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ANALYSIS OF ADHI PROTEIN EXPRESSION IN BACTERIAL SYSTEM

Nur 'Ain Najwa Bt Mohd Nor 24644

TP 593 N974 2812

Bachelor of Science with Honours (Resource Biotechnology)

2012

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ACKNOWLEDGEMENT

First of aU I would like to thank Allah for giving me strength and good health from

the start until the completion of this project.

My sincere and gratitude goes to my supervisor, Assoc. Prof. Dr. Hairul Azman

Roslan for his support and guidance in this project. I also wanted to thank to all

Postgraduate Student of Genetic Engineering Laboratory which are Nabella Holling,

Mastura Sani, Liyana Ismail, Jerry Gerunsin, Chai Suk Phin and Nurul Izzati for

supporting me in my project.

Not to forget to my co-supervisor, Dr. Azham Zulkhamain for giving me advices

regarding this project and special thanks to Assoc. Prof. Dr. Hasnain bin Md. Hussain for

providing Rosetta II (DE3) strains for this project and also to all lecturers in Faculty of

Resource Science and Technology especially in Department of Molecular Biology for all

the guidance and educations for this entire 3 years.

Thanks also go to all my friends especially my course mate for their support. The

most important is both of my parents whom always pray for me and gave me moral support

from the start until the end.

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• • I , .t ". PUlat Klailhaat Maklwnat Akademik r"\'TRSm MALAVSIA SAlUW~K

TABLE OF CONTENTS

Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. I

Table of Contents.. ................ ............... ..... .............. .......... ... ....... II

List of Abbreviations................................................................... IV

List of Tables and Figures............................................................. V

Abstract.................. .................... .... ...... .................. ................ 1

1.0 Introduction.................................................................... . 2

2.0 Literature Review ................... . ........................................ .. 5

2.1 Genetic Engineering ................................................. .. 5

2.2 ADH.................................................................. .. 6

2.3 Applications of Genetic Engineering ........... . .................. . 7

2.3.1 Transgenic Tomato and Safflower.................. ...... 7

2.3.2 Recombinant Gene Expressions........................... 7

2.4 Heterologous Expressions .......................................... . 8

2.5 Escherichia coli .. .................................................... , 8

2.6 Gene expression in Escherichia coli . ............................. . 9

2.7 T7 Expression System .............................................. . 10

3.0 Methods and Materials ............................ . ......................... . 12

3.1 Culturing XLI Blue................................................ .. 12

3.2 Plasmid Extraction .................................................. . 12

3.3 Polymerase Chain Reaction (PCR) analysis .................... . 13

3.4 Restriction Enzyme analysis ...................................... . 14

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3.5 Culturing Rosetta II (DE3) strains ............. . ................... . 14

3.6 Calcium Chloride (CaCh) Bacterial Competent Cell .......... . 14

3.7 Transformation ofpET41a+ in Rosetta II (DE3) strains ........ . 15

3.8 Colony Polymerase Chain Reaction (PCR) ..................... . 16

3.9 Optimization of Protein Expressions in XLI Blue and Rosetta II

(DE3)................. . .......... . ............. .. ........ . .... .......... 17

3.10 Protein Extraction............... . ...................... .............. 18

3.11 SDSPAGE.................................................... . ....... 18

3.12 Total Protein Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3.13 His-Tag Purification........ . . ..... ... ..................... . .. .... . ... 20

