Linköping University | Department of Physics, Chemistry and Biology Bachelor thesis, 16 hp | Educational Program: Physics, Chemistry and Biology

Spring term 2016 | LITH-IFM-G-EX—16/3194—SE

Does Sterol Carrier Protein-2 promote the expression of foreign proteins in Escherichia coli?

Isak Mikkola

Examinator, Jordi Altimiras Tutor, Johan Edqvist

2

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Department of Physics, Chemistry and Biology

Linköping University

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ISRN: LITH-IFM-G-EX--16/3194--SE

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Title

Does SCP-2 promote the expression of foreign proteins in Escherichia coli?

Författare

Author

Isak Mikkola

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Keywords

CAT∆9, Escherichia coli, GFP, inclusion body, pET-15b, SCP-2, solubility tag

Sammanfattning

Abstract

Expression of foreign proteins in host organisms usually results in the development of insoluble,

inactive proteins. Further, these proteins have a tendency to form aggregates termed inclusion

bodies. However, the formation of inclusion bodies can be avoided by fusing the gene encoding

the foreign protein to a highly soluble protein. In this report Sterol Carrier Protein-2 (SCP-2) is

reviewed as a possible solubility tag. The experiment was carried out by fusing SCP-2 to one of

two insoluble proteins, Green fluorescent protein (GFP) or a form of chloramphenicol acetyl

transferase (CAT∆9). The protein fusion was then inserted into the vector pET-15b, transformed in

Escherichia coli and the yield of actively expressed protein was measured. The results obtained

from this study, as evaluated by PageBlue staining and Western blot, are indicating that SCP-2

does not improve the solubility of GFP or CAT∆9. Nonetheless, the solubility of GFP has earlier

been increased by fusing it to the solubility tag maltose-binding protein (MBP). Producing more

soluble forms of CAT∆9 have also been tested but without success. Therefore the conclusion

drawn from this experiment is that SCP-2 does not work as a solubility tag, however more research

must be performed to conclude this with certainty.

Datum:

Date: 2016-06-01

3

Content

1 Abstract ............................................................................................... 4

2 Introduction ......................................................................................... 4

3 Material & methods ............................................................................ 6

3.1 Bacteria ............................................................................................. 6

3.2 Vector and genes .............................................................................. 6

3.3 DNA manipulations .......................................................................... 7

3.4 PCR ................................................................................................... 9

3.5 Expressing proteins .......................................................................... 9

3.5.1 Total cell protein fraction .............................................................. 9

3.5.2 Soluble cytoplasmic protein fraction .......................................... 10

3.5.3 PageBlue staining ........................................................................ 10

3.5.4 Western blot ................................................................................ 10

4 Results ............................................................................................... 10

4.1 Vector extraction and cleavage ...................................................... 10

4.2 Construction of fusion genes .......................................................... 11

4.3 Expression of fusion proteins ......................................................... 14

5 Discussion ......................................................................................... 15

5.1 Societal & ethical considerations ................................................... 17

6 Acknowledgement ............................................................................ 17

7 References ......................................................................................... 17

4

1 Abstract

Expression of foreign proteins in host organisms usually results in the

development of insoluble, inactive proteins. Further, these proteins have a

tendency to form aggregates termed inclusion bodies. However, the

formation of inclusion bodies can be avoided by fusing the gene encoding

the foreign protein to a highly soluble protein. In this report Sterol Carrier

Protein-2 (SCP-2) is reviewed as a possible solubility tag. The

experiment was carried out by fusing SCP-2 to one of two insoluble

proteins, Green fluorescent protein (GFP) or a form of chloramphenicol

acetyl transferase (CAT∆9). The protein fusion was then inserted into the

vector pET-15b, transformed in Escherichia coli and the yield of actively

expressed protein was measured. The results obtained from this study, as

evaluated by PageBlue staining and Western blot, are indicating that

SCP-2 does not improve the solubility of GFP or CAT∆9. Nonetheless,

the solubility of GFP has earlier been increased by fusing it to the

solubility tag maltose-binding protein (MBP). Producing more soluble

forms of CAT∆9 have also been tested but without success. Therefore the

conclusion drawn from this experiment is that SCP-2 does not work as a

solubility tag, however more research must be performed to conclude this

with certainty.

