Purification, functional characterization andcrystallization of the MntR manganese sensorfrom Saccharopolyspora erythraeaMalin Svensson

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Purification,

functional

characterization

and

crystallization

of

the

MntR

manganese

sensor

from

Saccharopolyspora

erythraea

Malin

Svensson

Degree project inbiology, Master ofscience (2years), 2020 Examensarbete ibiologi 30 hp tillmasterexamen, 2020 Biology Education Centre

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Metals are important for many cellular processes, they participate as cofactors in gene transcription, oxidation-reduction reactions, and in protection against oxidative stress. MntR is a manganese sensing protein from Saccharopolyspora erythrea that senses the concentration of manganese and regulates the expression of manganese transporter genes to maintain manganese homeostasis. These transporters are very important for the cell since manganese cannot be synthesized or degraded by the cell itself, so it must rely on the transporters to import and export manganese from the environment. For the cell to import enough metal, to maintain cellular processes but not so high so it becomes toxic, it needs metal sensing regulators like MntR. When there are sufficient levels of manganese ions in the cell, MntR is activated by the metal ions, binds to DNA and represses gene transcription of metal ion transporters. When the levels of manganese ions are low in the cell, DNA is detached from MntR and transcription of metal ion transporter genes can continue. The aim of this project was to optimize the protocol for expression and purification of MntR and to characterize MntRs metal-dependent DNA-binding activity. The result shows that MntR can be expressed and subsequent purified with high yield and purity. The result also shows MntRs metal-dependent DNA-binding activity. This report aims to provide information for future experiments and works on expression, purification and characterization of MntR.

List of abbreviations

IMAC- Immobilized metal ions affinity chromatograph SEC- Size exclusion chromatograph

IEX- Ion-exchange chromatograph CEX- Cation-exchange chromatograph EMSA- Electrophoretic mobility shift assay SAXS- Small angle X-ray scattering

SDS-PAGE- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis IPTG- Isopropyl β-D-1-thiogalactopyranoside

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1. Introduction

1.1 Model organism

Saccharopolyspora erythraea (S. erythraea) is a gram-positive actinomycete organism that is usually found in soil. It has a big and GC rich genome that enables them to grow in harsh environments where nutrients are scarce (Marcellin et al. 2013, Sayed et al. 2020). It is able to synthesize secondary metabolites, for example the antibiotic erythromycin A, which is a very useful antibiotic for fighting pathogenic gram-positive bacteria, and has been used to study antibiotic production for many years (Oliynyk et al. 2007, Marcellin et al. 2013, Liu et al. 2013).

S. erythraea has a complex life cycle which is still not fully understood. The initial growth phase is followed by a transition period, which is then followed by a secondary growth phase. The transition period is also known as the metabolic switch and it is a switch that leads to morphological changes, such as forming the aerial hyphae, and expression of the erythromycin gene cluster. The metabolic switch also leads to expression of genes associated with for example secretion and transport of metabolites (Marcellin et al. 2013, Yin et al. 2013).

Having such a complex life cycle makes S. erythraea a very interesting organism and in this project, it will be used as a model organism to study its manganese transport regulator (SeMntR).

1.2 Metal regulation

Many transition metals including iron, manganese, copper, cobalt and zinc are key players in cell biology. They participate in many cellular processes as cofactors in respiration, gene transcription, oxidation-reduction reactions, and in protection against oxidative stress (Dean et al. 2012).

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1.2.1 Oxidative stress

Iron (Fe) and manganese (Mn) are very important metals when it comes to cellular adaption to oxidative stress. They are both metals that can serve as cofactors for enzymes in removal of toxic byproducts like superoxide and hydrogen peroxide (Aguirre & Culotta 2012). These byproducts are reactive oxygen species (ROS) and if they accumulate within the cell, the cell can die. Fe2+_{is very reactive with peroxide and through Fenton reactions which is a catalytic}

process, peroxide can be converted into highly reactive hydroxyl radicals. Mn2+ has a higher reduction potential than Fe2+ and is thus less inclined to undergo Fenton reactions (Aguirre & Culotta 2012, Zhao & Drlica 2014). Since manganese is less prone to undergo Fenton reaction and thus do not generate reactive hydroxyl radicals, it can act as a cofactor for Mn superoxide dismutase (MnSOD) enzymes. MnSOD are enzymes whose main responsibility is to protect the cells from ROS in oxidative stress (Aguirre & Culotta 2012, Li & Yang 2018).

