• No results found

Expression of the Majastridin-like protein from Streptococcus pneumonia for crystallization and antibody production

N/A
N/A
Protected

Academic year: 2021

Share "Expression of the Majastridin-like protein from Streptococcus pneumonia for crystallization and antibody production"

Copied!
20
0
0

Loading.... (view fulltext now)

Full text

(1)

Örebro University

School of Science and Technology Josefin Persson

2009

Expression of the Majastridin-like protein from Streptococcus

pneumonia for crystallization and antibody production

(2)

2 Abstract

The F1 part of F0F1-ATP synthase in the proteobacterium Rhodobacter blasticus contains five different proteins, but when the DNA was sequenced a sixth gene was found in the operon. The protein that corresponds to the sixth gene has been named Majastridin. When an amino acid BLAST search is performed with the Majastridin sequence, protein sequences have been found that are similar to Majastridin in other bacterial strains, and one of them is

Streptococcus pneumonia. The hypothetical protein from Streptococcus pneumonia contains 242 amino acids and has a molecular weight around 30 kDa.

In this work the Majastridin-like protein from Streptococcus pneumonia was expressed in E. coli cells and purified with nickel affinity chromatography and size exclusion

chromatography. The result was verified with SDS-PAGE and western blot. The purified protein was then crystallized with the hanging drop method, where crystals were formed and optimization was made. The protein was also used to produce antibodies.

(3)

3

Index

1. Introduction ... 5

1.1. Aim ... 5

1.2. Function and structure of F0F1-ATP synthase ... 5

1.3. The Majastridin protein which corresponds to the URF6 gene ... 5

1.4. The function and structure of the Majastridin protein ... 5

1.5. Streptococcus ... 6

1.6. Hypothetical protein from Streptococcus pneumonia... 6

2. Materials and methods ... 7

2.1. Vector ... 7

2.2. Growth of E. coli cells ... 7

2.3. Purification of protein ... 8

2.3.1. Lysis of bacteria and release of the protein ... 8

2.3.2. Nickel affinity chromatography ... 8

2.3.3. Purification with Äktadesign... 9

2.4. SDS-PAGE ... 9

2.5. Western blot ... 9

2.6. Protein concentration determination ... 10

2.7. Concentration of protein ... 10

2.8. Cleavage with SUMO protease ... 10

2.9. Crystallization ... 11

(4)

4

3. Result and discussion ... 12

3.1. Purification of protein ... 12

3.2. Purification with Äktadesign ... 13

3.3 Cleavage with SUMO protease ... 15

3.4. Crystallization ... 16

3.5. Antibody production ... 18

4. Conclusion ... 18

5. Acknowledgments ... 18

(5)

5 1. Introduction

1.1. Aim

The aim of this work was to express a Majastridin-like protein from Streptococcus pneumonia in E. coli cells for crystallization with hanging drop method and rabbit antibody production.

1.2. Function and structure of F0F1-ATP synthase

F0F1- ATP synthase is an enzyme that is membrane bound and has similar function and structure in mitochondria, chloroplasts and bacteria [1].

In eukaryotes and prokaryotes the F0F1- ATP synthase function is to synthesize ATP and in prokaryotes the F0F1-ATP synthase can also create a proton gradient by breaking down ATP [2].

The enzyme has two parts, F0 and F1. In E. coli F1 contains five different subunits, α3β3γδε and F0 contains three different subunits, ab2c9-12 [2]. F0 is a transmembrane protein and its function is to translocate the protons through a transmembrane proton channel that is coupled to ATP synthesis. F1 is the catalytic and regulatory part of the enzyme and is protruding into the cytosol from the membrane [1].

1.3. The Majastridin protein which corresponds to the URF6 gene

The URF6 gene is a sixth open reading frame and was found in the proteobacterium Rhodobacter blasticus when the F1 part of F0F1-ATP synthase operon was sequenced. The gene is located between the γ and β subunits and the protein corresponding to the gene has been named Majastridin [3].

1.4. The function and structure of the Majastridin protein

The URF6 gene has been cloned and expressed in E. coli, the Majastridin protein has been purified and its structure has been determined using protein crystallography [3].