2 Introduction

Recombinant proteins expressed and produced in Escherichia coli

more often than not result in insoluble and inactive proteins (Kapust &

Waugh 1999). The foreign proteins expressed in E. coli tend to form

insoluble aggregates, so called inclusion bodies, due to the limitation of

E.coli to perform posttranslational modifications (Demain & Vaishnav

2009). To bypass this biological restriction it is sometimes possible to

fuse the gene encoding the insoluble protein to a highly soluble protein,

termed solubility tag (Bell et al. 2013). The idea of the solubility tag is to

enhance the folding of the recombinant protein and thus avoid

aggregations while also promoting a higher solubility (Kapust & Waugh

1999). New solubility tags with unique characteristics are emerging

frequently and they often share three main features: (1) minimal effect on

the biological activity and tertiary folding of the protein; (2) removal of

the tag is easy and specific; (3) applicable to a number of insoluble

proteins (Terpe 2003). Commonly used fusion tags include maltose-

binding protein (MBP) and Glutathione S-transferase (GST) (Kapust &

Waugh 1999, Scheich et al. 2003). MBP is particularly preferred as it also

protects the target protein from degradation by translocating the protein

to cellular compartments that contain less protease (Kapust & Waugh

1999, Costa et al 2014).

5

To further promote the protein purification an affinity tag can be

used in conjunction with the solubility tag as it binds antibodies targeted

at the protein. The selected affinity tag for this project is the hexahistidine

tag (His6), which is a small peptide sequence, often located N-terminally

on the vector (Terpe 2003). Smaller tags are not as immunogenic as

larger tags and the His6-tag is also used advantageously when purifying

proteins as it binds immobilized transition metals (Esposito & Chatterjee

2006, Waugh 2005).

Sterol Carrier Protein-2 (SCP-2) is a protein that naturally occurs

as a fusion protein in eukaryotic organisms. In vitro SCP-2 promotes the

transfer of sterols between membranes and it was long thought that the

protein acted as a substrate carrier involved in sterol metabolism (Pfeifer

et al. 1993). More recently it has been shown that SCP-2 lacks specificity

for sterols as it also binds other molecules such as fatty acids and

phospholipids (Edqvist & Blomqvist 2004). The protein is predominantly

localized in peroxisomes making it impractical to study in living cells

(Pfeifer et al. 1993). Although studied extensively the actual function of

SCP-2 in vivo is still not fully determined.

SCP-2 is a potential solubility tag as it naturally functions as a

fusion protein possible to produce in high concentrations in E. coli

(Edqvist & Blomqvist 2006). To assess the strength of SCP-2 as a

solubility tag the protein will be fused to one of two foreign and insoluble

proteins, green fluorescent protein (GFP) or a form of chloramphenicol

acetyl transferase (CAT∆9). CAT∆9 is not a wild-type protein as nine

residues have been deleted from the C-terminus transforming the soluble

chloramphenicol acetyl transferase (CAT) into the highly insoluble

CAT∆9 (Robben et al. 1993). A previous study by Kapust & Waugh

(1999) has shown that CAT∆9 is hard to express in E. coli even when

fused to a variety of solubility tags. GFP, on the other hand, has been

successfully expressed in a soluble form when fused to the solubility tag

MBP (Kapust & Waugh 1999).