4.0 Results and Discussions ... . ......... . ....... . ................ . ........ . ....... . 21

4.1 Plasmid Extraction ... . ........ . ...................................... . 21

4.2 Polymerase Chain Reaction analysis ... . ......................... '" 21

4.3 Restriction Enzyme analysis ................ . ................. . ...... . 22

4.4 Transformation of pETlAdhI in Rosetta II (DE3) ............. . . . 23

4.5 Colony PCR..... .. ........ . ........ . .................. . ............... . 25

4.6 Expressions of Adhl in XLI Blue and Rosetta ........... . 26

4.7 SDS PAGE analysis for expressions in XLI Blue........ . ... . ... . 27

4.8 SOS PAGE analysis for expressions in Rosetta .................. . 29

4.9 His-Tag purification ................... . ....... . ... . .................. . 33

5.0 Conclusions .............. .. ...... . ............................... . ............. . . 34

6.0 References .............................................. . ....... . ................ . 35

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ADHI

AGE

APS

DNA

IPTG

LB

PCR

SDSPAGE

TEMED

List of Abbreviations

Alcohol Dehydrogenase 1

Agarose Gel Electrophoresis

Ammonium Persulfate

Deoxyribonucleic Acid

isopropyl-!3-D-thiogalactopyranoside

Lysogenic Broth

Polymerase Chain Reaction

Sodium Dodecylsulfate Polyacrylamide Gel

Electrophoresis

Tetramethylethylenediamine

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1

Table I

Table 2

Table 3

Table 4

Table 5

Table 6

Figure I

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

List of Tables and Figures

Ingredients for Polymerase Chain Reaction

Polymerase Chain Reaction profiling

Ingredients for plasmid cutting

Optimization of protein expressions for XL I Blue strain based on different temperatures and at different incubation times

Ingredients for Polymerase Chain Reaction

Polymerase Chain Reaction profiling

A 1% agarose gel electrophoresis of plasmid extraction.

PCR product of plasmid extraction

A I % agarose gel electrophoresis

Transformation of plasmid pETIAdhl in Rosetta II (DE3) strains by using heat shock method

A I % agarose gel electrophoresis of colony PCR for transformation of pETlAdhi in Rosetta strains

SDS PAGE analysis of protein expressed in XLI Blue at 3TC with induction using IPTG

SDS PAGE analysis for protein expressed in XLI Blue at 3TC with induction using IPTG and 2 % sucrose

SDS PAGE analysis for protein e expressed in Rosetta II (DE3) at 30"C with induction using IPTG

SDS PAGE analysis for protein expressed in Rosetta II (DE3) at 30°C with induction using IPTG and 2 % sucrose

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Figure 10 SDS PAGE analysis for His-tag purification ofAdhl gene.

Figure 11 Map ofpET4Ia(+) plasmid

Figure 12 SDS PAGE analysis for protein expressed in XLI Blue at 35°C upon induction with IPTG

Figure 13 SDS PAGE analysis for protein expressed in XL 1 Blue at 30°C upon induction with IPTG.

Figure 14 SDS PAGE analysis for protein expressed in XLI Blue at 30°C upon induction with IPTG and 2 % sucrose.

Figure 15 SDS PAGE analysis for protein expressed in Rosetta at 35°C upon induction with IPTG.

Figure 16 SDS PAGE analysis for protein expressed in Rosetta at 3TC upon induction with IPTG.

Figure 17 SDS PAGE analysis for protein expressed in Rosetta at 35°C upon induction with IPTG and 2 % sucrose.

Figure 18 SDS PAGE analysis for protein expressed in Rosetta at 3TC upon induction with IPTG and 2 % sucrose.

VI

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ANALYSIS OF ADUl PROTEIN EXPRESSION IN BACTERIAL SYSTEM

NUR 'AIN NAJW A BT MOUD NOR Resource Biotechnology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Alcohol dehydrogenase (ADH) is a group of gene involved in the oxidation of alcohols. Adhi cDNA that was derived from sago palm has been cloned into a bacterial expression vector, pET41a+ in order to obtain a high level expression of this recombinant in expression host E.coli XLI Blue and Rosetta II (DE3). Vector pET4Ia+ that has Adhi gene was transformed into E. coli by using heat shock transformations. The aim of this study was to express Adhi protein in bacterial system and the main focus of this research was to address the issues that affecting the heterologous proteins. The protein expression was optimized at different parameters which were the temperature and incubation time. The expressed protein was analyzed by SDS PAGE based on the total protein staining by using Coomassie Blue staining. The protein was successfully expressed in Rosetta II (DE3) strains based on the result from His-Tag purification.

