CONSTRUCTION OF A STREP-TAG II MUTANT MALTOSE...
Transcript of CONSTRUCTION OF A STREP-TAG II MUTANT MALTOSE...
CONSTRUCTION OF A STREP-TAG II MUTANT MALTOSE BINDING
PROTEIN FOR REAGENTLESS FLUORESCENCE SENSING
SITI HALIMAH BINTI HASMONI
UNIVERSITI TEKNOLOGI MALAYSIA
CONSTRUCTION OF A STREP-TAG II MUTANT MALTOSE BINDING
PROTEIN FOR REAGENTLESS FLUORESCENCE SENSING
SITI HALIMAH BINTI HASMONI
A thesis submitted in fulfillment of the requirement for the award of the degree of
Master of Science (Bioscience)
Faculty of Bioscience and Bioengineering
Universiti Teknologi Malaysia
NOVEMBER 2012
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To my beloved parents, my family and my soulmate, my best friend; my husband
and not forgetting to our newborn bub, Muhammad Rafiqi.
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ACKNOWLEDGEMENT
First and foremost, I would like thank Dr Shafinaz, my supervisor, who had been
dedicated and committed to guide me all this while. Her patience, encouragement and
trust is truly appreciated and deeply cherished. Also, thanks to Prof Dr Anthony Cass,
for his advice and insights on this research. Next, I would like to express my gratitude to
Dr. Saiful Karsani and Dr. Goh Kian Mau is members of the research group. Thank you
for the opportunity and the wealth of information shared during discussions. Also, my
greatest gratitude goes to Prof Dr. Peter Klappa, who helps me solve the missing puzzle
of my research with his knowledge and wisdom.
Furthermore, I am thankful to Ministry of Higher Education for funding my
masters study. I would like to thank the Ministry of Higher Education again for
funding this project under Fundamental Research Grant Scheme (FRGS) Vote
Number 78309. A great appreciation for FBB,UTM for the research facilities they
provided and also thank you to PSZ library for easy access of many literatures
online.
Thank you to the technical supports from most of the companies that I
contacted with for their advices and help. Not forgetting my fellow postgraduates’
friends in this faculty for giving me their encouragements, helping hand and
motivation. Thank you for being there for me. Special thanks to the best
labmates/friends a researcher could for; Edot, Yok, Maya, Kak Su and Tiqah.
I would like to thank my supportive father and mother, my beloved sisters
and brothers for supporting me no matter what. I would like to extend my
appreciation to my husband who is always there for me through the ups and downs of
this journey. And lastly, I am grateful to Allah.
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ABSTRACT
Maltose binding protein (MBP) changes its conformational structure upon its ligand
binding.This molecular recognition element that transduces a ligand-binding event
into a physical one make MBP an ideal candidate for reagentless fluorescence
sensing. MBP gene, (malE) was amplified from a pMaL-C4x plasmid vector and was
fused to a Strep-Tag II pET-51b(+) vector. Strep-Tag II is a tag that will enable the
MBP to be unidirectionally immobilized on solid supports. A cysteine mutant of the
MBP was constructed by inverse PCR and the recombinant protein fusion was then
purified by affinity purification using Strep-Tactin resin. To sense maltose binding,
an environmentally sensitive fluorophore (IANBD amide) was covalently attached to
the introduced thiol group. The tagged mutant MBP (D95C) was successfully
generated and the protein was successfully purified with the expected molecular size
of ~42 kDa observed on the SDS PAGE. The fluorescence measurements of the
IANBD labeled of tagged mutant MBP (Strep-Tag II D95C) in the solution phase,
showed an appreciable change in fluorescence intensity with dissociation constant,
(Kd) of 7.6 ± 1.75 µM. Nonetheless, it could retain its ligand binding activity towards
maltose. However, immobilization of Strep-Tag II D95C on solid surface suffered
some limitation with the Strep-Tactin coated microwell plates because it did not give
any dependable results to support the ligand binding activity of the site directed
immobilized protein. Thus, this engineered mutant MBP (Strep-Tag II fused D95C)
could be potentially developed for biosensor application with further improvement in
protein immobilization method.