Expression of recombinant proteins can be done in a variety of

different expression systems such as yeast, bacteria, or insect cells. Most

commonly bacterial systems are used because of the ability to produce

proteins in large quantities at a low cost. However, as earlier described, at

the price of the easy appearance of inclusion bodies (Demain & Vaishnav

2009). Yeast on the other hand, being both a microorganism and a

eukaryote, provides more advanced folding pathways for heterologous

proteins in addition to the ability to properly fold and process proteins

(Verma et al. 1999). The main problem with using yeast systems is the

production of proteases, causing degradation of expressed proteins

(Verma et al. 1999). Insect cells are less common but sometimes used as

they are capable of performing post-translational modifications (Verma et

6

al. 1999).

The aim of this projects is to use SCP-2 as a solubility tag to

overcome the formation of inclusion bodies in a bacterial expression

system. The experiment is carried out by constructing a gene fusion

containing the solubility protein, SCP-2, fused together with an insoluble

partner, GFP or CAT∆9. The protein fusions are to be inserted in the

pET-15b vector, with an incorporated affinity tag (His6), and expressed in

E.coli BL21 (DE3) with the intention of producing a soluble, active

protein.

3 Material & methods

3.1 Bacteria

For this experiment two E. coli strains were used, DH5α and BL21

(DE3). The mutations in DH5α are: dlacZ Delta M15 Delta (lacZYA-

argF) U169 recA1 endA1 hsdR17 (rK-mK+) supE44 thi-1 gyrA96 relA1

(Taylor et al. 1993). The mutations in BL21 (DE3) are: lon-11 DE(ompT-

nfrA)885 DE(galM-ybhJ)884 LAMDE3 DE46 mal+([K-21]) (LamS)

hsdS10 (Wood 1966).

3.2 Vector and genes

The pET-15b vector (Fig. 1) was extracted from the E. coli strain pET-

15b DH5α, grown overnight (37 ᵒC, 200 rpm) in LB-medium (Thermo

Scientific) with an addition of ampicillin (100 μg/μl).

Single colonies of E. coli containing the plasmids pGEX SCPAt

(Edqvist et al. 2004) and pMDC123 (CAT∆9) (Carbonell et al. 2014)

were grown overnight (37 ᵒC, 200 rpm) in LB-medium with an addition

of ampicillin. The plasmid encoding GFP, psmRS-GFP (Davis & Vierstra

1996), was provided as such.

7

Fig. 1. The pET-15b expression vector with BamHI and NdeI sites (black arrows). Figure from Addgene (https://www.addgene.org/vector-database/2543/) accessed 15 may 2016.

.

3.3 DNA manipulations

Plasmid and vector isolations were done with a GeneJET™ plasmid

miniprep kit (Fermentas). The pET-15b vector was cleaved with the

enzymes BamHI (Biolabs 10U/μl) and NdeI (Promega 10U/μl) with an

addition of buffer D (Promega, 10x) and water. The reaction was

incubated at 37 ᵒC followed by an inactivation of enzymes at 65 ᵒC.

Purification of the BamHI/NdeI digested vector made with a GeneJET™

PCR Purification Kit (Fermentas) followed by a gel electrophoresis.

Purified PCR products were cloned into a pGEM®-T vector with a

pGEM®-T Vector System I (Promega). The vector with inserted PCR

products was transformed on LB/ampicillin/X-gal/IPTG plates.

The pGEM®-T vectors with inserts were isolated and a nanodrop

was conducted to confirm sufficient DNA concentrations. Purified

fragments of SCP-2, GFP and CAT∆9 were cleaved with restriction

enzymes (Tab. 2) followed by a PCR.

8

Table 2. Reagents for restriction cleavage of SCP-2, GFP and CAT∆9.

Gene Buffer Enzyme (N-terminal) Enzyme (C-terminal)

SCP-2 EcoRI/pGEM-T Buffer D NdeI EcoRI SCP-2 HindIII/pGEM-T Buffer 2 NdeI HindIII GFP/pGEM-T Buffer EcoRI BamHI EcoRI CAT∆9/pGEM-T Buffer 3 BamHI HindIII

An ethanol purification step was run to increase the SCP-2 DNA

concentration.

