CONSTRUCTION AND EXPRESSION OF HUMAN

ADIPONECTIN IN PROKARYOTIC AND EUKARYOTIC

EXPRESSION SYSTEMS AND THE STUDY OF ITS

EFFECT ON SELECTED BLOOD PARAMETERS AND

EXPRESSION OF RELATED GENES

HUSSIN ALWAN ROTHAN AL-MAAMURI

FACULTY OF SCIENCE

UNIVERSITY OF MALAYA

KUALA LUMPUR

2011

CONSTRUCTION AND EXPRESSION OF HUMAN

ADIPONECTIN IN PROKARYOTIC AND EUKARYOTIC

EXPRESSION SYSTEMS AND THE STUDY OF ITS EFFECT

ON SELECTED BLOOD PARAMETERS AND EXPRESSION

OF RELATED GENES

HUSSIN ALWAN ROTHAN AL-MAAMURI

THESIS SUBMITTED IN FULFILMENT OF

THE REQUIREMENT FOR THE DEGREE

OF DOCTOR OF PHILOSOPHY

INSTITUTE OF BIOLOGICAL SCIENCES

FACULTY OF SCIENCE

UNIVERSITY OF MALAYA

KUALA LUMPUR

2011

Abstract

i

Abstract

Adiponectin is one of the most bioactive substances secreted by adipose tissue

which is involved in protection against metabolic syndrome, artherosclerosis and type II

diabetes. Research into the use of adiponectin as a promising drug for metabolic

syndromes requires production of this hormone in high quantities. This may be achieved

using recombinant DNA technology, which would also allow the production of different

molecular forms as well as providing greater input in terms of understanding its

signalling pathway. This study was mainly targeted towards producing adiponectin

hormone as a recombinant protein by P. pastoris (P-ADP) as a cheap and convenient

eukaryotic expression system for potential application in pharmaceutical therapy. For

comparison, adiponectin was also expressed using the E. coli (E-ADP) as a traditional

prokaryotic expression system. Following successful expression, the relative bio-

properties of P-ADP was assessed in vivo in comparison with E-ADP. Additional

studies of the effect of P-ADP on the expression of the genes encoding glucagon,

insulin and leptin receptors were carried out.

Adiponectin gene was constructed in vitro by splicing its two exons using

overlap-extension PCR. Full length adiponectin was amplified by PCR and cloned into

pMAL™-p4 vector for expression in E. coli as periplasmic secreted protein. The fusion

protein was purified by amylose column after digestion with factor Xa. To express

adiponectin in P. pastoris, the full length adiponectin was amplified by cloning into

pGEM-T vector and then sub-cloning into pPICZαA vector to be expressed as

extracellular secreted protein. The 6xHis-tagged recombinant adiponectin was purified

by one step affinity chromatography using Nickel column. SDS-PAGE and western blot

were used to detect and analyse the recombinant proteins and Bradford assay was used

for protein quantification. Three experiments were designed to assess and compare the

effects of E-ADP and P-ADP on blood glucose and lipid profile using ICR mice as a

Abstract

ii

model system. Real-Time PCR was used to examine the changes in the regulation of

glucagon, insulin and leptin receptors after administration with P-ADP. The expression

of target genes was normalized with β-actin as endogenous gene and the data was

statistically analysed based on Δct values and RQ values using t-test. The results

showed that adiponectin gene was successfully constructed in vitro by overlap-

extension PCR and expressed by E. coli as a soluble periplasm protein and by P.

pastoris as a soluble extracellular protein. P. pastoris expression system was successful

in producing high molecular weight of adiponectin molecules and relatively high

quantity of recombinant protein (0.1 mg/ml) as compared with E.coli (0.04 mg/ml).