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ABSTRAK
Protein pengikat maltosa (MBP) mengalami perubahan struktur konformasi
semasa mengikat pada ligan. Molekul pengenalpastian yang menyebabkan transduksi
pengikatan ligan kepada bentuk fizikal menjadikan MBP calon yang sesuai sebagai
penderia pendaflour tanpa reagen. Gen MBP (malE) diamplifikasi daripada vektor
plasmid pMaL-C4x dan kemudian digabungkan dengan Strep-Tag II yang terdapat
pada vektor pET-51b(+). Strep-Tag II merupakan tag yang membolehkan MBP
disekatgerak secara seragam kepada penyokong pepejal. Mutan MBP yang
mempunyai satu residu sisteina telah dihasilkan melalui PCR berbalik dan gabungan
protein rekombinan ini ditulen melalui penulenan afiniti yang menggunakan resin
Strep-Tactin. Untuk mengesan pengikatan maltosa, flourofor yang sensitif pada
persekitaran (IANBD amida) telah diikat secara kovalen kepada kumpulan tiol yang
telah diperkenalkan pada protein itu. Mutan MBP yang bertag telah berjaya dijana
dan melalui pemerhatian SDS-PAGE protein ini telah berjaya ditulen dengan saiz
molekul ~42 kDa seperti yang dijangkakan. Ukuran pendaflour di dalam fasa larutan
bagi mutan MBP yang bertag dan berlabel dengan IANBD menunjukkan perubahan
ketara bagi keamatan pendaflour dengan pemalar penguraian Kd 7.6 ± 1.75 µM.
Walau bagaimanapun, aktiviti pengikatan ligan terhadap maltosa boleh dikekalkan.
Pemegunan protein Strep-Tag II D95C pada permukaan pepejal, berhadapan dengan
beberapa kelemahan apabila piring mikrotelaga yang bersalut Strep-Tactin
digunakan kerana ia tidak memberi keputusan yang dapat menyokong aktiviti
pengikatan ligan oleh protein yang dipegunkan pada tapak khusus. Oleh itu,
kejuruteraan mutan MBP gabungan Strep-Tag II-D95C berpotensi dibangunkan
untuk aplikasi biopenderia dengan lebih penambahbaikan dalam kaedah pemegunan
protein.
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TABLE OF CONTENT
CHAPTER TITLE PAGE
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENT vii
LIST OF TABLES xii
LIST OF FIGURES xiii
LIST OF APPENDICES xvi
LIST OF ABBREVIATIONS xvii
1 INTRODUCTION
1.1 Introduction 1
1.2 Research Objectives 3
1.3 Significance of Research 4
1.4 Scope of Research 4
2 LITERATURE REVIEW
2.1 Periplasmic Binding Protein (PBPs) 5
2.2 Maltose Binding Protein (MBP) 6
2.3 Reagentless Fluorescence Sensors Based on PBPs 9
2.3.1 Principles of Fluorescence 10
2.3.2 Thiol-reactive Fluorophore 12
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2.4 Engineering MBP for Reagentless Fluorescence Sensing 13
2.5 Tagged Proteins for Site-specific Immobilization 16
3 CONSTRUCTION OF STREP-TAG II MUTANT
MBP (D95C) FOR FLUORESCENCE SENSING
3.1 Introduction 19
3.2 Research Methodology 20
3.3 Materials 21
3.3.1 Chemicals and reagents 21
3.3.2 Bacterial strains 21
3.3.3 Plasmids 22
3.3.4 Enzymes 22
3.3.5 Culture media, buffers and stock solutions 22
3.4 Molecular Biology Methods 23
3.4.1 Competent cell preparation 23
3.4.2 Heat-shock transformation of cells 24
3.4.3 Plasmid extraction and purification 24
3.4.4 Restriction enzyme digestion of DNA 24
3.4.5 Agarose gel electrophoresis 25
3.4.6 DNA ligation 25
3.4.7 Construction of pET-51b (+)- malE 25
3.4.8 Screening for recombinant clones 27
3.4.9 Construction of pET-51b(+)-D95C 28
3.5 Results and Discussion 29
3.5.1 Construction of pET-51b (+)- malE 29
3.5.2 Construction of cysteine mutants of MBP (D95C) 34
3.5.2.1 Design of primer sequence for site-directed
mutagenesis 34
3.5.2.2 Identification of cysteine mutants by
restriction enzyme digestion 35
3.6 Conclusions 39
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4 EXPRESSION AND PURIFICATION OF STREP-TAG II
WILD TYPE MBP AND ITS MUTANT (D95C)
4.1 Introduction 40