T4 DNA ligase and restrictions enzymes were used according to

the manual provided by the enzyme supplier (Thermo Scientific) to ligate

SCP2 with GFP respectively CAT∆9 and insert them into the pET-15b

vector (Fig. 2).

Fig. 2. Cloning strategy of SCP-2/GFP and SCP-2/CAT∆9 into the pET-15b vector.

9

3.4 PCR

Amplification of the genes was done with PCR using 2 μl primers

(Thermo Scientific, Tab. 1), 5 μl 10X DreamTaq buffer, 1 μl 10 mM

dNTP mix, 0.4 μl DreamTaq DNA polymerase (5 U/μl), 0.5 μl template

DNA and 39.1 μl H2O for a total volume 50 μl for each reaction. The

program used was the following: 95 ᵒC for 3 min, then 35 cycles of 95 ᵒC

for 30 s, 55 ᵒC for 30 s and 72 ᵒC for 1 min. The PCR ended with 72 ᵒC

for 10 min. After the PCR a gel electrophoresis was made.

Table 1. Forward- (F) and reverse- (R) primers used in the plasmid amplification PCR.

Gene Primer sequence

SCP-2 NdeI/EcoRI F 5´-ACCAGACATATGATGGCGAATACCCAACTCAAATCCGA-3´ R 5´-TAGGACAGAATTCCAACTTTGAAGGTTTAGGGAAG-3´

SCP-2 NdeI/HindIII F 5´-ACCAGACATATGATGGCGAATACCCAACTCAAATCCGA-3´ R 5´-CAGTCAAAAGCTTCAACTTTGAAGGTTTAGGGAAG-3´

GFP F 5´-CAACCAAGAATTCATGAGTAAAGGAGAAGAACTTT-3´ R 5´-CGTACAACGGATCCTTATTTGTATAGTTCATCCATGC-3´

CAT∆9 F 5´-GGTAACAAAGCTTATGGAGAAAAAAATCACTGGA-3´ R 5´-CAAACAACGGATCCTTACTGTTGTAATTCATTAAGCA-3´

After the ligation of SCP2 with GFP and CAT∆9 a PCR with primers for

GFP and CAT∆9 was conducted followed by a gel electrophoresis.

3.5 Expressing proteins

The two ligations were transformed into the E. coli strain (BL21 (DE3)).

Single colonies from the transformation were inoculated into 5 ml

LB/ampicillin-medium and cultured overnight (37 ᵒC, 200 rpm). 2 μl

from the overnight culture, one for each DNA construction, were diluted

(1:20) in LB/ampicillin-medium. The optical density (OD600) of the

cultures were measured followed by a 3 hour incubation (37 ᵒC, 200 rpm)

at which point OD600 was measured again. The cultures were split up in

four E-flasks, two for each culture, where 1 mM IPTG was added to one

of the E-flasks while the other served as control. A second incubation for

3 hours (37 ᵒC, 200 rpm) was carried out.

3.5.1 Total cell protein fraction

A sample from each of the four E-flasks were transferred to an

Eppendorf-tube and centrifuged for 10 min (4 ᵒC, 10 000 rpm). The

supernatant was discarded and the pellet was re-suspended in cold

sodium phosphate buffer (20 mM, pH 7.4), with an addition of 30 μl

NuPAGE® LDS sample buffer (4X) and 10 μl DTT (0.5 M). The

10

samples were incubated for 10 min (70 ᵒC), cooled to room temperature,

quickly spun down in a table centrifuge and loaded on two protein gels.

One gel intended for staining with PageBlue and the other for Western

blot.