Keywords: heterologous expression, Adhl, pET4Ia+, XLI Blue, Rosetta

ABSTRAK

Alcohol dehydrogenase merupakan kumpulan gen yang terlibat didalam pengoksidaan alcohol. Adh cDNA diperoleh dari pokok sago di mana ianya telah diklon di dalam ekspresi vektor bakterial iaitu pET41a+ untuk membolehkan rekombinan ini di ekspres pada tahap yang tinggi di dalam hos XLI Blue dan Rosetta 11 (DE3). Vektor pET41a+ yang mempunyai gen Adhi telah dimasukkan kedalam E. coli menggunakan teknik kejutan haba. Tujuan utama eksperimen ini ialah untuk menghasilkan protin Adhi di dalam sistem bakteria, manakala Jokus projek ini adalah untuk mengenal pasti faktor yang mempengaruhi ekspresi protin heterologos. Protin telah diekspres berdasarkan dua parameter iaitu suhu dan masa inkubasi. Protin yang telah diekspres dianalisis menggunakan teknik SDS PAGE berdasarkan jumlah total protein menggunakan Coomassie Biru. Protin Adhi telah berjaya di ekpsress didalam Rosetta 11 (DE3) berdasarkan keputusan dari penulenan His-Tag

Kata kunci: Ekspresi heterologos, Adhl, pET4Ia+, XLI Blue, Rosetta

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1.0 INTRODUCTION

Alcohol dehydrogenase (Adhl) is a group of genes that is involved in the oxidation of

alcohols. Adh cDNA derived from sago palm has been cloned into a bacterial expression

vector, pET4Ia+. This study involved the characterisation of parameters for the detection

and heterologous expression of Adhl gene in XLI Blue cells and Rosetta II (DE3) strains

which are in the E. coli family.

Heterologous expression involves expressions and characterisation of genes to host

cells other than original source, for the synthesis of the encoded proteins. Proteins

isolation, especially the one from plant sources, can be very costly and lengthy. However,

heterologous expression can provide a convenient alternative. Host use for heterologous

expression may also provide a simpler system thus easier for the studies on proteins

functions. According to Yesilirmak and Sayers (2009), many studies have been done on

plant gene expression especially in bacteria, yeast, insect cell and Xenophus oocytes

presented the comparative advantages and disadvantaged of each system. There are several

factors influencing the choice of host that include the stability and folding characteristics

of proteins, post-translational modifications, simplicity of the host as well the efficiency of

expression systems (Yesilirmak & Sayers, 2009).

Alcohol dehydrogenase (ADH: alcohol: NAD+ oxidoreductase; E.C. 1.1.1.1.)

comes from a medium-chain dehydrogenase protein superfamily. Based on the research by

Dolferus et al. (1997), Adh is present in many life forms, with varied metabolic functions.

Addition to that, Adh and pyruvate decarboxylase are part of alcoholic fermentation

pathway which is a two-step pathway that convert pyruvate via acetaldehyde to ethanol. In

this project, Adhl genes that are already cloned into pET expression system have been

expressed. Escherichia coli containing the desired genes were induced by IPTG and

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sucrose to compare the intensity of the protein expressed with the controls without

inductions.

Choosing the best expressIon system and vector are the most crucial steps in

heterologous expression. The expression system is selected depending on the purpose of

study as to whether for the production of large quantities of proteins or investigation of

functional features of expressed protein (Yesilirmak & Sayers, 2009). In this project, we

used the pET system. pET vectors were originally constructed by Studier and colleagues

(PET System Manual, 1999). pET system is one of the most powerful system for cloning

and expressions of recombinant proteins in E. coli (PET System Manual, 2002). The

expression is induced by providing a source of T7 RNA polymerase in host cell. Once T7

RNA polymerase is induced, all the cell resources are converted to express the gene. The

cell can contain more than 50 % of total proteins after induction for a few hours. The

benefit of this system is that it can maintain the targeted genes to transcript silently in the

uninduced state (PET System Manual, 2002).