The optimum conditions of adiponectin production by P. pastoris were 0.5% of

methanol induction every 12 hours for 60 hours at 30°C. E-ADP and P-ADP were

biologically active in the lowering of blood glucose and triglyceride and increasing high

density lipoprotein. The ability of P-ADP in lowering blood glucose was significantly

higher than E-ADP. However, there was no significant difference on the effect on lipid

profile. P-ADP significantly down-regulates glucagon receptors and up-regulates leptin

receptors, whilst there was no significant effect on insulin receptors. Our results suggest

that P. pastoris expression system is better in producing high quantity, high biological

activity and easily purified recombinant adiponectin comparing with E. coli expression

system that can be used in large scale production of adiponectin as potential drugs for

metabolic syndromes.

iii

ACKNOWLEDGEMENTS

This project was supported through UMRG Research University grant RG022/09B10

and short term grant PS232/2008C, awarded to the author and his supervisor by the

University Malaya and Ministry of Higher Education, Malaysia. The author wishes to

thank these funding bodies for enabling this project to be completed. The author also

wishes to acknowledge Faculty of Science, University of Malaya for access and use of

all relevant facilities and many thanks to Center for Biotechnology Research in

Agriculture (CEBAR) for use of equipments and facilities. The main person who guided

me through all the steps in this work was my supervisor Associate Professor Dr.

Zulqarnain Bin Mohamed, who gave ideas, time, support, and encouraged me to do the

best; he deserves the highest respect and thanks. Many thanks and appreciation to Miss

Teh Ser Huy. She provided so much gracious help with the research. Much appreciation

is also expressed to Associate Professor Dr. Mahmood Ameen Abdulla who provided

help, and research associates for bioassay experiments. I would really like to thank all

my wonderful lab mates. Personally, I would like to thank my wife Rehab for her

patience and support, and also to my brothers and sisters.

Contents

iv

Contents Page

Abstract

Acknowledgements

contents

i

iii

iv

List of figures xi

List of tables xvi

Abbreviations xvii

Chapter 1: Introduction

1.1. Adiponectin 1

1.1.1. Adiponectin gene characteristics 6

1.1.2. Adiponectin protein characteristics 7

1.1.3. Glycosylation of adiponectin protein 11

1.1.4. Regulation of adiponectin gene expression 13

1.1.5. Adiponectin single nucleotide polymorphisms (SNPs) 17

1.1.6. Adiponectin biological activity 20

1.1.6.1. Anti-diabetic properties of adiponectin 22

1.1.6.2. Anti-atherogenic, anti-inflammatory and

cardioprotective properties of adiponectin

27

1.2. Expression of proteins in prokaryotic and eukaryotic expression

systems.

30

1.2.1. Expression in Escherichia coli 31

1.2.1.1. The features of E. coli expression system 32

1.2.1.2. Formation of inclusion bodies and protein

folding

34

Contents

v

1.2.2. Pichia pastoris expression system 38

1.2.2.1. Advantages of using P. pastoris as an

expression system

39

1.2.2.2. Pichia pastoris promoters 41

1.3. Objectives of study 42

Chapter 2: Materials and Methods

2.1. Construction of human adiponectin by overlap-extension PCR 44

2.1.1. Primers design 44

2.1.1.1. Primer design for expression of adiponectin in

P. pastoris

44

2.1.1.2. Primer design for expression of adiponectin in

E. coli

45

2.1.2. PCR reaction 47

2.2. Agarose gel electrophoresis 48

2.3. DNA extraction by gel extraction kit 48

2.4. Adiponectin expression by E. coli 49

2.4.1. Preparation of Luria-Bertani (LB) broth and agar media 49

2.4.2. Competent cells preparation of TB1 E. coli strain 50

2.4.3. Cloning of adiponectin into pMAL™ vector 50

2.4.4. Transformation into E. coli 53

2.4.5. Colony screening PCR 53

2.4.6. Small scale expression of adiponetin by E. coli 54

2.4.7.

Native sodium dodecyl sulfate poly-acrylamide gel

electrophoresis (SDS-PAGE) and SDS-PAGE without

54

Contents

vi

denaturing condition.