There have been doubts if the protein is involved in the synthesis, in the complex or in the regulation of F0F1-ATP synthase [3]. It is also unclear if the protein has any physiological role and if it has a physical connection with F0F1-ATP synthase or not [2].

(6)

6

When comparing the 3D structure of the crystallized Majastridin with other protein structures it was found to be similar to glycosyltransferases [3]. Glycosyltranferases are enzymes that transfer an activated sugar unit to other molecules, such as another sugar, a lipid or a protein [4]. An amino acid BLAST [5] search shows protein sequences similar to Majastridin from different bacterial strains and Streptococcus pneumonia is one of them. Majastridin from Rhodobacter blasticus and the corresponding hypothetical protein from Streptococcus pneumonia are 23 % identical (figure 1) [5].

Query 72 RCKGRYVIFCHEDVELVDRGYDDLVAAIEALEEADPKWLVAGVAGSPWRPLNHSVTAQAL 131 + KG Y+ F H+D+ D D +A +E+ + + VAGVAG ++ + + Sbjct 55 QAKGDYLFFVHQDISFQD---DFELAKLESYCR-NSIFGVAGVAGV---KNIEGKVV 104

Query 132 HISDVFGNDRRRG---NVPCRVESLDECFLLMRRLKPVLNSYDMQG--FHYYGADLC 183 S++F D + + P V+++DEC +++ + N + + G +H YG D Sbjct 105 SFSNIFHGDPKTKAAGKSISAPVEVDAIDECLIIIPKKVFSTNQFSIIGPTWHLYGTDYA 164

Query 184 LQAEFLGGRAYAIDFHLHHY--GRAIADENFHRLRQEMAQKYRR 225 LQ + + L H G+++ + N+ Q + +KY + Sbjct 165 LQMKLINSPVLVFPSELWHVSDGKSL-NLNYFDAIQWLLKKYSK 207

Figure 1. Amino acids BLAST search on the Majastridin protein (Query) from Rhodobacter blasticusand the corresponding hypothetical protein (Sbjct) from Streptococcus pneumonia. An identical amino acid is market with a letter, a similar amino acid is market with (+) and gaps are market with (-). The sequences are 23 % identical and 46 % similar in the middle of the sequences.

1.5. Streptococcus

Streptococcus belongs to gram positive bacteria. They are spherically shaped and found in pairs or chains. Hemolysis is the lysis of red blood cells [6]. The hemolytic properties are the base of three different groups of Streptococcus, alfa, beta and gamma [7, 8]. The alfa group gives a green zone around the cells when grown on blood agar plates, caused by incomplete lysis of red blood cells. The beta group gives a clear zone around the cells when grown on blood agar plates, caused by complete lysis of red blood cells. The gamma group includes the Streptococcus that has no hemolytic activity [6]. Of these three groups, the beta-hemolytic streptococci are further subdivided from the composition of

carbohydrates in the cell wall, called Lancefield serotyping. Streptococcus is pathogenic and can cause infections such as skin infections and blood poisoning. However, many of the Streptococci are non-pathogenic and are a part of the microbial flora through

(7)

7

inflammations in lungs, brain and ears is Streptococcus pneumonia that belongs to the alfa-hemolytic group of Streptococcus [7, 8].

1.6. Hypothetical protein from Streptococcus pneumonia

Q4K1H4 is a hypothetical protein from Streptococcus pneumonia [9] and has a molecular weight around 30 kDa [2]. The protein contains 242 amino acids and is a Majastridin-like protein [9].

2. Materials and methods 2.1. Vector

The vector (figure 2) that was used contained a kanamycin resistance gene, a His-tag gene and a SUMO gene fused to the gene of interest. The His-tag makes the purification steps easier and can be recognised by antibodies. SUMO and the His-tag can be removed by cleavage using a SUMO protease. The vector also has a T7lac promoter to induce production of the fusion protein [10]. The gene corresponding to the Majastridin-like protein from the bacteria Streptococcus pneumonia has been cloned into the pET SUMO vector (Invitrogen, Carlsbad, CA, USA) and the vector has been transformed into E. coli cells (BL21DE3, one shot) (Invitrogen) earlier and was stored in a freezer at -80°C.