There are two general categories of vectors which are transcription and translation

vectors (PET System Manual, 1999). Transcription vectors used for expression of target

genes that already carry their own prokaryotic ribosome binding site and A TG codon,

while translation vectors contain highly efficient ribosome binding site from phage T7

major capsid protein (PET System Manual, 1999).

For the expression of a gene, the recombinant plasmid is first transferred to a host

E. coli strain containing a chromosomal copy of the gene for T7 RNA polymerase for

protein production (PET System Manual, 1999). Escherichia coli have been used in this

experiment, as it is the most widely used hosts for the production of heterologous protein.

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(Baneyx, 1999). The most important is E. coli are efficient, cost effective and has high

level production of heterologous protein (Hannig & Makrides, 1998).

The focus of this research was to address the issues that affecting the heterologous

protein expression. The proteins expressions were optimized at different parameters which

were the temperature, incubation times and sucrose. Once the plasmid has been

transformed into E. coli, it was induced by IPTG and sucrose. Optimizations were

preceded by using different parameters.

The objective of this study was to analyse the protein expression of Adhl gene.

The focus objectives were:

1. To optimize the expressIOn by usmg different parameter; temperature

incubation times and sucrose.

2. To extract protein from Escherichia coli.

3. To compare the intensity of proteins expressed.

4

Pusat Khidauu Makiumat Akademik ., '.~

UNJVERSITI MALAYSIA SARAWAK

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2.0 LITERATURE REVIEW

2.1 Genetic Engineering

The science of genetics was started by the discovery of Deoxyribonucleotide (DNA) which

carries hereditary information. The DNA was first discovered by Friederich Miescher in

1896 but it was not taken seriously as the chemical basis of genes until it is discovered by

two scientists in [950s (Nair, 2008). Francis Crick and James Watson along with Rosalind

Franklin, discovered that DNA is a double helix, where two strands twisted around each

other like a spiral staircase. In 1961, Marshall Nirenberg and H. Gobind Khorana were

then carried out the deciphering of the genetic code (Nair, 2008). After that, scientists

started seeking to alter the genetic make up of living organisms by transferring genes from

one organism to another. The first recombinant DNA experiment was done by Walter

Gilbert in 1973 (Nair, 2008).

Genetic engineering was described as a technology that included genetic

manipulation, gene cloning, gene modification and recombinant DNA technology (Nicholl,

2008). It is defined as the technique of cutting foreign DNA at specific sites and inserting

the cleaved foreign DNA fragment, and cloned into another DNA molecule that are

similarly cut. The product obtained by inserting the foreign fragment is called recombinant

DNA. When this recombinant DNA is transformed into a host cell and allowed to multiply,

one can obtained millions of copies of foreign inserted DNA. This is described as DNA

cloning technique (Gupta, 2008). Transformation is important to genetic engineering. It is

defined as genetic integration of a bare, foreign DNA such as plasmid into bacterial cell

(Fitch, 2002).

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2.2 ADD

Alcohol dehydrogenase is a Zn-binding enzyme that serves as dimer and requires NAD (P)

co-factor to inter convert ethanol and acetaldehyde. It was reported for more than 50 years

ago that ADH involves in oxygen deprivation in plant (Strommer, 2011). During these

conditions, acetaldehyde is converted to ethanol whereby ADH acts in the terminal phase

of anaerobic glycolysis, or fermentation. NAD+ is regenerated and inadequate amount of

ATP is produced at a time when normal respiration is disturbed (Strommer, 2011).

In 1971, Schwartz was the first to report the activity of Adh in kernels and anthers

of maize. Recently, Adh gene expression and Adh activity has been found throughout plant

growing under normal condition or subjected to various stress condition. Most of the plants

analysed carried multiple Adh genes except for a few Arabidopsis species. It was reported

that in eudicots, the tissues-specific patterns of gene expression and enzymatic activities

provided signs of functional specialization (Strommer, 2011).