1.3 MntR

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Figure 1: Structure picture of MntR in Bacillus halodurans (PDB ID: 6KTB, Lee et al. 2019). MntR in B. halodurans contain two domains, the C-terminal dimerization domain (upper) and the N-terminal DNA and metal ion binding domain (lower). The metal ion binding sites are indicated by arrows. Several crystal structures of MntR have been determined in several bacterial species, see figure 1, like Bacillus subtilis, Bacillus halodurans (DeWitt et al. 2007, Lee et al. 2019), Escherichia coli (Tanaka et al. 2009) and Mycobacterium tuberculosis (Pandey et al. 2015). These previous studies have shown how the conformation of MntR changes when its metal binding sites bind to metal ions (Lieser et al. 2003, Lee et al. 2019).

1.4 Immobilized metal ions affinity chromatography (IMAC)

Immobilized metal ions affinity chromatography (IMAC) is a purification method that has been used for decades. It is mainly used to purify proteins that have a polyhistidine tag (His-tag) at their N-or C-terminus (Chang et al. 2017). Many metal ions have been used in this technique,

Cu2+_{, Mn}2+_{, Nd}3+_{, Ni}2+_{, Zn}2+_{and Fe}3+ _{to name a few. The reason why so many different metal}

ions that have been used is that every metal ion has different physical and chemical properties, and the nature of the ion affects the selectivity and affinity of the protein interaction (Blowers 2000). Histidine side chains have very strong interactions with immobilized metal ions. Histidine has an imidazole ring and that ring has electron donors that form bonds with the immobilized transition metal (Bornhorst & Falke 2000).

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1.5 Size Exclusion Chromatography (SEC)

Size exclusion chromatography (SEC) is just as its name implies, a technique for separating biomolecules by size, more exactly by their hydrodynamic radius. Whilst many other techniques use the molecules chemical properties for separation, SEC uses the molecules size difference. The chromatography column in SEC is packed with porous beads with controlled pore size, so that when biomolecules go through the column, the smaller molecules go through all the nicks and alleys of the beads whilst the bigger molecules are too big for these so they travel faster through the column (Fekete et al. 2014).

SEC allows the making of a calibration curve, this calibration is based on already known molecules, which can be used to estimate the molecular weight of an unknown molecule (Fekete et al. 2014).

1.6 Electrophoretic mobility shift assays (EMSA)

The electrophoretic mobility shift assay (EMSA) is a broadly used technique. EMSA is based on the discovery that molecules travel through an electrophoretic gel differently depending on size, charge or binding affinity (Hellman & Fried 2007). For example, the mobility through an electrophoretic gel is slower for a protein/nucleic acid complex than a free nucleic acid. EMSA can thus be used for separating different molecules, analyzing and characterizing them (Hellman & Fried 2007). Classically a solution of protein and nucleic acid is mixed together and then the sample is exposed to electrophoresis through a polyacrylamide gel (Hellman & Fried 2007, Altschuler et al. 2013). The DNA has usually been modified with a fluorescent probe, so that the samples that have bound to the nucleic acid can be determined with an imaging-system (Hellman & Fried 2007, Altschuler et al. 2013). The image that is obtained can be used to determine the binding affinity between the protein and the nucleic acid. Binding affinity is how strong the binding interaction between two molecules is. Binding affinity is measured by the equilibrium dissociation constant (Kd). Kd is the half activation, the

concentration at which 50% of the DNA is bound by the protein (Heffler et al. 2012). A low Kd

value means strong binding affinity, and the higher the Kd value is the weaker is the binding

affinity (Ma et al. 2018). The Hill slope equals to 1.0 when a monomer binds with no cooperativity to one site. When the Hill slope has a value higher than 1 it has positive cooperativity, this means that the binding of a ligand to one site affects the binding at a second site. If the value of the hill slope is less than one then it means it has negative cooperativity (Hulme & Trevethick 2010). Kd can be calculated with the following equation:

𝑦 = 𝑚𝑎𝑥 × 𝑥ℎ÷ 𝐾𝑑ℎ+ 𝑥ℎ

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1.7 Aims of the study

In this project the metal-sensor MntR from the model organism Saccharopolyspora erythraea will be studied. The crystal structures of MntR from other bacterial species have been determined but the knowledge about metal coordination and selectivity is limited and not yet fully understood.

• The main aim of this study was to optimize the protocol for heterologous production and purification of SeMntR.

• The second aim was to characterize SeMntRs metal-dependent DNA-binding activity. • The third aim of this project was to characterize the structure of SeMntR by X-ray

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2. Methods

2.1 Small scale MntR expression test

A small-scale expression experiment was performed to determine the optimal Isopropyl β-D

-1-thiogalactopyranoside (IPTG) concentration and incubation conditions for MntR expression in order to get maximum amount of the protein.

2.1.1 Transformation

An expression plasmid (pET28) encoding SeMntR with an N-terminal His tag that is cleavable by TEV protease was used for transformation. The pET28 plasmid also make the cells kanamycin resistant. The plasmid was transformed into electrocompetent Escherichia coli (E. coli) rosetta cells already containing the pRARE plasmid which encodes for rare tRNAs. The pRARE plasmid also make the cells chloramphenicol resistant.