2.4.8. Western blot 56

2.4.9. Scale up expression of adiponectin by E. coli 57

2.4.10. Protein purification by amylose resin column 58

2.4.11. Cleavage, denaturing and re-purification of recombinant

protein

58

2.4.12. Bradford assay 59

2.5. Adiponectin expression by Pichia pastoris 59

2.5.1. Cloning of adiponectin into pGEM-T vector 60

2.5.1.1. Ligation reaction 60

2.5.1.2. Transformation 60

2.5.1.3. Colony selection 61

2.5.1.4. Plasmid isolation 61

2.5.1.5. Restriction enzyme digestion of pGEMT-

adiponectin recombinant plasmid

63

2.5.2. Clone of adiponectin in pPICZαA vector 63

2.5.2.1. Preparation of Low Salt Luria-Bertani (LSLB)

broth and agar media

64

2.5.2.2. Preparation of Top10F E. coli competent cells 64

2.5.2.3. Ligation reaction 64

2.5.2.4. Transformation 65

2.5.2.5. Colony selection 65

2.5.2.6. Plasmid isolation 65

2.5.2.7. Linearization of the recombinant plasmid 66

2.5.3. Cloning into Pichia pastoris 66

Contents

vii

2.5.3.1. Preparation of P. pastoris broth and agar

media

66

2.5.3.2. Preparation of P. pastoris competent cells 67

2.5.3.3. Transformation 67

2.5.3.4. Selection of positive colonies 68

2.5.3.4.1. Colony selection via PCR 68

2.5.3.4.2. Colonies capacity of the Zeocin™

resistance

69

2.5.3.4.3. Differentiation between Mut+ and

MutS phenotypes

69

2.5.3.5. Preparation of expression media 69

2.5.3.6. Protein expression and optimization 70

2.5.3.7. Protein purification 71

2.5.3.8. Scale-up expression of recombinant

adiponectin

71

2.5.4. Bioactivity tests 72

2.5.4.1. Effect of recombinant adiponectin produced by

P. pastoris and E. coli on blood glucose and

lipids.

73

2.5.4.2. High and low doses effect of recombinant

adiponectin on blood glucose and lipid profile.