According to Strommer (2011), Adh genes have a long standing role in

evolutionary studies and it remained popular for such studies. It played a crucial role in the

development of evolutionary genetics and disciplines of population biology. Although

method for direct analysis of DNA became available, Adh genes remained popular for

analysis of evolutionary dynamics. Adh genes was supplanted by the availability of full­

genomes sequences, but because of lack information existing from previous studies, Adh

genes remam significant in genomic, transcriptomics, proteomics and metabolomics

researches.

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2.3 Application of Genetic Engineering

2.3.1 Transgenic Tomato and Safflower

There are many applications of genetic engineering such as transgenic tomato and

safflower. Roger Beachy from Washington University at St Louis and colleagues at

Monsanto Company has succeeded in producing tomato that resist the attack of tobacco

mosaic virus. Before they found the way to resist the virus, almost each year the yield of

the tomatoes were reduced. Beachy inserted the gene that encode for the protein that forms

the outer coat of the virus into the chromosome of tomato plant. As a result, the virus

cannot infect the tomato and in tum, the plant gives higher yields (New Scientist, (988).

Safflower plant was also genetically engineered. Scientists from Montana State

University has succeeded in developing safflower plants that are herbicide resistant

through genetic engineering. They insert the genes into crop plant that made the plant

tolerant to herbicide (Herren, 2002).

2.3.2 Recombinant Gene Expression

According to Lorence (2004), without the information provided by years of basic research

of E. coli, the successful application of gene expression would not have taken place with

such extraordinary speed. The use of E. coli is still not matched for gene expression work;

the foreign genes can be introduced into genome, its plasmid and viruses with relative easy

and predictability (Lorence, 2004).

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2.4 Heterologous expression

Heterologous expression is where the protein is inserted into a cell that does not nonnally

express the target protein. Heterologous is referred to the transferred protein that is derived

from a different species from the recipient. Heterologous expression system is easy way to

transfer foreign genetic materials into recipient cell via transfection and transduction. This

method cannot easily extend to mammalian membrane protein (MPs), because this protein

usually expressed in inclusion bodies. Therefore, they are usually impossible to purify

under non-denaturing conditions (Mus-Veteau, 2002). But, derivatives of E. coli are

selected to grow to high saturation cell density such as E. coli BL2l (DE3) strain,

CD4(DE3) and CD43(DE3). This can overproduce the proteins without toxic effect for the

host cell (Mus-Veteau, 2002).

2.5 Escherichia coli

Escherichia coli have been one of the most studied organisms and play crucial roles in

biological science, medicine and industry. The derivatives of the E. coli are not only used

as research model for the study of the restriction system, phage sensitivity and bacterial

evolution in laboratories, but also as a major workhorse for protein expression in

biotechnology industry (Lee, 2009).

E. coli is frequently used in most studies because it is well understood, inexpensive

to grow and relatively facile. Moreover, there are many cloning vectors available and also

have mutant host strains that differ in characteristics which increases the probability of

obtaining soluble protein (Kinsland, 2010). It is convenient to use E. coli as a host for

protein expression. It allows high level of heterologous gene expression and scalability of

experiments, low cost, fast growth, lack of post translational modification and has ability to

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express labelled protein. However, heterologous gene expression may also lead to the

production of insoluble or non-functional target protein (Tolia & Tor, 2006).

2.6 Gene expression in E. coli

Escherichia coli are easy to manipulate genetically, cheap to culture, the expression is fast

and can produce protein in a single day (Peti & Page, 2007). The wide variety of

commercial products available for E. coli expression system can affect the heterologous

protein production (Peti & Page, 2007). But, there are also disadvantages of E. coli as an

expression system where it is not capable of producing eukaryotic post-translational

modifications for example glycosylation (Peti & Page, 2007).

