Cloning and expression of superoxide dismutase from Sarcoptes scabiei in Escherichia coli
Luis Sanchez Lecaros
Department of Parasitology (SWEPAR), National Veterinary Institute, SE-751 89 Uppsala Sweden
Supervisors: Eva Pettersson and Jens Mattsson
ABSTRACT
Sarcoptes scabiei is a disease-causing parasitic mite of humans and animals that is prevalent worldwide. The parasite lives in burrows in the epidermis of its host. These burrows are formed by a combination of mechanical destruction by the mite and secretion of various factors.
The enzyme superoxide dismutase (SOD) catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. As such, it is an important antioxidant defense in nearly all cells exposed to oxygen. In this project, the enzyme was expressed in transformed Escherichia coli cells. The SOD cDNA from S. scabiei was ligated into two different expression vectors:
pPU16 and pET-14b. The S. scabiei SOD open reading frame reported here is 696 nucleotides long and yields a protein with a molecular weight of ∼ 69.5 kDa. Only one of the constructs was successfully created, using pPU16. The construct was designated pPU110 and has a sequence coding for a hexahistidine tag downstream of the SOD cDNA and has a sequence coding for the maltose binding protein (MBP) upstream.
The expression plasmid pPU110 was verified by DNA-sequencing and the tested in different expression experiments. Analysis using SDS-PAGE showed that recombinant fusion SOD could be readily expressed in E.coli.
KEYWORDS: recombinant protein, SOD, maltose binding protein, his-tag, defence _____________________________________________________________________
INTROD
he mite Sarcoptes scabiei (Arthropoda: Arachnida: Acari) is responsible for scabies or mange, an endemic skin disease that exist in many disadvantaged populations worldwide and affect wild and domestic animals. This parasite common
UCTION
has been identified in more than 40 different mammals including man. A name of this parasite is the “itch mite” and the name is derived from the severe prurities that it causes.
T
Secondary infections with pathogenic bacteria such as Streptococcus pyogenes is not
caused by the burrowing of this parasite. The most affected skin layer and where most of the damage is seen is called stratum corneum and corresponds to the upper part of the skin. This damage causes an infiltration of different kinds of immunological cells to the affected area such as neutrophils and macrophages and the immune response is extensive. The transmission of this parasite is by skin-to-skin contact and the transmission by sexual contact is not an exception. [1, 2].
The clinical manifestations of this parasite in humans are among others:
pruritic rash in anterior axillary folds, skin around the navel, etc. while in infants the palms, soles, face, neck and scalp may also be involved. By an early infection the symptoms are almost unrecognisable but 4-6 weeks later the host develops an immune response to the mite or its excrements. At this point the host’s been affected by purities and inflammations on the skin. A reinfection can immediately cause symptoms after just 1-3 days [3]. Special forms of S. scabiei show different symptoms. In humans the most affected are patients with a severe immunological deficiency like HIV-patients and organ transplant recipients [4]. S. scabiei is also of great relevance within veterinary medicine, especially in dogs and pigs, were the parasite is not only animal welfare problem but also has economic impact [5].
The clinical diagnosis must be confirmed by the microscopic demonstration of mites, eggs or its feces [6]. The skills of the physician or veterinarian are very important in the diagnosis of scabies and samples are prepared by removing parasites its burrows using a surgical blade, by biopsy, by skin scraping etc. [7]. Alternatively immunological methods such as immunoassays can be used to diagnose the disease parasite.
The result of every aerobic metabolism is the formation of reactive oxygen molecules that cause the well-known process of oxidative stress. Superoxide (O•2 ) and hydroxyl radicals ( OH) are the most important radicals containing unpaired electrons that increase their capacity to react with biomolecules [8]. Superoxide dismutases (SODs) catalyses the conversion of these highly reactive molecules to O2
2 2 2 2
2
•
and hydrogen peroxide (H O ). H O has a lower toxicity but it can spontaneously be converted to highly reactive radicals. Consequently, the enzymes catalase and glutathione peroxidase, which belong to the SOD-family, convert hydrogen peroxidase into H O [9]. Eukaryotes possess two major types of SOD: Mn SODs, which are found exclusively in mitochondria and CuZn enzymes that are localised either in the cytosol or peroxisomes.
