallization results
On the next three pages the growth progress of the three best crystallization conditions are visualized in figure 14, 15 and 16.
Figure 14 (A): Daily crystallization overview of the growth of needle crystals. The buffer for
condition A is 0.2 M Ammonium sulfate, 0.1 M Sodium trihydrate pH 4.6, 25% w/v polyethylene glycol 4,000. Day 1 shows some very small crystals, on day 2 the crystals showed a spectacular growth. Day 4 shows slight larger crystals, from day 4 onward the crystals do not show extra growth. The scales of the pictures are the same, images were taken by a Nikon DS FiC1 camera, the microscope lens used was the Nikon 10x/0.30 DIC L/N1 WD 16.
Figure 15 (B): Daily crystallization overview of the growth of small crystals and quasi crystals.
The buffer for condition B is 2.0 M Sodium chloride, 10% w/v polyethylene glycol 6,000.
Precipitation is visible on day 1, on day 2 tiny crystals and quasi crystals were formed. from day 4 onward the crystals don not show extra growth. The scales of the pictures are the same, images were taken by a Nikon DS FiC1 camera, the microscope lens used was the Nikon 10x/0.30 DIC L/N1 WD 16.
Figure 16 (C): Daily crystallization overview of the growth of different crystals in buffer B. The
buffer for condition C is B3A2: 0.5 M Sodium chloride, 0.01 M magnesium hexahydrate, 0.01 M hexadecyltrimethylammonium bromide. Day 1 shows many microcrystals, on day 2 all the crystals disappeared. On the pictures of day 4 and 5 some small needle crystals show, on day 6 the needle crystals disappeared again. On day 7 nothing changed. The scales of the pictures are the same, images were taken by a Nikon DS FiC1 camera, the microscope lens used was the Nikon 10x/0.30 DIC L/N1 WD 16.