E. coli, which are prokaryotic expression system, have many benefits that ensured

it remains a valuable host for the efficient, cost-effective high-level production of

heterologous protein. The proficient expression of different genes in E. coli is not a routine

matter as the structural characteristics of different genes and the transcribed mRNAs

preclude the adoption of generally applicable expression method, despite its many

advantages. There are lots of comprehensive review on gene expression in E. coli (Hannig

& Makrides, 1998). Based on Hannig and Makrides (1998), the level of gene expression in

E. coli can be affected by many different types of promoter. There are several criteria of

the promoters for high-level gene expression. First, in order to obtain protein production in

excess of 10-30% of the total cellular protein the promoter must be strong. Second, a

highly repressible promoter is particularly important for cases in which the protein is toxic

or can cause detrimental to the growth of the host cell. Third, it must be capable of

induction in a simple and cost effective manner. For example in this experiment,

Isopropyl- ~-D-thiogalactopyranoside (lPTG) will be used and act as effective inducer of

the powerful hybrid tac and trc promoters (Hannig & Makrides, 1998).

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2.7 T7 Expression System

pET is the most common vector series for bacterial protein expression. T7 expression

system was developed by F. William Studier and colleagues at Brookhaven National

Laboratory. T7 system is able to generate a high level of recombinant protein because T7

RNA polymerase is active and very selective for phage T7 promoters. In bacterial cells, T7

transcription can be directed at a single promoter within pET vector that carry the gene of

interest. Most of the T7 expression strains contain chromosomal DE3 prophage that carry

T7 RNA polymerase gene that is expresses by lacUV5 promoter. Some molecules of T7

RNA polymerase continuously expresses the protein and produce some amount of mRNA

in the absence of IPTG because lacUV promoter is not completely shut off by lacJ

(Samuelson, 2011).

However, this is not an acceptable condition. It can be solved by including lacJ

repressor gene on multicopy of pET vector. LacI repressor protein is produced in large

excess relative to its operator binding site present in the lacUV5 promoter. Another way is

to introduce T7-lac hybrid promoter to pET vector series. An effective method to control

the T7 expression is by coexpressing T7 lysozyme, the natural inhibitor of T7 RNA

polymerase. There are three types of lysozymes strains that frequently used which are

pLysS and pLysE that express wild-type T7 lysozyme from a low copy number plasmid

and in lysY strains which is an amidase, a negative variant of T7 lysozyme (Samuelson,

2011).

Addition of IPTG to the culture will increased the level of T7 RNA polymerase in

large excess and the target protein expression proceeds. If a membrane protein expression

plasmid does not produce transformants when using BL21 (DE3) or other T7 expression

strains, it should then be transformed into a lysozyme- strain. Those strains will yield

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nonnal colonies and express protein of interest at moderate high levels. The choice of

pLysE or pLysS should take into account downstream processing of cells (Samuelson,

2011).

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3.0 MATERIALS AND METHODS

3.1 Culturing XLI Blue

XLI Blue with the pET41a(+) vector was obtained from Genetic Engineering Lab. Ten

microliters of culture was pipetted from stock culture and was cultured in new lOm1 LB

media. From that, lOlll of 75 mg/m1 kanamycin antibiotic was added. It was then cultured

overnight in incubator with agitation at 37°C (Sambrook et at., 1989).

3.2 Plasmid Extraction

The plasmid extraction was conducted using GFI-P1asmid DNA Extraction Kit (Vivantis)

and according to the protocol provided, the bacterial culture containing the plasmid was

pelleted by centrifugation at 6000 rpm for 2 min. The supernatant was decanted

completely. Approximate]y 500 III of Solution 1 was added to the pellet and the cell was

resuspend completely by pipetting. The suspension was then divided into two and

transferred into 2 microcentrifuge tubes with each 250 III suspension. A 250 III of Solution

II was added in each micro centrifuge tube and gently mixed by inverting the tubes several

times (about 4-6 times) to obtain clear lysate. The tubes were then incubated on ice for

about 5 minutes.

A 400 III of Buffer NB was added into each tubes to neutralize the lysate and it was

gently mixed with by inverted the tubes about 6-10 times until a white precipitate formed.