This work has focused on the construction of two different vectors for the expression of SOD from S. scabiei. One includes a large fusion partner (pPU16, fig 1) that stabilises recombinant proteins and affinity purification tag (His6 – tag) (Mattsson et al, 2001). The second one (pET-14b, fig 1) has a short leader peptide including a His6 – tag. The His6 – tags are powerful tools for protein purification. The resulting recombinant SOD will be used to study the host – parasite interaction in future studies.
pPU16
6665 bp
mal E lacI
Amp-r rop
Histag Ptac lacI promoter
M13 origin pBR origin
pET-14b
4671 bp
T7-pro
ori
T7-ter
bla Bam HI (511)
Nde I (523)
Bam HI (2702) Pst I (2724)
Fig.1 Schematic diagram of expression vectors pET-14b and pPU16. The pET-14b plasmid has a t7 promoter (T7-pro) while pPU16 has a tac promoter (Ptac). The restriction sites shown are for cloning and control of insert.
2. MATERIAL AND METHODS
2.1 PCR-amplification of the superoxide dismutase cDNA from S.scabiei
Two master mixes were prepared using two different primer pairs: OP551- OP552 (designed with restriction sites for PstI and BamHI to allow the insertion of the cDNA into pPU16) and OP553-OP554 (designed with restriction sites for NdeI and BamHI to allow the insertion of the cDNA into pET14b). The master mixes contained: 0.2 mM of each dNTP, 10× Cloned Pfu-buffer, 2.5 units of Pfu Turbo DNA polymerase (Stratagene), 20 pmole of each primer and dH2O. Master mix I had the primer pair OP551 (forward primer 5´- CGG GAT CCA TGT TCC ATC AAA GGT TT-3´) and OP552 (Reverse primer 5´-AAC TGC AGT CCA ATG CTG ACA AAT TT-3´) while the master mix II contained: OP553 (Forward primer 5´-GGA ATT CCA TAT GAT GTT CCA TCA AAG GTT T-3´) and OP554 (Reverse primer 5´-CGG GAT CCC TAT CCA ATG CTG ACA AA-3´). From the master mixes 49 μL were transferred into individual PCR-tubes. For each primer combination, one negative control with dH2O was used. To the remaining four tubes 1 μL cDNA from S. scabiei was added. The following PCR program was used: an initial denaturation at 95°C for 2′ and then 95°C for 30″; 55°C for 30″ and 72°C for 1′ over 30 cycles, the run ended with an extension at 72°C for 10′ followed by cooling to 4°C.
For the post-PCR analysis 5 μL from the PCR-reaction was mixed with 2 μL gel loading solution (GLS) and 3 μL TE-buffer pH 8.0 (10 mM Tris HCl and 1 mM EDTA) and loaded on 1% agarose gel containing ethidium bromide. The gel was then photographed with a Kodak Electrophoresis Documentation and Analysis System 120. Afterwards the PCR products were purified with GenClean (Qbiogene).
2.2 Ligation of cDNA into pCR-Script Amp Sk (+) cloning vector.
The cDNA amplicon from S. scabiei was ligated into the cloning vector pCR- Script Amp Sk (+). A ligation-mixture for each cDNA was prepared. This mixture contained: 1 μL pCR-Script Amp Sk (+) cloning vector (10 ng/μL), 1 μL 10× pCR- Script buffer, 0.5 μL 10 mM rATP, 5.5 μL cDNA, 1μL SrfI (restriction enzyme, 5U/μL) and 1μL T4 DNA-ligase (4U/μL). The mixtures were mixed, incubated for 1 hour at RT and then heated to 65°C water bath before transfer to an ice bath.