4.2 Research Methodology 41
4.3 Materials 42
4.3.1 Chemicals and reagents 42
4.3.2 Culture media, buffers and stock solutions 42
4.4 Expression and purification of the recombinant protein 44
4.4.1 Recombinant protein expression of Strep-Tag II
Wild Type MBP and its mutant D95C 44
4.4.2 Spectroscopic determination of protein
concentration 45
4.4.3 Protein assay by Bradford reagent 47
4.4.4 Protein electrophoresis (SDS-PAGE) 47
4.4.5 Western blotting for detection of Strep-Tag II 48
4.4.6 Western blotting for detection of Maltose
Binding Protein 48
4.4.7 Recombinant protein purification via Strep-Tag II
affinity purification 49
4.4.8 Recombinant protein purification via
size exclusion chromatography 50
4.4.9 Ultrafiltration of the pooled fractions 51
4.5 Results and Discussions 52
4.5.1 Recombinant expression of Strep-Tag II
wild type MBP and its mutant (D95C) 52
4.5.2 Strep-Tag II affinity purification of Strep-Tag II
wild type MBP 57
4.5.3 Affinity purification of Strep-Tag II mutant
MBP (D95C) in room temperature 59
4.5.4 Size Exclusion Purification of Strep-Tag II
mutant MBP (D95C) 62
4.5.5 Affinity purification of Strep-Tag II mutant MBP
(D95C) in ice 65
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4.6 Conclusions 71
5 INTRINSIC TRYPTOPHAN OF STREP-TAG II WILD
TYPE MBP AND EXTRINSIC FLUORESCENCE
SENSING OF STREP-TAG II MUTANT MBP (D95C)
5.1 Introduction 72
5.2 Materials 73
5.3 Methodology 74
5.3.1 Intrinsic tryptophan fluorescence of Strep-Tag II
wild type MBP 74
5.3.2 Extrinsic fluorescence of Strep-Tag II mutant
MBP (D95C) 75
5.3.2.1 Fluorophore labeling 75
5.3.2.2 Extrinsic fluorescence measurement of
Strep-Tag II mutant MBP (D95C) 76
5.3.3 Immobilization of Strep-Tag II mutant
MBP (D95C) on functionalized surface 77
5.3.3.1 Immobilization of Strep-Tag II D95C on
Strep-Tactin coated microwell plates 77
5.3.3.2 Immobilization of Strep-Tag II D95C on
Strep-Tactin agarose. 78
5.3.3.3 Immobilization of Strep-Tag II D95C on
Streptavidin coated microwell plates 78
5.4 Results and Discussions 79
5.4.1 Intrinsic tryptophan fluorescence of Strep-Tag II
wild type MBP 79
5.4.2 Extrinsic fluorescence sensing of Strep-Tag II
mutant MBP (D95C) 81
5.4.3 Fluorescence response of immobilized
Strep-Tag II D95C 85
5.4.3.1 Immobilization of Strep-Tag II D95C on
Strep-Tactin coated microwell plates 86
5.4.3.2 Immobilization of D95C on
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Strep-Tactin agarose 89
5.4.3.3 Immobilization of Strep-Tag II D95C
on streptavidin coated microwell plates 90
5.5 Conclusion 93
6 CONCLUSIONS AND FUTURE WORK
6.1 Conclusion 94
6.2 Future Work 95
6.2.1 Protein modeling of Strep-Tag II mutant MBP 95
6.2.2 X-ray crystallography 96
6.2.3 Characterisation of binding interactions by
isothermal titration calorimetry (ITC) 96
6.2.4 Characterisation of secondary structure and
folding protein properties by circular
dichorism (CD) 97
6.2.5 Improved methods for immobilization of
Strep-Tag II mutant MBP (D95C) 97
REFERENCES 98
APPENDINCES A-E 109
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LIST OF TABLES
TABLE NO. TITLE PAGE
2.1 Types of thiol-reactive fluorophores 13
3.1 Primers sequence used for amplification of the
cytoplasmic malE gene 26
3.2 Primers sequence used for site-directed mutagenesis of the
MBP cysteine mutants D95C 28
4.1 The concentration (Molar) of crude lysate which
was calculated according to the Beer Lambert Law 54
4.3 The concentration (Molar) of protein sample which was
calculated according to the Beer Lambert Law 69
4.4 Purification table. The concentration and total protein
of protein samples using protein assay Bradford reagent. 70
5.1 Comparisons of the dissociation constant of D95C and wild type