Materials:

• Electrocompetent (E. coli) rosetta cells. • .

• SOC media: 2% (v/w) tryptone, 0.5% (v/w) yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose.

• Agar plates made from LB medium, supplemented with 50 μg/ml kanamycin and 34 μg/ml chloramphenicol.

Protocol:

SOC media was pre-warmed at 37 ºC and an electroporation cuvette was kept on ice. Electrocompetent cell tubes of 70 μl were picked from an -80 ºC freezer and thawed on ice. After thawed, 2 μl of plasmid solution (10 ng/μl) was added to each of the tubes. The cells were incubated on ice for 30 minutes. After incubation, the cells were transferred into the electroporation cuvettes. The electroporator was set with following settings:

• Exponential protocol. • 1800 V.

• 25 μF. • 200 Ohm. • 1 mm.

The transformation took between 4.5-5 ms. After that 450 μl of SOC media was added to each sample and then the samples were transferred to growing tubes. The growing tubes were then incubated on a shaker with 140 rpm at 37 ºC for 60 minutes. After incubation 400 μl of each culture was added onto two agar plates and incubated at 37 ºC overnight.

2.1.2 Pre-culture

Materials:

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• LB-medium: 1% (v/w) Peptone, 0.5% (v/w) Yeast extract, 0.5% (v/w) NaCl. • 1000x Kanamycin, 50 mg /ml, and chloramphenicol, 34 mg /ml.

Protocol:

Four growing tubes were prepared with 4 ml LB-medium supplemented with antibiotics in each tube. Single colonies were picked from the plate and inoculated into the media. Tubes were incubated on a shaker at 110 rpm and 37 ºC overnight.

2.2.3 Expression and cell harvest

Materials:

• Pre-culture.

• LB media: 1% (v/w) Peptone, 0.5% (v/w) Yeast extract, 0.5% (v/w) NaCl. • 1000x Kanamycin, 50 mg/ml, and Chloramphenicol, 34 mg/ml.

• 1 M MnCl2.

• 1 M IPTG. Protocol:

Eight main cultures were prepared in eight growing tubes with 7 ml LB-media supplemented with antibiotics and 200 μl of pre-culture as well MnCl2 to a concentration of 1 mM was added.

Fifteen minutes after adding MnCl2, the tubes were induced and incubated, see table 1. The

tubes were put on a shaker at 120 rpm. The cell cultures were centrifuged at 4000 x g for 15 min at 4 ºC. The supernatant was removed, and the cells were stored at -20 ºC. To analyze the expression of SeMntR on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), all the cell pellets were resuspended with 250 μl of ddH2O and samples of 20 μl were taken from every tube. Every sample was then mixed with 4x SDS loading dye. Tubes were incubated on a heating block at 95 ºC for 10 min. The samples were loaded on a Mini-PROTEAN TGX Precast Gel and run for 30 min at 200 V.

2.2 Large scale MntR expression

The large-scale protein expression protocol was developed on the bases of the small-scale expression test.

Transformation was performed as described before.

2.2.1 Overnight pre-culture

Materials:

• Agar plates, made from LB media, with colonies.

• TB media: 1.2% (w/v) Peptone, 2.4% Yeast extract (w/v), 0.5% (w/v) Glycerol, 10% Salt buffer, 10% P-buffer.

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Protocol:

The pre-culture was prepared late in the day to prevent overgrowth. Two 250 ml Erlenmeyer flasks were prepared with 60 ml of TB-media supplemented with antibiotics in each. Single colonies of medium size were picked from the plate and inoculated into the media. Flasks were incubated on a shaker at 110 rpm in 37 ºC overnight.

2.2.2 Expression and cell harvest

Materials:

• 8 x 800 ml TB media: 1.2% (w/v) Peptone, 2.4% Yeast extract (w/v), 0.5% (w/v) Glycerol.

• Salt buffer: 100 mM NH4Cl, 20 mM MgSO4x7H2O.

• P-buffer: 0.17 M KH2PO4, 0.72 M K2HPO4 pH 7.

• 100 ml overnight culture.

• 1000x Kanamycin, 50 mg /ml, and chloramphenicol, 34 mg /ml. • 1 M MnCl2.