2.3 Electroporation
In two 1.5 mL micro centrifuge tubes 40 μL of electro competent XL-1 Blue MRF´ cells were mixed with 2 μL ice-cool ligation mix. The samples were transferred to an electroporation cuvette, avoiding the formation of bubbles. The mixtures were electroporated at 1.7 kV. Immediately after the pulse 960 μL LB-medium was added and then mixtures were incubated on a shaker at 37°C for 1h . Transformed cells were isolated on selective LB/Amp-plates containing 5´-bromo-4-chloro-3-indolyl-β-D- galactopyranoside (X-gal, Sigma) and isopropylthiogalactosid (IPTG) for selection of positives and negatives colonies. These plates were incubated overnight (ON) at 37˚C.
2.4 Colony PCR
Single white colonies were resuspended in 50 μL dH2O in 1.5 μL micro centrifuge tubes and at the same time, inoculated on LB/amp-plates. The tubes were then boiled for 5 minutes and centrifuged for ∼ 1 minute at 13500 rpm. A master mix for 15 × PCR-reactions was prepared as follow: 37.5 μL 10×PCR buffer (with 15 mM MgCl2), 7.5 μL 0.2 mM dNTP, 15 μL 20 pmol/μL T7 (forward primer. 5´-GTA ATA CGA CTC ACT ATA GGG C-3'), 15 μL 20 pmol/μL T3 (reverse primer. 5´-AAT TAA CCC TCA CTA AAG GG-3´) 1.5 μL Taq polymerase (5U/μL; Applied Biosystems) and 283.5 μL dH2O. To 14 PCR tubes 24 μL master mix was added.
Two of these tubes were used as negative controls with 1 μL dH2O instead for DNA.
The following PCR-program was used for this reaction: (96°C for 15″; 45°C for 20″;
72°C for 2′) over 30 cycles before cooling to 4°C. The PCR product was analysed in a 1% agarose gel containing 0.5 μg/mL ethidium bromide.
Based on the result from gel analysis a single colony was resuspended in 100 mL LBAmp-medium and incubated on shaking at 37°C ON. The plasmid purification was then performed using the Wizard® Plus Midipreps DNA purification system. The plasmids were eluted in 300 μL preheated (at 65°C) TE-buffer pH 8.0 and the concentration of DNA was finally measured by spectrophotometry at 260 nm.
2.5 DNA Sequencing
The DNA was sequenced by the dideoxynucleotide chain termination method using Big Dye Terminators (BDT) v3.0 Cycle Sequencing Kit (Applied Biosystems).
For this reaction one tube were used for every primer. These tubes contained: 2μL BDT 3.1, 3μL AB1 5× buffer, 5μL 20 pmol/μL primer (T7 to one of these tubes and
T3 to the other) 10μL dH2O, 1μL PCR-product. The sequencing reaction was run according to the following program: (96°C for 10″; 45°C for 5″and 60°C for 4′) over 25 before cooling to 4ºC. The reaction products were purified and concentrated with 95% EtOH (ethanol) and NaAc pH 4.6. Finally, the resulting pellet was resuspended in 11 μL hidi (formamid, Applied Biosystems) before the sequencing analysis was performed. The analysis was performed with ABI PRISM 3100 Genetic Analyser (Applied Biosystems) and Vector NTI suite of programs (Invitrogen).
2.6 Construction of expression plasmids – restriction enzyme digestion
To prepare the cDNA the following components were added to a micro tube: 6 μg pCR-Script-SOD cDNA (33 µl), 10 μL OPA (One Phor All buffer, GE Healtcare), 1 μL PstI and 6 μL dH2O were added. To prepare pPU16 the following components were added to a micro tube: 1 μg pPU16 (3 μL), 1 μL BamHI, 8 μL 0.4-2 ×OPA and 28 μL dH2O. The mixtures were incubated at 37°C for 30 min. Thereafter 1 μL of BamHI was added to the first tube and 1μL PstI was added to the second tube and then the tubes were incubated again 1 hour at 37°C. The cleavage result was analysed in a 1% agarose gel as above. The cleaved DNA was finally purified from a preparative gel with Gel-M™ Extraction system (Viogene)
2.7 Ligation of SOD gene into expression plasmid.
Three ligation mixes were used. Each mixture contained: 1 μL plasmid (cleaved pPU16), 1 μL 10× T4 DNA ligase buffer, 1 μL DNA ligase and 6 μL dH2O.