MBP 83
5.3 Optimization of immobilization of D95C onto streptavidin
microwell plates. 91
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LIST OF FIGURES
FIGURE NO. TITLE PAGE
2.1 A three dimensional model of a maltodextrin binding
protein with bound maltose (1ANF) 6
2.2 A model of MBP transport system 7
2.3 Schematic model of MBP bound to Tar that induces the
chemotaxis of the bacteria 8
2.4 Jablonski Diagram 11
3.1 Workflow of construction of pET-51b(+)-malE 20
3.2 Workflow of construction of cysteine mutants of MBP (D95C)
by site-directed mutagenesis 21
3.3 pET-51b(+) Multiple Cloning Site 26
3.4 Agarose gel electrophoresis showing the size of
the amplified PCR product. 30
3.5 Agarose gel electrophoresis showing the size of the pET-51b(+)-
malE construct and the Restriction enzyme test of the construct 31
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3.6 The contig assemblies of forward and reverse sequence
of pET-51b(+)-malE construct using DNA Baser v.2.75.0. 32
3.7 ORF finder for the contig assemblies of forward and reverse
sequence of pET-51b(+)-malE 33
3.8 Construction of MBP cysteine mutant (D95C) 34
3.9 Agarose gel electrophoresis showing the size of the product
of inverse PCR of pET-51b(+)-D95C 35
3.10 Agarose gel electrophoresis of screening mutant MBP
(pET-51b(+)-D95C) with restriction enzyme FspI 36
3.11 Agarose gel electrophoresis of diagnostic restriction enzyme
of BamHI and HindIII on the wild type and mutant 37
3.12 ORF finder for the contig assemblies of forward and reverse
sequence of pET-51b(+)-D95C. 38
4.1 Workflow the expression and purification of Strep-Tag II
wild type MBP and its mutant (D95C). 41
4.2 SDS- PAGE (12%) of the crude lysate of Strep-Tag II wild type
and mutant (D95C) proteins. 53
4.3 A: SDS-PAGE of crude lysate of the wild type and mutant
D95C. B: Western blot of MBP for crude lysate of the wild type
and mutant D95C. C: Western blot of Strep-Tag II for crude lysate
of the wild type and mutant D95C. 55
4.4 Chromatogram of Strep-Tag II affinity purification using
Akta-Prime Plus 57
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4.5 SDS- PAGE of the crude lysate and pooled of the fractions
collected during elution of Strep-Tag II purification 58
4.6 (A) Western blot of maltose binding protein for the crude lysate
and fractions collected during elution of Strep-Tag II purification.
(B) Western blot of Strep-Tag II for the crude lysate and
fractions collected during elution of Strep-Tag II purification 58
4.7 Elution peak of Strep-Tag II affinity purification using
Akta-Prime Plus 59
4.8 SDS- PAGE of the crude lysate D95C and the fractions
collected during elution of Strep-Tag II purification of D95C 60
4.9 (A) Western blot of maltose binding protein for the crude lysate
and fractions collected during elution of Strep-Tag II purification.
(B) Western blot of Strep-Tag II for the crude lysate and
fractions collected during elution of Strep-Tag II purification 61
4.10 Elution peak of Strep-Tag II affinity purification using
Akta-Prime Plus 63
4.11 SDS- PAGE of the affinity purification product and the
fractions collected during elution of Strep-Tag II purification
of D95C 63
4.12 SDS- PAGE of the crude lysate of D95C and pooled
fractions of affinity and gel filtration purification 64
4.13 SDS- PAGE of the crude lysate of D95C and observed the
presence of inclusion bodies from the pellet of lysed cell 65
4.14 SDS- PAGE of the crude lysate of D95C and pooled
fractions of affinity and gel filtration purification 66
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4.15 (A) Western blot of Strep-Tag II for the crude lysate and
fractions collected during elution of Strep-Tag II purification.