• 100 mM IPTG. Protocol:

Salt buffer (100 ml) and P-buffer (100 ml) were added to each of the flasks containing TB-media. Eight 2.8 L baffled Fernbach-flasks were prepared with 1 L prewarmed TB-media supplemented with 1 ml of 1000x kanamycin and chloramphenicol in each flask. 10 ml of the overnight culture was added to each of the flasks and then the flasks were put on a shaker at 120 rpm at 37 ºC until OD600 reached 0.4. Manganese was added to the cultures to a final

concentration of 1 mM, the cultures were incubated for another 15 minutes before being induced with IPTG to a final concentration of 0.1 mM. After induction the flasks were put on a shaker at 120 rpm at room temperature overnight.

2.2.3 Cell harvest

Materials:

• 8 x 1 L Overnight culture Protocol:

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2.2.4 Cell lysis

Materials:

• Lysis buffer: 200 mM NaH2PO4 · H2O, 500 mM NaCl, 10 % Glycerol with pH 6. • 100 mM PMSF protease inhibitor.

• 5 mM MgSO4.

• DNase and Lysosome.

• Frozen cells from previous step. Protocol:

One Falcon tube of cells was thawed and resuspended with 1:10 of lysis buffer. The cells were vortexed until homogenized. A small amount of Dnase and lysosome was added to the solution, as well as 100 mM PMSF and 5 mM MgSO4. The resuspension was poured into a 250 ml beaker

kept on ice and 40 ml of lysis buffer was added. The cells were lysed by running the solution through a cell disruptor at 35 kPsi one time. After being disrupted, the cells were centrifuged at 18000 rpm in an SS.34 rotor for 1 hour at 4 ºC. Supernatant was poured into 50 ml Falcon tubes and stored on ice.

2.3 Large scale MntR purification

The first purification step was Immobilized metal ions affinity chromatography (IMAC) using Ni2+-NTA resin with a Bio-Rad NGC chromatography system. This was performed in order to find out at what imidazole concentration MntR was best eluted.

2.3.1 Immobilized metal ions affinity chromatograph (IMAC) on an NGC chromatography system

Materials:

• Lysis buffer: 200 mM NaH2PO4 · H2O, 500 mM NaCl, 10 % Glycerol pH 6. • 1 M imidazole.

Protocol:

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2.3.2 TEV cleavage & Reverse IMAC on NGC chromatography system

Material:

• Lysis buffer: 200 mM NaH2PO4 · H2O, 500 mM NaCl, 10 % Glycerol pH 6.

• Buffer A: 200 mM NaH2PO4 · H2O, 500 mM NaCl, 10 % Glycerol, 80 mM Imidazole. • 1 M imidazole. • 75 μM TEV protease. • 14.3 M β-mercaptoethanol. • 500 mM EDTA. • 20% EtOH. Protocol:

The sample was diluted with lysis buffer to decrease the imidazole concentration from 400 mM to 80 mM. This was done in order to perform a reverse IMAC and also because TEV is not stable at high concentrations of imidazole. The protein concentration was determined with a NanoDrop spectrophotometer at A280. EDTA was added to the solution to a final concentration

of 0.5 mM and β-mercaptoethanol was added to a final concentration of 14 mM. Lastly TEV was added in a molar ratio of 1:100 (TEV:Protein). The sample was incubated overnight at room temperature. The next day the concentration of β-mercaptoethanol was diluted with buffer A to a concentration of 5 mM. The Ni-NTA columns were equilibrated with lysis buffer at a flow rate of 2 ml/min. The sample was loaded into the columns with a 50 ml superloop. The run was started with lysis buffer with a flow rate of 1 ml/min. Increased imidazole concentration up to 100 mM in 10 CV (column volumes). All the uncleaved proteins and contaminants were eluted with 400 mM imidazole. Protein concentration was determined with a NanoDrop spectrophotometer at A280 and those with highest concentration were pooled together. The

sample was transferred into spin concentrators and the sample was concentrated, aiming for a concentration of 20 mg/ml. Samples of 20 μl were taken from all the fractions and prepared for SDS-PAGE by being mixed with 4x SDS loading dye, and incubated on a heating block at 95 ºC for 10 min. The samples were loaded on a Mini-PROTEAN TGX Precast Gel and run for 30 min at 200 V.

2.3.3 Immobilized metal ions affinity chromatograph (IMAC) batch purification

Based on the IMAC performed on an NGC chromatography system a second purification was performed with a new cell pellet, without the NGC chromatography system.

Materials:

• Buffer A: 200 mM NaH2PO4 · H2O, 500 mM NaCl, 10 % Glycerol, 80 mM Imidazole. • Buffer B: 200 mM NaH2PO4 · H2O, 500 mM NaCl, 10 % Glycerol, 300 mM Imidazole. • 6 ml Ni-NTA beads.

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This was performed in a similar way as on the NGC chromatography system with the following differences:

All following steps were performed at room temperature. Gravity columns with 3 ml of Ni-NTA beads in each were equilibrated with 3 times CVs of lysis buffer. The supernatant of the cell lysate was added to the columns. The columns were washed 10 times the CV with buffer A. Protein was eluted with 5 times CVs of buffer B.