The cDNA-insert was then added to two of these reaction mixtures at different concentrations: 1 μL undiluted and 1 μL diluted 1:10. The negative control contained 1 μL dH2O instead of insert DNA. All reactions were incubated at 16°C for ∼ 17 h.
The ligation product was introduced in competent E. coli (XL-1 Blue MRF′) by electroporation as previously described. Then the transformed cells were isolated on five selective LBAmp-plates and incubated ON at 37°C.
A new Colony PCR was performed to identify positive clones. Singles colonies were used and the following master mix was prepared: (33 × PCR-reaction) 82.5 μL 10×PCR buffer, 16.5 μL dNTP, 33 μL 20 pmol/μL MalE (forward primer.
5´GGT CGT CAG ACT GTC GAT GAA GCC-3´), 33μL 20 pmol/μL pPU16rev (reverse primer. 5´AGG CGA TTA AGT TGG GTA-3´.), 3.3 μL Taq polymerase, 623.7 μL dH2O. PCR program: (96°C for 15″; 45°C for 20″; 72°C for 2′) over 30 cycles before cooling to 4°C. The PCR products were analysed on agarose gel as above.
Five positive clones were inoculated into 10 ml LBAmp medium with ampicillin and incubated ON at 37°C on a shaking platform. The plasmids were then purified using the Wizard® Plus Minipreps DNA purification system (Promega) as described in manufacture’s manual.
The purified plasmid was sequenced using the primers MalE and pPU16Rev as previously described.
2.8 Test expression of superoxide dismutase
One of the correct clones was inoculated in 5 mL LBAmp-medium and incubated on a shaker at 37°C ON. The following morning 200 μL of this culture were transferred to 10 mL fresh LBAmp-medium. The cells were grown on a shaker at 37°C until the optical density reached ∼ 0.5 at 600 nm. A sample of 1 mL from the culture was transferred to a 1.5 mL micro centrifuge tube and centrifuged at 13500 rpm for 2 min. The supernatant was discarded and the resulting pellet was resuspended in 50 μL 1×SDS-PAGE sample buffer (0.5 M Tris-Hcl pH 6.8, glycerol, 20 % SDS, 0.1 % BFB, DTT). The remaining culture was induced for protein expression by the addition of IPTG to a final concentration of 0.3 mM. This culture was incubated at 37°C on a shaker during 2 hours. Afterwards 0.5 mL f was transferred to a 1.5 mL micro centrifuge tube and centrifuged at 13500 rpm for 2 minutes. The supernatant was discarded and the pellet resuspended in 100 μL 1×SDS- PAGE sample buffer.
2.9 Protein separation by Sodium Dodecyl Sulfate-Poly Acrylamid Gel Electrophoresis (SDS-PAGE)
A 12% SDS-PAGE separation gel was prepared from 0.375M Tris-HCl pH 8.8, 12% acrylamide; 0.1% SDS; 0.1% ammonium persulphate (APS) and 10μL N,N,N´,N´-tetramethyl-ethylenenediamine (TEMED). A 4% stackingel was prepared by combining 0.1M Tris-HCl pH 6.8; 4% acrylamide; 0.1% SDS; 0.1% ammonium persulphate (APS) and 20μL TEMED. The gel were run at 200 V for about 30 min in 1× running buffer (25mM Tris, 250mM glycine and 0.1% SDS). Afterwards the gel were stained with Coomassie Brilliant Blue solution (0.1% Coomassie Brilliant Blue, 0.1% Hac and 0.4% MeOH) ON and destained with destaining solution (0.1% Hac and 0.4% MeOH). Finally the gels were photographed with Kodak Electrophoresis Documentation and Analysis System 120.