(8)

8 2.2. Growth of E. coli cells

Seven tubes with 10 ml LB-medium (10 g/l trypton, 5 g/l yeastextract, 0.58 M NaCl) and 0.05 mM kanamycin were mixed and bacteria cells were added. These tubes were

incubated over night at 37°C and 170 rpm. Six of these tubes (10 ml LB-medium with kanamycin and bacteria cells) were placed in six 5l-flasks containing 1000 ml autoclaved LB-medium (10 g/l trypton, 5 g/l yeastextract, 0.58 M NaCl, 0.03 M betaine). The medium also contained kanamycin to a final concentration of 0.05 mM. The LB-medium was placed for further growth at 37°C and 170 rpm until OD600 (optical density) was between 0.5 and 0.8. When the value was reached, IPTG was added to a final

concentration of 0.1 mM and the LB-medium was incubated for three hours. IPTG is inducing the T7lac promoter which is placed just before the gene of the fusion protein. After the induction, the LB-medium was transferred to 1000 ml centrifuge tubes and centrifuged at 4 °C and 10000 rpm for 15 minutes. The pellet, which contained the bacteria with the protein, was placed in liquid nitrogen. The bacterial pieces were then placed in a Falcon tube and stored in the freezer (-20 °C) for further analysis.

2.3. Purification of protein

2.3.1. Lysis of bacteria and release of the protein

To break bacterial cells an X-press was used. The bacteria were then suspended in wash buffer (50 mM sodium phosphate pH 8, 0,3 M NaCl, 10 mM imidazole) and PMSF was added (25 μl/ml from 40mM stock solution) to avoid protein degradation. To the mixture DNase (Roche Diagnostics GmbH, Mannheim, Germany) was added to break down DNA. Then a sonication was performed (70%, 3 cycles and 1 minute) to lyse bacterial

membranes. Sonication is a method that releases ultra sounds that breaks and destroys the bacterial membranes. The suspension was ultra centrifuged for 50 minutes at 4°C and 45 000 rpm. The destroyed membrane and bacterial components will form a pellet and the soluble protein will be released in the supernatant.

2.3.2. Nickel affinity chromatography

The protein was purified with nickel affinity chromatography. To the protein suspension, His-Select® Nickel Affinity Gel (Sigma-Aldrich, Saint Louis, MO, USA) was added, but first the buffer, (ethanol) in which His-Select was stored, was removed and replaced with

(9)

9

wash buffer (50 mM sodium phosphate pH 8, 0.3 M NaCl, 10 mM imidazole). The protein suspension and Select was incubated over night at 4 °C and the protein with the His-tag will attach to the nickel charged affinity gel. In the column the protein will stay bound to the gel during wash until elution. The column was washed with three different wash buffers. All three buffers contained 50 mM sodium phosphate pH 8 and 0.3 M NaCl, but had different concentrations of imidazole (5 mM, 10 mM and 15 mM). Before starting with the elution the absorbance was measured. A280 of the wash buffer must be stable and near 0. During the elution the protein will change place with imidazole in the elution buffer and the protein will be eluted. The elution buffer contained 50 mM sodium phosphate pH 8, 0.3 M NaCl and 250 mM imidazole. The elution was finished when the absorbance A280 was stable and near 0. All the purification steps were performed at 4 °C. The used His-Select gel was stored in 20% ethanol after purification [11].

2.3.3. Purification with Äktadesign

To further purify the protein, size exclusion chromatography was performed using Äktadesign. A HiLoad 26/60 Superdex 200 prep grade column [12] was used with a buffer containing 0.05 M sodium phosphate pH 7.2 and 0.15 M NaCl. 0.8-1 ml sample was loaded and the eluate fraction size was 10 ml. A limit on the column pressure was set to 0.5 MPa and the flowrate was 2.5 ml/min. The length of elution was set to 1.5 column volume and A280 was measured throughout the analysis.

2.4. SDS-PAGE

A 10 % polyacrylamide gel was made [13] and 1xSDS buffer (5xSDS buffer; 15 g/l Tris, 72 g/l glycin and 5 g/l SDS) was used. The loading buffer (90µl) was mixed with

2-mercapetoethanol (10 µl), which breaks disulfide bounds. 10 µl of the sample and 10 µl of the loading buffer was mixed and heated for 5 minutes in a microwave oven to denature the proteins. The samples were loaded (10 µl) and as reference 2 µl PageRuler Plus Prestained Protein Ladder (Fermentas, Maryland, USA) was used. The electrophoresis was run for 30-45 minutes at 150 V. The gel was then cleaned with milli-Q water and stained with Coomassie Brilliant Blue (Fermentas). After one hour the stain was removed and the gel was destained with milli-Q water over night.