2.3.4 TEV cleavage & Reversed IMAC batch purification

Material:

• Buffer A: 200 mM NaH2PO4 · H2O, 500 mM NaCl, 10 % Glycerol, 80 mM Imidazole. • Buffer C: 200 mM NaH2PO4 · H2O, 500 mM NaCl, 10 % Glycerol, 500 mM Imidazole. • 75 μM TEV protease.

• 14.3 M β-mercaptoethanol. • 500 mM EDTA.

• 20% EtOH. Protocol:

This was performed in a similar way as on the NGC chromatography system with following differences:

The Ni-NTA columns were equilibrated 3 times CVs with buffer A and the protein solution was loaded. Buffer C was added to the columns to elute uncleaved proteins and contaminants.

2.3.5 Size exclusion chromatography (SEC)

Material:

• SEC-Buffer: 800 mM NaCl, 200 mM Na2SO4.

Protocol:

The size exclusion chromatography was performed on a ÄKTA purifier (GE healthcare) with a HiLoad 16/60 Superdex 75 column. The column was equilibrated with 1.5 CV of SEC- Buffer with a flow rate of 1.5 ml/min. After equilibration, the injection loop was rinsed with SEC-buffer and sample of 300 μl was loaded and run on the column with a flow rate of 1 ml/min. Starting at 40 ml, 2 ml fractions were collected until 95 ml. Fractions were analyzed on an SDS-PAGE and the fractions with pure MntR were pooled together and concentrated with spin concentrators. Sample was flash freezed and stored at -80 ºC.

2.4 Protein crystallization

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• SeMntR with concentration 18.22 mg/ml.

• Protein buffer: 200 mM Phosphate pH 6.0, 500 mM NaCl, 10% Glycerol, 80 mM Imidazole.

• SeMntR dsDNA. Protocol:

MntR was incubated with 1 mM MnCl2 and 150 μM wt DNA for 15 minutes at room

temperature. Crystallization plates were setup with the help of a pipetting robot (Mosquito, Rigaku) and the commercial crystallization screens (Shotgun and Morpheus I, Molecular Dimensions). Crystals was observed using a polarization filter.

2.5 Electrophoretic mobility shift assays

Material:

• 4 % native DNA acrylamide gels: 2.7 ml 30% acrylamide 0.8% bis-acrylamide, 13.2 ml dH2O, 4 ml 5x TAKA buffer, 100 μl 10% ammonium persulfate, 10 ul TEMED. • SeMntR dsDNA.

• 5xTAKA buffer: 75 mM Tris-acetate pH 7.3, 20 mM Potassium acetate, H2O, adjust pH to 7.3 with glacial acetic acid.

• SeMntR with concentration 14.7072 pmol/ul. • Poly dIdC.

Protocol:

Gels were prepared and pre-run at 4 ºC for 30 minutes at 20 mAmp. Samples were prepared with 5X TAKA buffer, 30 nM DNA, 30 nM MnCl2, 30 nM Poly dIdC, 50% Glycerol, different

concentrations of SeMntR protein and ddH2O to a final volume of 15 μl. Samples were

incubated in the dark for 30 minutes. The samples were loaded on the gels and run for 30-60 minutes at 4 ºC in the dark. The gels were visualized on a BioRad gel imager. The intensity of the bands was calculated with the program ImageJ and the values were used to calculate the Kd,

hill slope (h) and the max (maximum bound DNA), using the program Matlab with following equation:

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3. Results

The aim of the project was to produce and purify SeMntR in a high yield and high purity so that downstream experiments like EMSA and crystallization could be performed. The first step towards that was to perform an experiment showing the best induction and incubation conditions to obtain a high yield of SeMntR.

3.1 Expression

A small-scale expression test was performed to find out which IPTG concentration and at what temperature the highest yield of SeMntR protein could be attained. The samples were treated with different concentrations of IPTG, incubation times and temperatures, see table 2. From analyzing the SDS-PAGE the best treatment seemed to be 0,1 mM and 0.5 mM IPTG incubated at 20 ºC for 18 h, see figure 2.

I decided for future expression to use 0.1 mM IPTG for induction and to incubate the cell culture at 20 ºC. SeMntR was also overexpressed uninduced, see figure 2 lane 1 & 6, this is most likely because the promoter is leaking.