73

2.5.4.3. Oral glucose tolerance test 74

2.5.5. Assessment of genes expression 74

2.5.5.1. Total RNA extraction 74

2.5.5.2. DNase treatment 75

Contents

viii

2.5.5.3. cDNA preparation 76

2.5.5.4. Real-Time PCR 78

2.5.4.4.1. Primers design and validation 81

2.5.4.4.2. Data analysis of Real-Time PCR 84

Chapter 3: Results

3.1. In vitro construction of adiponectin 86

3.2. Expression of adiponectin gene in E. coli 91

3.2.1. Restriction enzyme digestion 93

3.2.2. Selection of recombinant E. coli colonies 95

3.2.3. Sequencing analysis 98

3.2.4. Recombinant adiponectin analysis 100

3.2.5. Scale up expression of adiponectin by E. coli 103

3.2.6. Recombinant adiponectin purification 103

3.3. Expression of adiponectin by Pichia pastoris 108

3.3.1. Cloning of adiponectin in pGEM-T vector 110

3.3.2. EcoRI and NotI digestion 114

3.3.3. Cloning of adiponectin in pPICZαA 118

3.3.4. Sequencing data 121

3.3.5. Transformation into Pichia pastoris 124

3.3.6. Colony selection 126

3.3.7. Selection of Mut+ phenotype with high insertion copy

number colonies

129

3.3.8. Small scale expression of adiponectin 131

3.3.9. Optimization of adiponectin expression 134

Contents

ix

3.3.10. Analysis of recombinant adiponectin protein produced by

E. coli and P. pastoris

140

3.4. Biological activity assessment of recombinant adiponectin 142

3.4.1. Comparison of adiponectin bioactivity produced by P.

pastoris and E. coli

142

3.4.2. Effect of the high and low doses of P-ADP on blood

glucose and lipid profile

145

3.4.3. Oral glucose tolerant test 148

3.5. Effect of P-ADP administration on the expression of glucagon,

insulin and leptin receptors

150

3.5.1. Total RNA extraction and DNase treatment 150

3.5.2. RT- PCR 152

3.5.2.1. Expression of glucagon receptors 152

3.5.2.2. Expression of insulin receptors 157

3.5.2.3. Expression of leptin receptors 162

Chapter 4: Discussion

4.1. In vitro gene construction 168

4.2. Expressions of adiponectin in prokaryotic and eukaryotic expression

systems

172

4.2.1. Expression of adiponectin by E. coli 173

4.2.1.1. Adiponectin protein solubility 175

4.2.1.2. Adiponectin protein purification 177

4.2.2. Adiponectin expression by Pichia pastoris 178

4.2.2.1. Production of adiponectin as extracellular 179

Contents

x

secreted protein

4.2.2.2. Optimization of protein expression 180

4.2.2.3. Protein solubility 182

4.2.3. Comparison between adiponectin expression in E. coli and

P. pastoris

182

4.3. Adiponectin bioactivity 184

4.3.1. Effects of E-ADP and P-ADP on blood glucose 185

4.3.2. P-ADP potential in lowering blood glucose 187

4.3.3. Effects of E-ADP and P-ADP on lipid profile 189

4.4. Effect of P-ADP administration on related gene expression 191

4.4.1. Effect of P-ADP administration on the expression of

glucagon receptors

192

4.4.2. Effect of P-ADP administration on the expression of insulin

receptors

193

4.4.3. Effect of P-ADP administration on the expression of leptin

receptors

193

4.5. Summary and conclusion 195

References 198

Appendices

Appendix A Coference abstracts and presentations

Appendix B Awards received

Contents

xi

List of figures

xi

List of figures Page

Figure 1.1 Summary of the diversity of cell-signalling responses to

adipokines

2

Figure 1.2 Research interest in adiponectin, a protein secreted by adipose

tissue

4

Figure 1.3 The relationships between plasma adiponectin levels, obesity

and insulin resistance

5

Figure 1.4 Structure and adiponectin domains 8

Figure 1.5 Multimer formation of adiponectin 10

Figure 1.6 Structural characteristics of adiponectin protein. 12

Figure 1.7 Regulation of adiponectin synthesis and function. 14

Figure 1.8 PPAR agonists thiazolidinediones (TZDs) ameliorate insulin

resistance and diabetes by both adiponectin-dependent and -

independent pathways.

16

Figure 1.9

Figure 1.10

Figure 1.11

Figure 1.12

Figure 1.13

Figure 1.14

Figure 1.15

Figure 2.1

Figure 2.2

Diagram of polymorphic variants of the adiponectin gene.

Main target organs and bio-activities of adiponectin.

The hypothesis of adiponectin effect on metabolic syndromes.

Proposed structure of adiponectin receptors.

Signal transduction by adiponectin receptors.

Adiponectin functions as anti-atherogenic factor.

The pathway of protein folding and secretion in E. coli

The design of RT-PCR experiment.

Melt curves of each gene of interest and an endogenous gene.

19

21

23

26

26

29

37

80

83

Figure 3.1 In vitro construction of ADPs. 88

Figure 3.2 In vitro construction of ADPws. 89

Figure 3.3 Amplification of ADPs and ADPws using different primers for 90

List of figures

xii

cloning into P.pastoris and E.coli.

Figure 3.4 The map of pMAL™-p4x and pMAL™-c4x vectors explaining

enzymes restriction sites.

92

Figure 3.5 Restriction enzyme digestion for cloning into E. coli 94

Figure 3.6 Agarose gel electrophoresis of colony screening ADPs. 96

Figure 3.7 Agarose gel electrophoresis of colony screening for ADPws. 97

Figure 3.8 Sequencing data of ADPs. 99

Figure 3.9 SDS-PAGE analysis of adiponectin expression before and after

induction with IPTG.

101

Figure 3.10 Western blot analysis of adiponectin expression before and after

induction with IPTG.

102

Figure 3.11 Purification by amylose resin column. 104

Figure 3.12 SDS-PAGE for optimization of Factor Xa digestion. 106

Figure 3.13 SDS-PAGE for second purification by hydroxyapatite column

and amylose resin column.

107

Figure 3.14 The map of pPICZα A plasmid that was used to clone the gene

of interest into P. pastoris.

109

Figure 3.15 PGEMT-vector map. 111

Figure 3.16 Agarose gel electrophoresis of colony selection by PCR using

M13 forward and reverse primers to detect existence of ADPws.

112

Figure 3.17 Agarose gel electrophoresis of colony selection by PCR using

M13 forward and reverse primers to confirm existence of ADPs.

113

Figure 3.18 Agarose gel electrophoresis of adiponectin-pGEMT

recombinant plasmid.

115

Figure 3.19 Agarose gel electrophoresis of restriction enzyme digestion to 115

List of figures

xiii

generate ADPs with two sticky ends.

Figure 3.20 Agarose gel electrophoresis of restriction enzyme digestion to

generate ADPws with two sticky ends.