3.5.2 Soluble cytoplasmic protein fraction

The remaining cultures were transferred from the E-flasks to falcon tubes

and centrifuged for 10 min (22 ᵒC, 4000 rpm). The supernatant was

discarded and the pellet re-suspended in cold sodium phosphate buffer

(20 mM, pH 7.4). The cell membranes were lysed by sonication at an

interval of 10 s with sonication pulses and 30 s without, for a total of 60 s

sonication (amplitude of 20 %) (Branson Digital Sonifier®). Fluid from

the sonicated samples were transferred to Eppendorf-tubes and

centrifuged for 10 min (22 ᵒC, 14 000 rpm). The supernatant was then

transferred to a new Eppendorf-tube with an addition of 30 μl NuPAGE®

LDS sample buffer (4X) and 10 μl DTT (0.5 M). The samples were

incubated for 10 min (70 ᵒC), cooled to room temperature, quickly spun

down in a table centrifuge and loaded on two protein gels. One protein

gel intended for staining with PageBlue and the other for Western blot.

3.5.3 PageBlue staining

The staining was made according to instructions that accompanied the

PageBlue™ Protein Staining Solution (Thermo Scientific).

3.5.4 Western blot

The primary antibody used in the Western blot was Pierce™ 6x His

Epitope Tag (Thermo scientific) and the secondary antibody was

Pierce™ Antibody Horseradish Peroxidase (Thermo scientific). The

antibodies were diluted 10 000:1 with 20 ml PBST and 2 μl antibody.

Detection of light emission from the completed Western blot was made

with a CCD-camera (LAS-4000 Mini, Fujifilm, Tokyo) with an exposure

time of 10 s.

4 Results

4.1 Vector extraction and cleavage

The pET-15b vector, extracted from the E. coli strain pET-15b DH5α,

was analyzed with gel electrophoresis after restriction cleavage and

purification (Fig. 3). The restriction cleavage was done at the sites

BamHI and NdeI to prepare for insertion of SCP-2, GFP and CAT∆9

(Fig. 2). When comparing the purified pET-15b vector with a non-

purified vector, the purified pET-15b shows a much clearer band

11

Fig. 3. Gelelectrophoresis showing the purified pET-15b (left) and the non-purified pET-15b (right) at

6000 bp (GeneRuler™ 1 kb DNA

ladder).

indicating less background disturbance. The bands corresponds to a size

of about 6000 bp as deducted from the ladder.

4.2 Construction of fusion genes

The genes encoding SCP-2, GFP and CAT∆9 isolated from available

plasmids were amplified by PCR followed by a PCR purification. To

improve the ligation efficiency of the genes into the pET-15b vector in

later stages a cloning into a pGEM-T vector was performed. The pGEM-

T vector with inserted SCP-2/EcoRI, SCP-2/HindIII, GFP and CAT∆9

were then transformed on Agar/ampicillin/X-gal/IPTG plates (Fig. 4).

The transformation was successful as all the plates displayed white

colonies. Single white colonies were selected and cultured for isolation of

the pGEM-T vectors with inserts.

10 000 6000

1000

12

A B C D

The pGEM-T vectors containing the inserted genes were isolated and a

restriction cleavage was done to obtain purified fragments. The fragments

were analyzed with a restriction analysis (Fig. 5). The genes that were

properly isolated and amplified are those showing two distinct fragments.

The larger of the two fragments is the pGEM-T vector at around 3000 bp

while the smaller fragment is the isolated gene encoding SCP-2 (400 bp),

GFP (750 bp) or CAT∆9 (750 bp).

Fig. 4. Agar/ampicillin/X-gal/IPTG plates showing the transformation of pGEM®-T vector with inserted SCP-2/EcoRI (A), SCP-2/HindIII (B), GFP (C) and CAT∆9 (D). White colonies were selected and cultured.

13

Fig. 6. The ligation of SCP-2/GFP and SCP-2/CAT∆9 inserted in the pET-15b vector and amplified by PCR. The vector at 6000 bp (red arrow) and the GFP and CAT∆9 inserts at 750 bp (green arrow). White arrows indicate samples used for protein expression.