(10)

10 2.5. Western blot

To perform a western blot a 10 % polyacrylamide gel was run as described in section 2.4. The gel was then transferred to a nitrocellulose membrane (Amersham Bioscience, Buckinghamshire, England). The blotting was run for 90 minutes at 40 V in 1xtransfer buffer (25 mM Tris, 192 mM glycin, 20 % methanol).

Before binding of antibodies, the membrane must first be blocked in TBST (20 mM Tris-HCl pH 7.2, 150 mM NaCl, 0.05 % Tween 20) with 5% milk powder for one hour at room temperature in a Falcon tube. Then the primary Anti-His antibody, produced in mouse (1:6000) (Sigma-Aldrich), was added and incubated for one hour at room temperature. After one hour the membrane was washed with TBST buffer for 2x5 minutes. Then TBST containing 5 % milk powder and the secondary Anti-mouse-AP antibody, produced in rabbit (1:30000) (Sigma-Aldrich), was placed in the Falcon tube with the membrane and incubated for one hour at room temperature. The secondary antibody will bind to the primary antibody which has bound to the protein. The membrane was washed once more with TBST buffer for 2x5 minutes and washed with alkaline phosphate (AP) buffer (100 mM Tris-HCl pH 9.0, 150 mM NaCl and 1 mM MgCl2). To detect the protein a color reaction was performed. 0.5 ml 4-nitroblue tetrazolium chloride (NBT) (Invitrogen) and 0.5 ml 5-bromo-4-chloro-3-indolylphosphate (BCIP) (Invitrogen) were mixed with 4 ml milli-Q water and incubated with the membrane. When the protein had been stained the reaction was stopped by adding milli-Q water. Then the membrane was dried over night.

2.6. Protein concentration determination

To determine the concentration of the protein, the Bradford method was used. The concentration was calculated from a calibration curve that was plotted prior to use. The formula that was used was: y = 0,2594x, where y = A595 and x = concentration in mg/ml. As blank 800 µl milli-Q water was used and the protein was measured undiluted (5 µl sample and 795 µl milli-Q water). To the blank and sample, 200 µl coomassie reagent (BioRad Laboratories, Hercules, CA, USA) was added and the sample was measured at the wavelength 595 nm on a spectrophotometer after 5 minutes. At this wavelength the color of the reagent absorbs light and the protein concentration can be calculated [14].

(11)

11 2.7. Concentration of protein

The protein was concentrated using an Amicon tube with 10 kDa cut-off (Millipore, Billercia, USA) in a centrifuge at 4°C, 4000 rpm for 15-30 minutes. During the

concentration the buffer was changed if needed to a buffer containing 100 mM Tris-HCl pH 8 with 100 mM NaCl [15].

2.8. Cleavage with SUMO protease

The reason for cleavage using the SUMO protease was to remove the His-tag and the SUMO protein that was used during the purification steps, so that only the Majastridin-like protein would be obtained. Before the reaction was performed on all protein, the cleavage was performed on a smaller amount of the protein. 7.4 µl protein with a

concentration of 2.7 mg/ml, dissolved in 100 mM Tris-HCl pH 8 and 100 mM NaCl, was mixed with 10x SUMO protease buffer with 1.5 M NaCl (20 µl) (the protease need the salt for the reaction), SUMO protease (5 µl) (Invitrogen) and milli-Q water (167.6 µl) and the mixture was incubated at 30°C. Control samples were collected after 0, 1, 2, 4, and 6 hours for a gel electrophoresis analysis [10].

2.9. Crystallization

For crystallization the hanging drop method was used, which is a vapor diffusion

technique. The protein and salt concentration is lower in the drop than in the container and water will diffuse to the container from the drop until equilibrium. This means that the protein concentration will slowly increase and go from under saturation to super saturation where crystals can be formed [16]. To see under what conditions the protein will form crystals, a kit, Crystal Screen HR2-110 (Hampton Research, Aliso Viejo, CA, USA), was used. The kit contained 50 different reagents. If the protein will form crystal in any of these reagents optimization will be made around that condition. 24-well plates and 18 mm cover glass were used. 0.5 ml of the reagent was placed in the well and 2 μl from the reagent was placed on the cover glass with 2 μl of the protein sample, which had a concentration around 10 mg/ml. The plates were placed in room temperature for crystal formation.