The tubes were then centrifuged at 13000 rpm for 10 minutes. The supernatant obtained

was then transferred into column assembled in a clean collection tubes. The columns were

then centrifuged at 10000 rpm for 1 minute. The flow through was discarded. The columns

were washed with 700 III Wash Buffer and centrifuged at 10000 rpm for 1 min. The flow

through was again discarded. The columns were re-centrifuged at 10000 rpm for 1 minute

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to remove any residual ethanoL Each column was placed into clean microcentrifuge tube

and 50 ~l of Elution Buffer was added directly into each column and were standing for 1

minutes. The columns were centrifuged at 10000 rpm for 1 minute to obtain the plasmid.

The plasmids obtained in all tubes were then pooled into another microcentrifiged tube.

The plasmid was stored at -20°C. The plasmid was then analysed on 1% agarose gel

electrophoresis.

3.3 Polymerase Chain Reaction (PCR) Analysis

Polymerase Chain Reaction (PCR) was done to amplify single copy of DNA segment into

billions of identical copies. The content of PCR is listed in Table 1. Negative control was

prepared to compare with the result. In this research, complete sequence of AdhJ was

obtained through PCR according to the PCR profiling in Table 2. The PCR product was

analysed on 1 % agarose gel electrophoresis.

Table 1: Ingredients for Polymerase Chain Reaction

Ingredients Volume Green Taq Polymerase 7.5 ~l 3'XhoJ Primer 1.5 ~l 5'NdeJ Primer 1.5 ~1 Distilled Water 3.5 ~l Plasmid 1.0 ~l Total 15.0 ~l

Table 2: Polymerase Chain Reaction profiling

Cycles Temperature Time

1 cycles Initial Denaturation 94°C 3 min { Denaturation 94°C 30 sec

35 cycles Annealing 62°C 30 sec Elongation 72 °c 45 sec

1 cycles Final Elongation 72°C 5 min

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3.4 Restriction Enzyme analysis

Restriction enzyme was conducted to check the plasmid and gene fragment. The ingredient

used is listed in Table 3. The plasmid was incubated for overnight and the result was

analysed on 1 % agarose gel electrophoresis.

Table 3: Ingredients for Plasmid cutting

Ingredients Restriction Enzyme Xhol Restriction Enzyme Ndel Buffer Orange Plasmid pET 41 a+ Total

3.5 Culturing Rosetta II (DE3)

37°C (Sambrook et al., 1989).

3.6

Volume

Rosetta strain was a gift from Assoc. Prof. Dr Mohd Hasnain Hussain from the Proteomics

Lab. A 10 III of the culture was pipetted into 10 ml of new LB media with 3 III of 34

mglml Chloramphenicol antibiotic. The culture was incubated overnight with shaking at

Calcium Chloride (CaCh) Bacterial Competent Cell Preparation

Approximately 100 III of Rosetta strain from the overnight culture was transferred into a

falcon tube containing 10 ml of new LB media without any antibiotics and was allowed to

grow at 37°C with shaking until OD600 reached approximately 0.45-0.5 taking about 1

hour and 30 minutes. A falcon tube was then cooled on ice for about 20 minutes. The cell

suspension was centrifuged at 3500 rpm at 4°C for 5 minutes. The supernatant obtained

was discarded and the cell was washed gently by re-suspending them in 25 ml iced-cold

100 mM CaCh (Sambrook et al., 1989).

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The cell suspension was kept on ice for about 10 minutes and was re-centrifuged

again at 3500 rpm at 4°C for 5 minutes. The supernatant was discarded again and the cell

pellet was re-suspended with 2.5 ml iced-cold 100 mM CaCho The cell was incubated on

ice for about 1 hour (Sambrook et ai., 1989). For long term storage of the ceH, 20% (v/v)

pure glycerol was added into the cell suspension. This competent cell was stored at -80°C

for further used.