Fig. 5. Restriction analysis showing purified genes of SCP-2/EcoRI, SCP-2/HindIII, GFP and CAT∆9 after cloning and restriction cleavage. The pGEM-T vector at 3000 bp (red arrow), GFP and CAT∆9 at 750 bp (green arrow) and SCP-2 at 400 bp (blue arrow).

SCP-2/EcoRI and GFP respectively SCP-2/HindIII and CAT∆9 were

cleaved from the pGEM-T vector and isolated with PCR. The extracted

genes were then ligated and inserted into the pET-15b vector. PCR using

primers for GFP and CAT∆9 and a gel electrophoresis was done to

evaluate the outcome of the ligation (Fig. 6). The larger fragment is

showing pET-15b around 6000 bp and the smaller fragment is showing

the GFP and CAT∆9 insert at 750 bp. SCP-2 is not visible on the gel as

only primers for GFP and CAT∆9 were used in the PCR.

SCP2 EcoRI

SCP2 HindIII GFP CAT∆9

3000

750 400

1000

6000 10 000

SCP-2/GFP SCP-2/CAT∆9

10 000 6000

750 1000

14

S

C

S S S T T T T

C C C IPTG IPTG IPTG IPTG

250

100

55

15

35

4.3 Expression of fusion proteins

The two ligations chosen from the previous step (Fig. 6) were

transformed into the E. coli strain (BL21 (DE3)) and single colonies were

selected and cultured followed by measurements of OD600. To actively

express GFP and CAT∆9 in E. coli the T7 promoter requires an inducer,

IPTG, which inhibits the lac repressor (Chaudhary & Lee 2015).

Therefore IPTG was added to half the samples whereas the rest served as

controls. Total cell protein fraction and the soluble cytoplasmic protein

fraction were extracted. The fractions, and thus the expression of GFP

and CAT∆9, were analyzed with PageBlue staining and Western blot.

The PageBlue staining resulted in bands from the soluble cytoplasmic

protein fraction and total cell protein fraction in all samples except one

control (Fig. 7).

The Western blot and the subsequent light emission detection gave no

observable results as the only thing detected was the ladder (Fig. 8).

Fig. 7. The PageBlue staining of the soluble cytoplasmic protein fraction (S) and total cell protein fraction (T) with controls (C) and IPTG included. Ladder indicates sizes in kilodalton (kDa).

15

5 Discussion

The aim of this report was to investigate whether or not the protein SCP-2

would work as a suitable solubility tag capable of fusing to the highly

insoluble proteins GFP and CAT∆9. Further, the protein fusion was to be

inserted in a vector and finally expressed in an E. coli strain that naturally

did not express GFP and CAT∆9. To be able to investigate the outcome

of the fusion, the construct consisting of SCP-2 fused to GFP or CAT∆9,

was inserted in the vector pET-15b. The construct was then transformed

into the E. coli strain BL21 (DE3). Analysis of the results were done with

PageBlue staining and Western blot.

The PageBlue staining did not show any bands, indicating that no

protein had been expressed (Fig. 7). This could be concluded since both

the soluble cytoplasmic protein fraction and total cell protein fraction

showed similar bands as the respective control on the stained gel.

Therefore it is not possible to draw any conclusions if SCP-2 did work as

a solubility tag. The absence of bands in the Western blot also indicated

no expression of the construct (Fig. 8).

The E. coli strain BL21 (DE3) requires an inducer, in this case

IPTG, to express heterologous proteins (Chaudhary & Lee 2015).

Therefore if the protein expression would have worked, unique bands

were to be expected on the samples containing IPTG. Without an addition

of IPTG the fusion should not work and therefore the controls were added

Fig. 8. Light emission from the Western blot as detected by a CCD camera (LAS-4000 Mini, Fujifilm, Tokyo). Soluble cytoplasmic protein fraction (S), total cell protein fraction (T), controls (C) and IPTG-samples. Ladder indicates sizes in kilodalton (kDa).