Table 2: Experimental setup for the small-scale expression test. Nr. Temperature OD600 at

induction

Incubation time IPTG concentration

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Figure 2: SDS-PAGE representing the small-scale expression test. Lane 1: BioRad Precision Plus Protein Dual Color. Lane 2: Uninduced at 37ºC for 3 h. Lane 3: 0,1 mM IPTG at 37ºC for 3 h. Lane 4: 0,5 mM IPTG at 37ºC for 3 h. Lane 5: 1 mM IPTG at 37ºC for 3 h. Lane 6: Uninduced at 20ºC for 18 h. Lane 7: 0,1 mM IPTG at 20ºC for 18 h. Lane 8: 0,5 mM IPTG at 20ºC for 18 h. Lane 9: 1 mM IPTG at 20ºC for 18 h. Lane 10: 0,1 mM IPTG at 37ºC for 3 h. Lane 11: 0,5 mM IPTG 37ºC. Lane 12: 1 mM IPTG at 37ºC for 3 h. Lane 13: 0,1 mM IPTG at 20ºC for 18 h. Lane 14: 0,5 mM IPTG at 37ºC for 3 h. Lane 2-9 was loaded with 5 ul and lane 10-14 was loaded with 10 ul.

3.2 Purification

After finding out at what conditions SeMntR is highly expressed an IMAC and reverse IMAC was performed with an NGC chromatographic system, this was done in order to find out at what concentration imidazole SeMntR is best eluted. This was done in order to optimize the purification protocol, as to get the highest possible purity of SeMntR in later purifications so that downstream experiments like EMSA and crystallography could be performed. The reverse IMAC was performed in order to remove TEV and the His-tag. The SeMntR had precipitation problems, after switching out the sulfate to phosphate in the lysis buffer the protein didn’t precipitate anymore.

From analyzing the SDS-PAGE it seemed like SeMntR is well eluted at 400 mM imidazole, see figure 2 lane 6. Did not perform a SEC on this sample since it was an experiment for finding out the best imidazole concentration. There is a clear shift in the bands of the samples with and without TEV, see figure 3 lane 6 & 7, showing that the digestion was successful and that the his-tag was cleaved and removed.

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Figure 3: SDS-PAGE on the fractions collected from the IMAC on NGC chromatographic system. Lane 1: BioRad Precision Plus Protein Dual Color. Lane 2: IMAC FT 40-60 mM imidazole. Lane 3: IMAC FT 40-60 mM imidazole. Lane 4: IMAC Wash 80 mM imidazole. Lane 5: IMAC Wash 80 mM imidazole. Lane 6: IMAC elute 400 mM imidazole. Lane 7: TEV Dig. Lane 8: Rev IMAC 0 mM imidazole 9: Rev IMAC 40-60 mM imidazole. Lane 10: Rev IMAC 80 mM imidazole. Lane 11: Rev IMAC 100 mM imidazole. Lane 12: Rev IMAC 400 mM imidazole. Lane 13: TEV Dig. Lane 14: Rev IMAC 0 mM imidazole. Lane 15: Rev IMAC 400 mM. Lane 2-7 was loaded with 3 ul and lane 8-14 was loaded with 5 ul.

For the second purification of SeMntR, I performed an IMAC and reverse IMAC batch purification with 400 mM imidazole to elute SeMntR, however it seemed to also elute other proteins and/or impurities, see figure 4 lane 7 & 10. SeMntR was not eluted in a high enough purity, the gel show that there is not a clear band in the lanes 7 and 10 which should contain the SeMntR and many other bands is visible in those lanes showing that there is a lot of other proteins/impurities in the sample. I decided to perform an SEC on this sample in an attempt to make it purer. I decided to proceed with the lower concentration of 300 mM imidazole for further purification experiments to try and avoid eluting molecules that are not SeMntR.

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Figure 4: SDS-PAGE on the fractions collected from the IMAC batch purification. Lane 1: BioRad Precision Plus Protein Dual Color. Lane 2: Lysis sample. Lane 3: IMAC FT. Lane 4: IMAC Wash 80mM imidazole. Lane 5: IMAC Elute 400 mM imidazole. Lane 6: IMAC Elute diluted. Lane 7: IMAC Elute concentrated. Lane 8: TEV Digestion 9: Rev IMAC FT 400 mM imidazole. Lane 10: Rev IMAC FT concentrated. Lane 11: Rev IMAC Elute 500 mM. Lane 12: Rev IMAC Elute 500 mM. Lane 13: TEV Dig. Lane 14: Rev IMAC FT 400 mM. Lane 15: Rev IMAC FT concentrated. Lane 2-11 was loaded with 2 ul and lane 12-15 was loaded with 5 ul.

The size exclusion chromatogram showed three peaks with the approximate sizes 140 kDa, 59 kDa and 20 kDa, as seen in figure 5. The molecular weight of SeMntR with his-tag is 27.5 kDa and without is 25.3 kDa, so the third peak could indicate presence of SeMntR.