2.10 Large-scale expression of Superoxide dismutase
The SOD-pPU16 expression plasmid, designated pPU110, was transformed into E. coli strain BL21 (DE3) for large-scale expression of the recombinant protein.
A single colony was inoculated in 20mL LBAmp medium and incubated ON at 37°C on a shaker. Afterwards 2.5 mL from the ON culture was transferred to 250 mL Minimal medium with casamino acids (MM/CA-medium) (50% glukos, 1 mg/mL biotin, 20 mg/mL Thiamin, 1g/5 mL (NH4)2SO4, dH2O, 4% Heavy metal stock, 10%
Casamino acids, 5× MM/CA phosphate buffer, ampicilin) and then incubated at 37°C until the optical density reached ∼ 0.8 –1.00 at 600 nm. The culture was then cooled for 5 minutes at 22°C in a water bath. From this culture 1 mL was transferred to a micro centrifuge tube and centrifuged at 17000 rpm for 2 minutes. The supernatant was discarded and the pellet resuspended in 50μL 1× SDS-PAGE sample buffer.
IPTG was added to the remaining culture to a final concentration of 0.5 mM, and incubated on a shaker ON at RT.
2.11 Purification of SOD
The cells from the large-scale expression were lysed by sonication with amplitude of 3, medium power, with 6 pulses. Each pulse was 15 s long. The cell lysate was centrifuged at 4°C at 9000 rpm for 30 min and the supernatant was filtered through a 0.45 μm filter. The recombinant protein was then purified using a peristaltic pump and an affinity chromatography column (1 mL HiTrap chelating HP column (GE healthcare)). Firstly the column was pre-loaded with Ni2+ ions as suggested in the manufacture’s protocol. In the next step, the column was equilibrated with 10mL start buffer, pH 7.44 (Phosphate buffer 8× stock solution pH 8.8; 10mM Imidazol pH 7.4).
The equilibration of the column was performed with a flow rate of 4mL/min. In the third step the protein solution was loaded onto the equilibrated column with a flow rate of ∼ 1-2 ml/min. The flow through was collected in a falcon tube. After loading the protein, the column was washed with 10mL start buffer, pH 7.44 (Phosphate buffer 8× stock solution pH 8.8; 10mM Imidazol pH 7.4) with a flow rate of ∼ 2.0- 2.5mL/min. The flow through of the wash was collected in micro centrifuge tubes (1.5 ml wash product in each tube). Finally, the elution of the recombinant protein was performed with 5mL elution buffer pH 7.52 (Phosphate buffer 8× stock solution pH 8.8; 0.5M Imidazol pH 7.4; dH2O). The eluted proteins were collected in micro centrifuge tubes as well. The result of this purification was analysed on a 10% SDS- PAGE separation gel.
3. RESULTS AND DISCUSSION
The aim of this study was to clone and express superoxide dismutase originating from S. scabiei. For this purpose two different expression plasmids from pPU16 and pET14b was used. Unfortunately, there were several factors that made hampered the design of a pET14b-based system and by the end of this project the expression could only be done with the pPU16-based construct pPU110.
3.1 PCR-amplification of the superoxide dismutase cDNA from S.scabiei
Initially a PCR-amplification was done on cDNA from S. scabiei and in order to produce SOD cDNA suitable for sub-cloning.
1 2 3 4 5 6 7
700 bp→
Fig.3 Analysis of the PCR amplification on a 1% agarose gel. Lane 1: M: Marker (100bp); lanes 1 and 2 are the same gene amplified with the primer pair: OP551-OP552 and the line 3 and 4 are the same gene amplified with the primer pair OP553-OP554. Lane 5 and 6 are negative controls. The gene was amplified with duplicate tests.
The result of the PCR was amplicons of the correct size of about 690 bp. The concentrations measured after the purification of the DNA from this gel (fig 3) gave the following results: sample 1 from lane number 1: 0.0936 μg/μL; sample 2 from lane number 3: 0.108 μg/μL.