(12)

12 2.10. Antibody production

To denaturate the protein 100 µl protein sample, which had a concentration around 10 mg/ml, was suspended in 2 ml 50 mM Na2H2PO4 pH 7 and 8 M urea and incubated for one hour at 4°C. Urea was replaced by centrifugation with an Amicon tube with 10 kDa cut-off (Millipore) to 10 mM Na2H2PO4 pH 7. The sample was then freeze-dried over night and sent for rabbit antibody production (David Biotechnologie, Regensburg, Germany) [17].

3. Result and discussion 3.1. Purification of protein

The Majastridin-like protein from Streptococcus pneumonia was purified with nickel affinity chromatography and figure 3 shows the result from the purification. On the same samples a western blot was performed (figure 4) which show that the Majastridin-like protein has been produced, but there are still other proteins present in the sample. These proteins have probably histidines that have bound to the nickel gel and have been detected by the antibodies. The fusion protein has a size of around 40 kDa, where SUMO protein is 13 kDa [10] and the Majastridin-like protein is around 30 kDa [2]. This is shown in figure 2, 3 and 4 where a band between 35 and 55 kDa is observed. In the flowthrough from the purification (figure 3) a lot of the fusion protein is found, why the flowthrough was repurified on the nickel affinity chromatography (figure 5).

1 2 3 4 5 6 7 L

Figure 3. SDS-PAGE on the fractions from purification of the protein. From left; (1) flowthrough, (2) wash

I with 5 mM imidazole, (3) wash II with 10 mM imidazole, (4) wash III with 15 mM imidazole, (5) wash IV with 15 mM imidazole, (6) eluate I and (7) eluate V eulted with 250 mM imidazole and (L) ladder.

35 55

(13)

13

L 1 2 3 4 5 6 7

Figure 4. Western blot on fractions from purification of the protein. From left; (L) ladder, (1) eluate V

eluted with 250 mM imidazole, (2) eluate I eluted with 250 mM imidazole, (3) wash IV with 15 mM imidazole, (4) wash III with 15 mM imidazole, (5) wash II with 10 mM imidazole, (6) wash I with 5 mM imidazole and (7) flowthrough.

1 2 3 4 5 6 7 L

Figure 5. SDS-PAGE of purified flowthrough from purification of the protein. From left; (1) flowthrough,

(2) wash I with 5 mM imidazole, (3) wash II with 10 mM imidazole, (4) wash III with 15 mM imidazole, (5) wash IV with 15 mM imidazole, (6) eluate I eluted with 250 mM imidazole, (7) eluate V eluted with 250 mM imidazole and (L) ladder.

3.2. Purification with Äktadesign

As shown in figure 3, 4 and 5 the protein purification with nickel affinity chromatography does not purify the protein completely. So, the Majstridin-like protein was further purified with size exclusion chromatography. Figure 6 shows the result from the size exclusion step. The first peak corresponds to the Majastridin-like protein which is the largest band

35 55 35

(14)

14

on the gel (figure 3) and will be eluted first during size exclusion. The other three peaks correspond to proteins that have been separated from the Majastridin-like protein and these proteins are smaller. Figure 7 shows an SDS-PAGE on the four peaks and figure 8 shows a western blot on the four peaks.

For crystallization and antibody production fraction B2 and C2 were chosen and concentrated to around 10 mg/ml. Fraction B2 has two bands (figure 7 and 8), one

between 35 kDa and 55 kDa which correspond to the Majastridin-like protein. The second band is between 70 kDa and 100 kDa, which can be a dimer (80 kDa) of the Majastridin-like protein. Fraction C2 has only one band (figure 7 and 8) between 35 kDa and 55 kDa, which correspond to the Majastridin-like protein.

Figure 6. Diagram of size exclusion chromatography with Äktadesign on the protein sample. The first peak

is the fusion protein and the second, third and fourth peak are other proteins that were separated from the fusion protein.