116

Figure 3.21 Agarose gel electrophoresis of pPICZαA plasmid digestion with

EcoRI and NotI.

117

Figure 3.22 Colony screening PCR using α-factor forward primer and

3’AOX1 reverse primer to detect ADPws.

119

Figure 3.23 Colony screening PCR using α-factor forward primer and

3’AOX1 reverse primer to detect ADPs.

120

Figure 3.24 Sequencing data of ADPws after cloning in pPICZαA. 122

Figure 3.25 The sequence of ADPs after cloning in pPICZαA. 123

Figure 3.26 Recombinant plasmid linearization with SacI restriction enzyme. 125

Figure 3.27 Colony PCR screening using α-factor forward primer and

3’AOX1 reverse primer to detect ADPws into yeast colonies.

127

Figure 3.28 Colony screening PCR using α-factor forward primer and

3’AOX1 reveres primer to detect ADPs into yeast colonies.

128

Figure 3.29 Multi insertion of expression cassette into yeast cells. 130

Figure 3.30 SDS- PAGE analysis of the supernatant collected from cultures

inoculated with yeast colonies carrying ADPws.

132

Figure 3.31 SDS- PAGE analysis of the supernatant collected from cultures

inoculated with yeast colonies carrying ADPs.

133

Figure 3.32 The growth rate of yeast during time course expression. 135

Figure 3.33 SDS-PAGE analysis of the time course for adding methanol and

culture harvesting every 12 hours after methanol induction.

136

Figure 3.34 Western blot analysis of the time course for adding methanol

and culture harvesting every 12 hours after induction.

137

List of figures

xiv

Figure 3.35 SDS-PAGE without denaturing condition showed less oligomers

for E-ADP compared with P-ADP.

141

Figure 3.36 Effect of P-ADP and E-ADP on the levels of blood glucose. 143

Figure 3.37 Effect of P-ADP and E-ADP on lipid profile. 144

Figure 3.38 Effect of the high and low doses of P-ADP on blood glucose

levels.

146

Figure 3.39 Effect of the high and low doses of P-ADP on blood lipid

profile.

147

Figure 3.40 Oral glucose tolerance test. 149

Figure 3.41 Total RNA extraction by TRIzol™ method. 151

Figure 3.42 Total RNA after DNase treatment. 151

Figure 3.43 Expression of glucagon receptors: The comparative ΔCt value of

the biological groups that contain control or treatment groups.

153

Figure 3.44 Expression of glucagon receptors: The mean of biological

group’s ΔCt value for liver, abdominal fat tissue and kidney for

treatment and control groups.

154

Figure 3.45 Expression of glucagon receptors: Relative quantification

differences between treatment and control groups.

156

Figure 3.46 Expression of insulin receptors: The comparative ΔCt value of

the biological groups that contain control or treatment groups.

158

Figure 3.47 Expression of insulin receptors: The mean of biological group’s

ΔCt value for liver, abdominal fat tissue, skeletal muscle and

kidney for treatment and control groups.

159

Figure 3.48 Expression of insulin receptors: Relative quantification

differences between treatment and control groups.

161

Figure 3.49 Expression of leptin receptors: The comparative ΔCt value of the 163

List of figures

xv

biological groups that contain control or treatment groups.

Figure 3.50 Expression of leptin receptors: The mean of biological group’s

ΔCt value for liver, abdominal fat tissue and kidney for

treatment and control groups.

164

Figure 3.51 Expression of leptin receptors: Relative quantification

differences between treatment and control groups.

166

Figure 4.1 In vitro construction of adiponectin gene by overlap-extension

PCR

170

List of tables

xvi

List of tables Page

Table 2.1 Sequences of primers used to express adiponectin in P. pastoris. 46

Table 2.2 Sequences of primers used to express adiponectin in E. coli. 46

Table 2.3 The components of the ligation mix. 52

Table 2.4 The components of 2X RT master mix. 77

Table 2.5 Thermal cycle conditions of RT reaction. 77

Table 2.6 The components of RT-PCR reaction 79

Table 2.7

Table 2.8

Table 3.1

Table 3.2

Table 3.3

Table 3.4

Thermal cycle conditions of RT-PCR reaction

Forward and reverse primers for each gene of interest and an

endogenous gene.