3.2 Ligation of cDNA into the cloning vector
The ligation of the amplicon into the cloning vector is a process that used to produce DNA for subsequent cloning step. By this procedure one can be ensured to have cDNA that will be easy digest and have the correct restriction enzyme ends for future manipulations.
To clone cDNA in a plasmid vector can be a fairly challenging procedure because of the instability of the new recombinant plasmid vectors. There are risks that the new constructed plasmid can be lost during the cell division or inhibit the growth of the cell and, in some cases even kill it. Structural rearrangement of the host cell has also been observed [10]. Fortunately, in this study, the cloning of the SOD-gene into pCR-Script worked without any problems and both white (positive, suggesting that the cells carried the insert cloned in pCR-Script) and blue (negative) colonies could be observed.
3.3 Electroporation
The transformation of E. coli cells was a very important stage in this project.
The cells are very sensitive to heat so during the most steps of this procedure, the cells were kept on ice. This increases the chance to have good transformation frequencies.
In some experiments, I had problems with arching: the electroporation cuvette
“explodes”. This is due to for example, bubbles in the sample or if the current is too high because of a high ion-strength. When this happens the cells are killed and a new transformation is needed.
3.4 Colony PCR
From the twelve single white colonies that were used in this trial, just one clone was positive.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M
←700 bp
Fig. 4. Result of the colony PCR analysed on a 1% agarose gel. The band in the lane number 3 is positive. Relevant molecular size of the bands is indicated to the right. Lanes 1-6 are genes amplified with the primer pair OP551-OP552 and from the lane 7 to 12 the genes were amplified with the primer pair OP553-OP554. Lane 13 and 14 are negative controls.
The SOD-gene from S. scabiei was originally amplified with two primer pairs, with different restriction sites suitable for pPU16 and pET14-b, respectively. In this gel, lane number 3 represents a positive clone, suggesting that the ligation of the gene into pCR-Script gave a satisfactory result and that the fragment corresponds to a cDNA suitable for ligation into pPU16. Because the ten week time-limit of this project no further attempts were made to work with the pET14-b system and all time was focused on the cloning and expression using pPU16.
3.5 Purification of plasmids
From the plate containing the positives colonies (fig.2) a single colony was inoculated in 100 mL LB-medium. During this project, the cultivation of the bacteria was done in the same way when plasmid purification would be performed. In all cases the growth of the cells gave good results, with a large amount of cells. Unfortunately, this procedure took, in some cases, more time than expected. An inexperienced student could of course be of some influence to the whole procedure, but sometimes, the ON cultures gave unexpectedly low concentrations of plasmid DNA.
3.6 The DNA sequencing
The sequencing of cDNA established that the insert length was 696 bp. Furthermore, after the cloning into pPU16 we could also confirm that the insert was in-frame with the rest of the sequences and that no PCR-induced errors had been introduced in the cDNA. Thus, the resulting expression plasmid was called pPU110 and was constructed from pPU16 and carried the SOD in-frame with the MBP and the his-tag.
3.7 Test and large expression of SOD.
For the test expression, the cell cultivation was done in 100 mL LBAmp- medium and incubated on a shaker at 37°C ON and then from this solution, 200µL were transferred to 10 mL fresh medium and the same incubation was done, until the OD (optical density) would reach about0.5. The graph below shows the growth of the recombinant bacteria. Because bacteria have a exponential growth, in the first minutes of this incubation the cells were multiplied in a very slow speed but with time, the bacteria concentration began to increase quickly and the desired OD was be reached in a very short time. After a first sample was taken the bacteria was induced to express the recombinant protein.
Bacterial growth in LB-medium
0 0,1 0,2 0,3 0,4 0,5 0,6
0 20 40 60 80 100
Measurements (min)
Absorbance at 260 nm
Serie1
Fig 5. Growth-curve of the recombinant E. coli cells. The second measurement was done ∼ 1 hour after the first. The remaining three measurements were done with a time interval of ∼10 minutes.