3.7 Transformation

A heat shock transformation was conducted. Begin starting transformation; water bath was

preheated to 42°C exactly. Ten microliters of plasmid added into micro centrifuge tube that

has been precooled in ice. Rosetta II (DE3) competent cell was removed from the freezer

and placed in 50% ice/deionized water bath for 5 minutes. The competent cell was gently

mixed. The 100 III of competent cell was then added into microcentrifuge tube containing

plasm ids on ice by using wide-bore pipette tips. The mixture was mixed gently. The

mixture was left on ice for about 20 minutes. The cells were heat-shocked for exactly 45

seconds at 42°C. The cells were returned to ice for 2 minutes (Sambrook et at., 1989).

Approximately 1 ml of LB media was added into the transformation and was mixed

by flicking gently. The mixture was incubated in incubator-shaker at 37°C for 90 minutes.

About 30 ml LB agar was prepared with the addition of 30 III of 75 mg/ml kanamycin and

9.5 III of 34 mg/ml chloramphenicol antibiotics. When the agar plates were dried and the

transformation has been incubated for 90 minutes in the shaker, 100 III of the

transformation culture was spread onto the agar by using a sterile pipette tips. Three plates

were prepared and the plates were sealed with parafilm strip and placed in a 37°C

incubator for overnight (Sambrook et ai., 1989).

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3.8 Colony PCR

A single colony was picked and transferred into 1.5 ml micro centrifuge containing I ml

LB media, 1 ~l of 75 mg/ml kanamycin and 0.4 ~l of 34 mg/ml chloramphenicol

antibiotics. The mixture was incubated for about 2 hour in an incubator-shaker at 37°C.

About 200 ~l from the mixture was stored for further used. The remaining mixture was

centrifuged at 3500 rpm for 5 minutes. The supernatant obtained was discarded and the

pellet was resuspend by using 200 ~l distilled water. The mixture was preceded to colony

PCR (Sambrook et ai., 1989).

The ingredients were prepared in Table 4 and the negative control was prepared

without the addition of the template. The PCR was run according to the profiling in Table

5. The PCR products was analysed on 1 % agarose gel electrophoresis.

Table 4: Ingredients for Polymerase Chain Reaction

Ingredients Volume

Green Taq Polymerase 7.5 III 3'Xhol Primer 1.5 III 5'Ndel Primer 1.5 ~l Distilled Water 3.5 III Template 1.0 III

Total 15.0 III

Table 5: Polymerase Chain Reaction proflling

Cycles Temperature Time

I cycles Initial Denaturation 3 min

Denaturation 30 sec 35 cycles

{ Annealing 30 sec Elongation 45 sec

1 cycles Final Elongation 5 min

16

· .. 3.9 Optimization of Protein Expression in XLI Blue and Rosetta II (DE3)

Protein expression was optimized according to temperature and incubation times. For

temperatures, 30°C, 35 °C and 37°C were selected. While for incubation times, 2 V2

hours, 5 hours and 24 hours incubation times were selected. The proteins of Adh] were

expressed by the addition of IPTG and 2% sucrose. Set 1 was induced with IPTG only,

while set 2 induced with IPTG and 2% sucrose.

Table 6: Optimization of protein expression based on different temperatures and at different incubation times

~Incubation tim 30°C 35 °C 37°C

2 Yz hours

5 hours

24 hours

On day 1, XLI Blue with pET/AdhJ was cultured overnight at 37°C in 10 ml LB

media with 1 0 ~l of 75 mg/ml kanamycin antibiotics. On the next day, 500 ~l from the

culture overnight was pipetted and transferred into 30 ml LB media together with 30 ~l of

75 mg/ml kanamycin antibiotic. The cultures were grown with shaking at 37°C until

00600 reached between 0.5 - 0.6. Once the OD600 reached the targeted, IPTG solution

was added into the cultures meanwhile the control cultures were not added. The cultures

were grown to the desired temperatures. A control was prepared which was the expression

ofempty pET to compare with expression of pET / Adhl.

The samples of the cultures were collected about 5 ml at 2 V2 hours, 5 hours and 24

hours according to the parameters. The parameters were repeated with induction by IPTG

and 2% sucrose. Protein expression parameters were repeated by using Rosetta II (DE3)

strains.

17