The differences were observed in the optical density, total cells

mass and protein concentration at different time points after

methanol induction.

The results of real time PCR and the results of statistical analysis

for mean ΔCt values and relative quantification values (RQ)

using t-test statistical analysis.

The results of real time PCR and the results of statistical analysis

for mean ΔCt values and relative quantification values (RQ)

using t-test statistical analysis.

The results of real time PCR and the results of statistical analysis

for mean ΔCt values and relative quantification values (RQ)

using t-test statistical analysis.

79

82

139

155

160

165

Abbreviation

xvii

Abbreviations

3T3-L1

Cell line derived from 3T3 cells used in biological research

on adipose tissue.

C degree Celsius

g microgram

g/ml microgram per millilitre

l microlitre

M micro Molar

3T3-F442A preadipocyte cell lines

ADPs adiponectin DNA fragment with signal peptide sequence

ADPws adiponectin DNA fragment without signal peptide

sequence

AMPK 5' adenosine monophosphate-activated protein kinase

AOX Alcohol oxidase

APS

TEMED

ammonium persulfate

N, N, N', N'-tetramethylethylenediamine

Arg Arginine

BMGY Buffered Glycerol-complex Medium

BMMY Buffered Methanol-complex Medium

bp base pair

BSA bovine serum albumin

BW Body weight

CaCl2 calcium chloride

cDNA complementary DNA

Abbreviation

xviii

CDS coding sequence

CHO Chinese hamster ovary

CHOL total cholesterol

Ct Cycle time

CV coefficient of variation

Cys cysteine

DEPC Diethylpyrocarbonate

DNA deoxyribonucleic acid

dNTPs Deoxyribonucleoside triphosphate

e.g for example

E-ADP recombinant adiponectin expressed in E. coli

EDTA ethylenediaminetetraacetic acid

ERp44 endoplasmic reticulum folding assistant protein

et al. et alii (and other people)

EtBr ethidium bromide

FLD1 formaldehyde dehydrogenase gene

g/L gram per litre

Glu Glutamic acid

Gly Glycine

HDL high density lipoprotein

His Histidine

HMW High molecular weight

hrs hours

i.e. that is

IGF-1 Insulin like growth factor 1

Ile Isoleucine

Abbreviation

xix

IPTG isopropyl-beta-dthiogalactopyranoside

kb kilo bases

kDa kilo dalton

KOAc potassium acetate

lbs/sq.in.

LB

pounds per square inch

Luria-Bertani broth

LDL low-density lipoproteins

LMW Low molecular weight

LSLB low salt Luria-Bertani broth

M molar

MBP maltose binding protein

mg/L milligram per litre

mg/ml milligram per millilitre

Mg2+

magnesium ion

min minute

mM milli Molar

mRNA messenger RNA

Mut- methanol utilization minus

Mut+ methanol utilization plus

MutS methanol utilization slow

NaCl sodium chloride

NaOH sodium hydroxide

ng nanogram

Ni nickel

nm nanometer

O2 oxygen

Abbreviation

xx

OD600 optical density at 600nm

P-ADP recombinant adiponectin expressed in P. pastoris

PAGE polyacrylamide gel electrophoresis

PAOX1 promoter from the alcohol oxidase 1 gene

PBS phosphate buffered saline

PCR polymerase chain reaction

Pfu Pyrococcus furiosus

PPAR peroxisome proliferator-activated receptor

Pro proline

RNA ribonucleic acid

RNase A ribonuclease A

rpm revolutions per minute

RQ relative quantification

RT reverse transcription

RT-PCR Real-Time PCR

s second

sdH2O sterile distilled water

SDS sodium dodecyl sulfate

SEM standard error means

Ser Serine

SNPs single nucleotide polymorphisms

Stdev standard deviation

Taq Thermus aquaticus

TBE Tris Borate EDTA

TBS Tris buffered saline

TBST Tris buffered saline-Tween 20

Abbreviation

xxi

TCA trichloroacetic acid

TE Tris-EDTA

TG triglyceride

Tm Melting temperature

TNF-α Tumor necrosis factor-alpha

TZDs thiazolidinediones

U unit

UV ultraviolet

V volume

X-Gal bromo-chloro-indolyl-galactopyranoside

YPD Yeast Extract Peptone Dextrose