A protein separation was performed by a 12% SDS-PAGE and it was clear that IPTG induced the cells to produce the recombinant protein (lane 2, fig 6). The results suggested that the expression levels of SOD had a satisfactory result.
M 1 2
∼70 kDa →
Fig 6. Result of the test expression of SOD from pPU110 (Coomassie stained 12% SDS-PAGE separation gel). The line 1 shows uninduced expression of SOD (without IPTG) and the lane number 2 shows induced expression with IPTG additive. Relevant molecular size shows to the right.
The large-scale expression of this recombinant protein was basically done in the same way but the cells were grown in a defined medium. The MM/CA-medium normally guarantee a higher expression of the recombinant protein compared to other richer media. The cells grow slower and it can take several hours to reach the target OD. In this case, the desired optical density at 600 nm was ∼ 0.8 – 1.0. Several measurements were done and it took ∼ 4:30 hours to reach correct cell concentration.
with a OD of 0.852 (fig 7).
Bacterial growth in MM/CA medium
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9
0 50 100 150 200 250 300
Measurements (min)
Absorbance at 660 nm
Serie1
Fig 7. Growth curve of the cells in MM/CA-medium. The figure shows the time of every measurement.
At the point when protein expression was induced samples were taken and later analysed by a 10% SDS-PAGE.
M 1 2 3 4
∼ 70 kDa→
Fig 8. Result of the large-scale expression of SOD from pPU110 (Coomassie stained 10% SDS-PAGE separation gel). The lanes 1 and 2 show uninduced expression (without IPTG) and lanes 3 and 4 show induced expression with IPTG. Relevant molecular size shows to the right.
3.8 Protein purification
During the last thirty years, different purification systems have been developed for the purification of recombinant proteins. Most of these have fusion proteins to improve the purification of the recombinant protein under study. The fusion-protein-system aids in the efficient purification and recovery of the
recombinant proteins from cell extracts and other organic solutions. These systems involve structures called “tags” that are composed of amino-terminal proteins. These tags aid in the purification through binding them to a high affinity matrix [11]. This structures or “tags”, which are bound to the N-terminus of the recombinant protein of interest, can even increase its solubility [12, 13].
The purification of SOD produced from pPU110 was performed with affinity purification by means of the His6-tag interacting with a HiTrap Chelating HP column.
When this protein was expressed, a His-tag was also produced at the same time with SOD. The His-tag binds to the Ni2+-ions on the column and by this way, the protein can be captured and purified [14]. The affinity purification with HiTrap Chelating HP columns gave very good yield. SOD could be purified but unfortunately, a total purification from others cell proteins could not be observed (fig 9)
M 1 2 3 4 5 6 7 8 9
∼ 70 kDa→
Fig. 9. Purification of SOD using HiTrap Chelating HP column (12% SDS-PAGE separation gel, Coomassie stained). Lane 1 shows the sonication product, lane 2 is the flow through fraction, lanes 3 and 4 are the wash fractions 1 and 7 respectively and the lanes 5 to 9 represent the final eluted protein fractions. Relevant molecular size shows to the right.
In lanes 6 and 7 (fig 9) SOD can be observed as a bold band but unfortunately, other cell proteins continued its passage through the column and were also eluated with our recombinant protein. This suggests that additional steps have to be taken into consideration before the enzyme can be studied in more detail.
4. ACKNOWLEDGMENT
I wish to thank my supervisor Eva Pettersson for all help I got during the performance of this project. Thank you for answer my questions, for all the advices I got from you and of course, for your infinite patience. Thank you for never being far away from a smile when I felt that my world would go under every time I did something wrong in the laboratory. I really appreciate it.
I want to thank my other supervisor Jens Mattsson for gave the opportunity to do my degree project in the department of parasitology at SVA. Thank you for all your advices and scientific support.
Annie Engström and Katarina Näslund have also being a big help in the performance of this project. Thank you both for your support in the lab.
Finally I would like to thank everyone in the department of parasitology for a really nice time.
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