(B) Western blot of maltose binding protein for the crude
lysate and fractions collected during elution of
Strep-Tag II purification 67
4.16 A: SDS-PAGE (12%) of crude lysate of the wild type
and mutant D95C. B: Western blot of MBP for crude lysate
of the wild type and mutant D95C. C : Western blot of
Strep-Tag II for crude lysate of the wild type and mutant D95C 68
5.1 Maltose binding curves of intrinsic tryptophan of wild type
MBP 80
5.2 Fluorescence emission spectra of IANBD labeled D95C 81
5.3 Maltose binding curves of labeled IANBD D95C 82
5.4 Glucose binding curve of the labeled IANBD D95C 84
5.5 Illustration of unidirectional immobilization of Strep-Tag II
fused D95C on functionalized surface 85
5.6 Maltose binding curves of Immobilized labeled IANBD of
D95C 90
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LIST OF APPENDICES
APPENDIX TITLE PAGE
A Preparation reagents for SDS PAGE 109
B Standard curve for Bradford assay 111
C Molecular weight of protein standard of unstained
protein marker (Fermentas) 112
D Molecular weight of protein standard of Strep-Tag II
perfect protein markers (Merck) 113
E Raw data of fluorescence measurement for
immobilization Strep-Tactin coated microwell plate 114
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LIST OF ABBREVIATIONS
ºC - Degree celcius
µg/ml - Microgram per millilitre
µl - Microliter
µM - Micromolar
bp - Basepair
BLAST - Basic local alignment search tool
CaCl2 - Calcium chloride
cm - centimeter
DMSO - Dimethyl sulphoxide
DNA - Deoxyribonucleic acid
dNTP - Deoxyribonucleotides
dsDNA - double stranded Deoxyribonucleic acid
DTT - Dithiothreitol
EDTA - Ethylenediaminetetraacetic acid
ELISA - Enzyme-linked immunosorbent assay
g - gram
HRP - Hydrogen peroxidase
IANBD - N,N’-dimethyl-N-(iodoacetyl)-N’-(7-nitrobenz-2-oxa-
1,3-diazol-4 -yl)ethylenediamine
IPTG - Isopropyl β-D-1-thiogalactopyranoside
K2HPO4 - Dipotassium hydrogen phosphate
KCl - Potassium chloride
Kd - Dissoication constant
kDa - kilodalton
KH2PO4 - Potassium dihydrogen phosphate
KOAc - Potassium acetate
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LB - Luria Bertani
LBA - Luria Bertani Agar
M - Molar
MgSO4.7H2O - Magnesium sulfate heptahydrate
mM - Milimolar
MnCl2 - Manganese chloride
MOPS - 3-(N-morpholino)propanesulfonic acid
NaCl - Sodium chloride
nm - Nanometer
OD - Optical density
PAGE - Polyacrylamide Gel Electrophoresis
PBS - Phosphate buffer saline
PCR - Polymerase chain reaction
PMSF - Phenylmethanesulfonyl fluoride
RbCl - Rubidium chloride
rpm – Rotation per minute
SDS - Sodium dodecyl sulfate
TAE - Tris base, acetic acid, EDTA buffer
UV - Ultraviolet
V - Voltage
v/v - Volume per volume
w/v - Weight per volume
CHAPTER 1
INTRODUCTION
1.1 Introduction
Biosensor is an analytical device that functions by coupling a biological
sensing element with a detector system using a transducer (Chauhan et al., 2004). It
involves exploiting the recognition and detection system of a biology component for
a target molecule or macromolecule with a transducer that converts the biological
recognition event into an output signal (Collings et al., 1997). The signal can be
electrical, optical or thermal and is converted by a suitable transducer into a
measurable electrical parameter such as electrical or current. Since its establishment,
biosensors have been widely used in diagnostics, pharmaceutical research,
agriculture, food safety, environment and industrial monitoring (Luong et al., 2008).
There have been some limitations in producing biosensors because each
device is unique and requires an amount of time and optimization. This is because
developments of most biosensors involve the identification of natural specificity of a
biological component for an analyte and the discovery of a suitable signal transducer
that is adapted to the macromolecule (Chauhan et al., 2004). Hence, protein
engineering techniques are being used to overcome this issue where signal
transduction properties of biological molecules are being modified to adapt the
detector instrumentation rather than adapting the detector instruments to the unique
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requirements of each natural molecule. This is achieved by integrating a functional
group that gives simple signal-transduction mechanism such as optical or electrical,
to the protein itself (Hellinga and Marvin, 1998).
Development of sensor devices requires incorporation of sensing proteins into
a detector element by encapsulation or surface immobilization on a suitable material
for interfacing with detectors (De Lorimier et al., 2006). It is crucial for the protein
to be immobilized for reagentless biosensing and for further application in biosensor
such as protein microarray. Thus, with protein engineering techniques, enzymes and
proteins can be chemically modified to make them more stable and more specific
with more regular interface for immobilization. A protein may be modified so that its
active site points outwards from the sensor surface once the protein is immobilized
because the active sites need to be accessible to the analyte (Collings et al., 1997).