220

100

60

40

20

S S S S T T T T

C C C C IPTG IPTG IPTG IPTG

16

for comparison.

The pET-15b vector that was purified early in the experiment do

show a band in the expected region of about 6000 bp (Fig. 3, left),

indicating a functional vector. In the following steps the genes encoding

SCP-2, GFP and CAT∆9 were isolated and cloned into the pGEM-T

vector and also these results are promising (Fig. 5). This can be

concluded as the gel is showing bands that are representative for the

pGEM-T vector together with smaller bands representing the isolated

genes. The cloning of the genes into a pGEM-T vector was made to

improve the efficiency of the ligation to pET15-b. The pGEM-T vector

contains single 3´-terminal thymidines at both ends. These thymidines

provides compatible overhangs for the PCR products as well as reduces

background from non-recombinants (Zhao et al. 2009). The added

overhang should positively affect the ligation of PCR products to the

expression vector.

The next step where SCP-2 were ligated to GFP and CAT∆9 also

shows promising results (Fig. 6). The gel is clearly showing the vector at

around 6000 bp and the inserted GFP and CAT∆9 at around 750 bp,

which are the expected sizes. A major flaw in the method was that the

PCR performed to confirm the insert was done with primers for GFP and

CAT∆9, but not for SCP-2. Therefore it is not possible to verify that

SCP-2 is inserted in the vector, even though GFP and CAT∆9 are present.

To conclude with certainty that the ligation was inserted appropriately, a

second PCR should have been performed with primers for SCP-2.

Prior to the start of this experiment no earlier studies were found

where SCP-2 was used as a solubility tag in E. coli. However GFP and

CAT∆9 have earlier been fused to the solubility tag MBP in a study by

Kapust & Waugh (1999). In their study they managed to increase the

production of actively expressed GFP by doing so. They also tried to fuse

CAT∆9 to a variety of different solubility tags such as MBP, glutathione

S-transferase and thioredoxin without success. The results from this

report differs as GFP was not successfully fused to SCP-2. Regarding

CAT∆9 they do present the same results as this study.

Unfortunately no conclusions can be made about the functionality

of SCP-2 as a solubility tag from the results presented in this report. This

was made clear as both the Western blot and PageBlue staining indicated

that no expression of proteins was achieved. Due to the fact that there

was no PCR with primers for SCP-2 confirming a correct insertion into

the pET-15b vector, more elaborate studies with SCP-2 are required

before drawing any conclusions about the functionality as a solubility tag.

17

5.1 Societal & ethical considerations

The development and usage of solubility tags could provide a solution to

overcome the major problem of producing biologically active

recombinant proteins in heterologous systems (Kapust & Waugh 1999).

Traditionally the recombinant proteins form insoluble aggregates, so

called inclusion bodies, when expressed in heterologous systems. To

produce proteins such as medicines, at a large scale and low cost, the E.

coli system is the preferred choice for now. As an example E. coli was

the first heterologous system used to produce insulin, which is used to

treat diabetes (Swartz 2001). To achieve a large scale production of

various proteins through heterologous systems, it is important to solve the

inclusion body problem. As solubility tags are not universally applicable

to all proteins, there currently exists a demand to examine and review

new potential solubility tags. The formation of inclusion bodies is also

associated with neuronal degeneration and organ failure in genetic

diseases (Gundersen 2010, Kopito RR 2000).

The ethical considerations linked to this project is the production of

genetically engineered bacteria. However, the genetic changes made in

this experiment is not expected to pose a threat as they are not aimed at

inducing resistance to medicines or such. The use of the bacteria was

contained to the laboratory and handled with care.

6 Acknowledgement

I want to thank my co-worker Amanda Lundén, who I have been working

with throughout this project. The second person I am grateful to is my

supervisor, Johan Edqvist, for guiding me through this project as well as

providing help whenever needed.

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