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-Figure 5: Size exclusion chromatogram. The size corresponding to the peak is indicated. The molecular weight was determined from a linear interpolation of a calibration run that was done in 25 mM Tris, pH 7.5, 150 mM NaCl on bovine serum albumin (66 kDa) and α-chymotrypsin (25 kDa). The size exclusion chromatography was performed on a ÄKTA purifier (GE healthcare) on a HiLoad 16/60 Superdex 75 column with SEC-buffer.

The fractions for each peak were analyzed on an SDS-PAGE, see figure 5, and showed very faint bands of SeMntR, see figure 6. Fractions 10-13 were pooled together and concentrated as much as possible, but resulted in very low concentration of SeMntR, only 5.5 mg/ml which is most likely too low for crystallization.

-10 0 10 20 30 40 50 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 mAU 280 ml

Size Exclusion Chromatogram

140 kDa

59 kDa

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Figure 6: SDS-PAGE of the fractions collected during the size exclusion chromatograph. Fractions from lane 10-13 were pooled together and concentrated to 5.5 mg/ml.

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Figure 7: SDS-PAGE on the fractions collected during IMAC batch purification. Lane 1 is the ladder: BioRad Precision Plus Protein Dual Color. Lane 2 is the lysis sample. Lane 3 is the flowthrough fraction. Lane 4 is the wash fraction. Lane 5-15 is the elution fractions.

Figure 8: SDS-PAGE of the fractions collected during IMAC. Lane 1is the ladder: BioRad Precision Plus Protein Dual Color. Lane 2 is elution fraction. Lane 3 is concentrated elution fraction. Lane 4 is the TEV digestion fraction. Lane 5 is the reversed IMAC flowthrough. Lane 6 is the reversed IMAC elute. All the lanes were loaded with 3 ul of sample.

3.3 Crystallization

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Figure 9: Picture with polarization filter of protein crystals from a shotgun screen with 1.5 M lithium sulfate, 0.1 M sodium HEPES pH 7.5.

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3.4 DNA-binding

Electrophoretic mobility shift assays were performed to determine the binding affinity of SeMntR to S. erythraea wildtype (wt) DNA. The wt DNA binding motif (red) sequence is:

GTACAGTTTTCGCCGTGGCGAAATCTCGAG.

I first performed an EMSA with wt DNA and, as can be seen in figure 11, SeMntR bound to wt DNA.

Figure 11: EMSA gel showing MntR with wt DNA. The protein concentration is gradually increasing from left to right.

To determine at what affinity SeMntR binds to the wt DNA, I calculated the equilibrium dissociation constant (Kd), hill slope (h) and the maximum bound DNA (max) with Matlab, as

mentioned in method. The Kd value was calculated to be 822 nM, the h value 3.98 and the max

value 1.0.

The hill slope was greater than 1.0 so the graph shows a sigmoidal curve, see figure 12. This means that the ligand has more than one binding site and it has positive cooperativity.

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Figure 12: Graph showing intensity of bound DNA in relevance to protein concentration. It is a sigmoidal curve, meaning that the ligand has more than one binding site and has cooperativity. I wanted to test the specificity of SeMntR/DNA binding, so I performed an EMSA with mutant DNA (MBS-P6027-S1). MBS-P6027-S1 has a mutation (green) in the beginning of the sequence: GTACAGCGCGCGCCGTGGCGAAATCTCGAG. As can be seen in figure 13, SeMntR did not bind to MBS-P6027-S1 at all.

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Figure 13: EMSA gel showing SeMntR with MBS-P6027-S1. The protein concentration is gradually increasing from left to right.

I wanted to further test the specificity of SeMntR/DNA binding, so I performed an EMSA with another mutant (MBS-P6027-S2), that has a mutation (green) at the end of the sequence: GTACAGTTTTCGCCGTGGCCGCGTCTCGAG. As can be seen in figure 14, SeMntR did not bind to MBS-P6027-S2 at all.

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Figure 14: EMSA gel showing SeMntR with MBS-P6027-S2. The protein concentration is gradually increasing from left to right.

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4. Discussion

4.1 Expression

By growing the bacteria to an OD600 of approximately 0.4 before inducing with IPTG, a quite

large amount of SeMntR could be obtained. Letting the cells reach a OD600 between 0.4- 1.0 is

important because at this cell density the bacteria is in the exponential growth phase (Kelley et al. 2010). IPTG affects the cell growth negatively so if the cell culture is induced too early the overexpression of the protein will inhibit the cell growth (Kelley et al. 2010). Most of them are in the log-phase where they upregulate their protein production and inducing makes it possible to maximize the amount of protein before the cells slow down their protein production, cell culture is overgrown, or cells start dying. MntR overexpressed in E. coli has mostly been induced at an OD600 of 0.4-0.6 and with an IPTG concentration of 0.1- 1 mM (Schmitt 2002,

Baumgart & Frunzke 2015, Huang et al. 2017, Lee et al. 2019). An IPTG concentration test therefore felt necessarily to see at what IPTG concentration SeMntR would be overexpressed at a high level.