Thus, in this research, rational protein engineering techniques will be applied to
molecular engineer proteins for unidirectional immobilization on functionalized
surfaces and to investigate its protein activity. Moreover, it will assess the
applicability of immobilized protein for biosensing application for high throughput
analysis of analyte.
The protein used in this research is maltose binding protein (MBP) which is
in the superfamily of periplasmic binding protein (PBP) that is extensively studied
as receptors for sensor applications (Hellinga et al., 1998). This is because PBP
superfamily has a remarkable adaptablity for their cognate ligands and can be
designed to bind nonnatural ligands (Marvin and Hellinga, 2001). The structure of
PBPs both with and without their ligands bound, has been described as a ‘Venus
Flytrap’ where the two lobes of the protein will close upon the ligand, entrapping it
(Gilardi et al., 1994). This molecular recognition element that transduces a ligand-
binding event into a physical event makes it suited for biosensor applications such as
reageantless fluorescent biosensing. MBP will be altered genetically to construct a
regeantless biosensor so that a reporter group (fluorophore) may be covalently linked
to MBP. The fluorophore will respond to the ligand binding event of the labeled
protein and this response is measured by fluorescence intensity changes.
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It is worth noting that, to the extent of our knowledge, all of the reported works
describing signal transduction by fluorophore labeled PBPs have been studied
without their linkage to solid supports (Brune et al., 1994; Gilardi et al., 1994;
Marvin et al., 1997; Hellinga and Marvin, 1998; Marvin and Hellinga, 2001). In this
research, protein immobilization will be done by exploiting the affinity tag by
genetic engineering. Affinity tags are widely used in biotechnology to assist
purification of recombinant protein. The MBP will be fused to an affinity tag, Strep-
Tag II to aid in purification and site-directed immobilization of the MBP onto
functionalized solid surfaces. Strep-Tag II consists of eight amino acids fusion tag
that will bind to Strep-Tactin protein (Merck Biosciences, 2007). The Strep-Tag II
will act as a handle that will enable the attachment of MBP to a Strep-Tactin surface
on microwell-plates for optimal ligand binding.
1.2 Research objectives
The ultimate aim of this work was to construct tagged fusion proteins for site
directed immobilization onto surfaces with a view to investigate their potential for
biosensor applications. To achieve this, the objectives were as follows:
1. To mutate the Maltose Binding Protein (MBP) for the development of a
reagentless fluorescence sensing system for maltose.
2. To overexpress and purify Strep-Tagged II fusion MBP and its mutant via
affinity chromatography
3. To determine the ligand binding activity of fluorophore-labeled mutant MBP
via fluorescence intensity measurements.
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1.3 Significance of Research
The immobilization of proteins onto solid surfaces remains a critical aspect in
the development of biosensors. Furthermore, to the extent of our knowledge, all of
the reported works describing signal transduction by fluorophore labeled PBPs have
been studied without their linkage to solid supports. Thus, in this research, MBP was
first mutated to construct a reagentless biosensor for maltose sensing. The Strep-Tag
II fused to the mutated MBP enabled the attachment of the ligand binding protein
onto a functionalized solid surface. The ability of the immobilized protein to sense
ligand binding was subsequently assessed.
1.4 Scope of Research
To fulfill the objectives of this research, there were four main experimental
steps in laboratory work that needed to be done. Firstly, the construction of plasmids
containing wild type malE gene and its variant were done. This involved
amplification of malE gene, site-directed mutagenesis of malE gene and cloning. The
next step was expression and purification of wild type MBP and its mutant; which
involved optimizing protein expression to produce the optimal amount of protein and
purification of the protein by affinity purification. The third step was to analyze the
ligand binding characteristics of wild type MBP and its mutant via fluorescence.
Fluorescence measurements involved labeling the mutant MBP with fluorophore and
analyzing the ligand binding characteristic of MBP via fluorescence. The final step
was to analyze the ligand binding characteristics of the immobilized protein via
fluorescence. In this final step, the labeled mutant protein was immobilized onto a
functionalized microwell plate and its ability to sense maltose via fluorescence
intensity changes was determined.
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