B2 C2 A3 B3 A4 B4 C4 D4 L

Figure 7. SDS-PAGE on the peaks in the diagram (figure 6) from size exclusion chromatography of the

protein. From left; B2, C2, A3, B3, A4, B4, C4, D4 and ladder (L).

35 55 Majastridin-like protein, 40 kDa

(15)

15

L D4 C4 B4 A4 B3 A3 C2 B2

Figure 8. Western blot on the peaks in the diagram (figure 6) from size exclusion chromatography of the

protein. From left; ladder (L), D4, C4, B4, A4, B3, A3, C2 and B2.

3.3. Cleavage with SUMO protease

The SUMO cleavage was performed to remove the His-tag and the SUMO protein from the Majastridin-like protein, but as shown in figure 9 the cleavage did not work. If the reaction would have worked, two bands should have been visible on the SDS-PAGE. One at 13 kDa [10] which correspond to the SUMO protein and one at 30 kDa [2] which correspond to the Majastridin-like protein. But on the gel a weak band can be observed between 35 kDa and 55 kDa, which is the Majastridin-like protein with the SUMO protein attached. A reason to why the protease did not cleave can be that too little protease was used for the cleavage reaction or that the cleavage site is hidden by the protein. Since the Majastridin-like protein’s first amino acid is methionine [9], the SUMO protease cut the protein less effectively than if the first amino acid would have been a serine, the amino acid which gives the most effective and optimal cleavage reaction. Therefore, the PCR primers could be redesigned to introduce a serine at the beginning of the Majastridin-like protein [10].

35 55 70 100

(16)

16

0 1 2 4 6 L

Figure 9. SDS-PAGE on the cleavage of the protein using SUMO protease for different time points. From

left; 0h, 1h, 2h, 4h, 6h and ladder (L).

3.4. Crystallization

The Majastridin-like protein from Streptococcus pneumonia was crystallized with the hanging drop method. After one day, several crystals had been formed but only one condition was optimized for further crystallization. The condition that was chosen was where most and best-looking crystals had formed after one day. The crystals had formed with the protein that corresponds to fraction B2, which had two bands in figure 7 and 8. The crystals were thin, triangular and had formed three different groups within the crystallization drop (figure 10). Two of these groups were similar and the third had crystals that were clearer and fewer. In the same drop a large single crystal was also formed after three weeks (figure 10 and 11).

35 55

(17)

17

Figure 10. Picture of the crystals within the crystallization drop.

Figure 11. Picture of the large single crystal within the crystallization drop.

The condition that was optimized was 0.2 M potassium chloride, 0.1 M sodium acetate pH 4.6 with 20 % v/v iso-propanol. In earlier work where Majastridin from Rhodobacter blasticus was studied the final crystallization conditions were 100 mM sodium citrate, pH 6.0 with 75 mM magnesium acetate [3]. The mix was remade with the original content to try to repeat the crystal formation and the salt concentration, pH and iso-propanol

concentration were also varied around the original concentrations to see if a better condition could be created. Streakseeding was also performed under the original conditions (0.2 M potassium chloride, 0.1 M sodium acetate pH 4.6 with 20 % v/v iso-propanol) to see if better crystals could be formed.

Groups of crystals

(18)

18

After one day, crystals had started to form in the optimization experiment. This time the crystals were formed in the mix that was made by us and not under conditions of the kit first used. The crystals are similar to the first crystals; thin, triangular and had formed one group but with fewer crystals within the group.

Several optimization experiments on the same condition were made before the crystal formation could be repeated. Also, no crystals had been formed in those conditions where the salt concentration, pH and iso-propanol concentration were varied. One conclusion is that the crystals formed best under the original condition or in a condition very close to it. Another reason to why it was hard to repeat and form crystals can be that the Majastridin-like protein is fused with the SUMO protein, since the SUMO cleavage did not work.

3.5. Antibody production

The freeze-dried Majastridin-like protein was sent for rabbit antibody production to David Biotechnologie in Regensburg, Germany. The antibodies have not been tested on the Majastridin-like protein yet since the production is not finished.