SeMntR was overexpressed uninduced, as can be seen in figure 2 lane 1 and 6, this is most likely because of a leaky promoter. A lot of promoters are not tightly regulated and will show some degree of expression even without an inducer. The lac promoter does not have full transcriptional control and is thus known as being leaky (Rosano & Ceccarelli 2014). Since the pET28-TEV-SeMntR plasmid used in this project uses the T7 system for protein expression, and the T7-system is controlled by the lac promoter, the proteins is produced at a low level even without the IPTG inducer. Still in order to produce protein at a high level, the inducer is necessary.

4.2 Purification

The purification experiment shows that SeMntR can be obtained with high purity from IMAC alone. However, in the first batch purification, see figure 4, SeMntR was not attained with high purity. A purified sample which still contain impurities can be further purified with a size exclusion chromatography to further get rid of unspecific proteins that has a lower molecular weight.

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performing more SEC experiments would be interesting to see if a high amount of protein would be lost again, or just a small amount gets lost and it is worth it for the higher purity. Some parts of the method for purifying SeMntR can still be optimized even further. During the purification it was shown that the buffer that was used first, which contained sulfate, made the sample precipitate. After switching out the sulfate to phosphate it did not precipitate anymore. It most likely precipitated because the sulfate made the protein to aggregate. The lysis buffer I used had a pH of 6, it would be interesting to see if different pH would make a difference. A higher pH could result in higher protein solubility and stability. When MntR from other organisms has been overexpressed, it has mostly been lysed in a buffer with a pH of 7- 8 (Schmitt 2002, Baumgart & Frunzke 2015, Pandey et al. 2015, Huang et al. 2017, Lee et al. 2019). A buffer optimization should be performed to see how it affects the stability of the protein and to avoid precipitation.

4.3 Crystallization

There are crystals formed in the crystallization plates. They could be found under a polarization filter, so they are birefringent. Birefringent means that they turn the light going in, so it is split into two rays that then take a slightly different path going out. This can be observed using a polarization filter (Singer et al. 2004). In figure 9 small light reflecting crystals can be seen, they are too small to fish, so they can’t be sent for diffraction. The crystal in figure 10 is from a shotgun screen with 0.2 M calcium chloride dihydrate and since the phosphate is already present in the protein solution this could be a calcium-phosphate crystal. In order to find out what kind of crystal has formed it would have to be fished and be shot with x-rays, to observe the diffraction.

4.4 EMSA

The image of the EMSA gel shows that SeMntR binds to the wt dsDNA. When EMSA was performed with mutant DNA it did not bind at all. This shows that SeMntR has a specific binding sequence that will not bind if there is a mutation either at the beginning of the sequence or at the end of the sequence. This shows that the whole sequence is important for DNA-binding.

The value for Kd I got was quite high, meaning that it takes quite a high concentration of

SeMntR before 50% of the wt DNA is bound. In other words, it has low affinity. It could be that the low affinity is because there are still some impurities left in the protein sample. When staining the gel with coomassie I could see that a lot of the protein stayed in the wells. The reason could be because the protein aggregated or because of the low affinity. If protein is not bound to DNA, it will not move in the gel. I performed the EMSA at 4 ºC and the temperature could also possibly affect this, so future EMSAS could preferably be performed at room temperature, since SeMntR is stable in room temperature.

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5. Conclusion

SeMntR can be obtained at high purity and with high concentration from IMAC and reverse IMAC alone, it is not necessary to perform a SEC. More experiments with SEC would however be interesting to see if the sample gets even purer. Another thing to consider could be to perform an ion-exchange chromatography (IEX), like a cation-exchange chromatography (CEX) instead of SEC.

There are still some parts that could be optimized in the method, for example the lysis buffer and the SEC buffer, and performing an experiment with different chemicals and pH would be a good idea for the future.

There is more work to be done to characterize SeMntR. a SAXS experiment would have been very interesting to perform and is something that should be considered for future experiments.

6. Acknowledgements

I would like to thank my supervisor Julia Griese for letting me do this project.

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Purification, functional characterization andcrystallization of the MntR manganese sensorfrom Saccharopolyspora erythraeaMalin Svensson

Purification,

functional

characterization

and

crystallization

of

the

MntR

manganese

sensor

from

Saccharopolyspora

erythraea

Malin

Svensson

Table of contents

List of abbreviations

1. Introduction

2. Methods

3. Results

Size Exclusion Chromatogram

4. Discussion

5. Conclusion

6. Acknowledgements

References