4. Conclusion

The Majastridin-like protein from Streptococcus pneumonia has been expressed in E. coli cells and then purified with nickel affinity chromatography and size exclusion

chromatography. The purified protein has been crystallized using the hanging drop method and was used to rabbit antibody production. For the future, the cleavage reaction with the SUMO protease has to be optimized and better conditions for crystal formation have to be created so that the protein structure can be determined, which gives a clue to the function of the Majastridin-like protein from Streptococcus pneumonia.

5. Acknowledgments

Elin Grahn, PhD, Örebro University (Life Science Center), for tutoring me during this work and all the feedback on the report.

(19)

19

Professor Åke Strid, PhD, Örebro University (Life Science Center), for letting me be a part of your laboratory group.

Irina Kalbina, PhD, Örebro University (Life Science Center), for helping me to prepare the sample for antibody production and answering my questions.

Ingrid Lindh, PhD student, Örebro University (Life Science Center), for helping me with antibody production.

Andreas Ottosson, Johanna Sundin, Anna Gustavsson and Jan Bauer, for your cooperation and daily laboratory work.

(20)

20

6. References

1. Falk G, Walker E. J, DNA sequence of a gene cluster coding for subunits of the F0 membrane sector of

ATP synthase in Rhodospirillum rubrum, Biochem. J. 254, (1988), 109-122

2. Borsché M, Kalbina I, Arnfelt M, Benito G, Karlsson B.G, Strid Å, Occurrence, overexpression and partial purification of the protein (majastridin) corresponding to the URF6 gene of the Rhodobacter blasticus atp operon, Eur. J. Bichem. 255, (1998), 87-92

3. Enroth C, Strid Å, Crystal structure of a protein, structurally related to glycosyltransferases, encoded in the Rhodobacter blasticus atp operon, Biochim. Biophys. Acta 1748, (2008), 379-384

4. Wikipedia, http://en.wikipedia.org/wiki/Glycosyltransferase 2009-02-27

5. NCBI Blast, http://blast.ncbi.nlm.nih.gov/Blast.cgi 2009-03-02

6. Wikipedia, http://en.wikipedia.org/wiki/Hemolysis_(microbiology) 2009-03-05

7. Wikipedia, http://en.wikipedia.org/wiki/Streptococcus 2009-02-11

8. Wikipedia, http://sv.wikipedia.org/wiki/Streptokocker 2009-02-11

9. NCIB, http://www.ncbi.nlm.nih.gov/protein/122682326 2009-03-03

10. Invitrogen, Carlsbad, CA ,USA, ChampionTM pET SUMO Protein, Instruction Manual

11. Sigma-Aldrich, Saint Louis, MO, USA, His-Select Nickel Affinity Gel, Technical Bulletin

12. GE Healthcare Biosceince AB, Uppsala, Sweden, High Performance Columns, Instructions

13. Biorad, Mini-PROTEAN® 3 Cell, Instruction Manual

14. Fermentas, http://www.fermentas.com/catalog/electrophoresis/bradford.htm 2009-03-05

15. Millipore, http://www.millipore.com/techpublications/tech1/pf1055en00 2009-02-25

16. Rhodes G, Crystallography Made Crystal Clear, Elsevier Inc. (2006), 37-40

References

Related documents

The binding of Slr and M1 protein, the wild type strain AP1, and mutant strains MB1 and MC25 strains to immobilized collagen I was investigated using surface plasmon resonance..

 We have solved the crystal structure of the variable domain of the AgI/II adhesin SspB from S. We conclude that the overall structure of the SspB variable domain

The focal company being one of the stakeholders in the supply chain would like to have an overview on how E-commerce has impacted the American and European markets,

There were some effective interventions including abdominal massage, health guidance, avoid the source of infection, equipment management and oral care could be used by nurses to

When looking at protein structure it is common to study it on four separate levels. The first level is the primary structure, which is the actual sequence of linked amino acids.

Purification of 5g of cells with EcLepB_V77_his, purified according to protocol version twelve resulted in a protein concentration in elution of 0.1mg/ml corresponding to

This pub- lication of the Stora Brunneby hoard should be regarded as a first step towards the evaluation of the LEO databases, now that the relationship between Scandinavian

Figure 30: The averaged DAMMIF model result out of 20 runs with the experimental data from the monomeric peak for PerR treated with EDTA and zinc.. The mesh represents the