Title Glycosylation and glycoengineering of recombinant antibody fragments in Escherichia coli ...

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Glycosylation and glycoengineering of recombinant antibody
Title
fragments in Escherichia coli
Author(s) O' Connor, Iain
Publication
2016-11-14
Date
Item record http://hdl.handle.net/10379/6158
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Glycosylation and glycoengineering of recombinant
antibody fragments in Escherichia coli

A thesis submitted to the National University of Ireland,
Galway for the degree of Doctor of Philosophy by:

Iain O' Connor, B.Sc.

Recombinant Protein Production Laboratory,
Discipline of Microbiology, School of Natural Sciences
College of Science, National University of Ireland, Galway

Research Supervisor: Dr. J. Gerard Wall
Head of Discipline: Dr. J. Gerard Wall

November 2016

Table of contents

Table of contents i
List of abbreviations vi
List of figures ix
List of tables xii
Acknowledgements xiii
Declaration xiv
Abstract xv
Chapter 1: General Introduction 1
1.1 Recombinant protein production 2
1.2 Glycosylation in recombinant hosts 4
1.3 E. coli as an expression host 6
1.4 Glycosylation in E. coli 10
1.5 Antibody sources 13
1.6 Antibody structure 15
1.7 Antibody fragments 17
1.8 Antibody fragment isolation and expression in E. coli 19
1.9 Immobilisation approaches 21
1.10 Immobilised antibody fragments 24
1.11 Scope of this thesis 25
Chapter 2: Materials and Methods 27
2.1 Materials 28
2.1.1 Suppliers and reagents 28
2.1.2 Bacterial strains, plasmids and primers 29
2.1.3 Antibiotics 33
2.1.4 Antibodies, lectins and detection reagents 33
2.1.5 Molecular weight markers 33
2.1.6 General multi-component buffers 33
2.1.7 Protein purification buffers 34
i
2.1.8 Culture media 34
2.1.8.1 Lysogeny broth (LB) 34
2.1.8.2 ZYP-5052 34
2.1.8.3 Pichia pastoris media 35
2.2. Methods 35
2.2.1 Bacterial growth and protein expression 35
2.2.2. Electrocompetent cell preparation 35
2.2.3 Transformation by electroporation 36
2.2.4 Transformation by heat-shock 36
2.2.5 Protein expression by induction via IPTG 36
2.2.6 Auto-induction 37
2.2.7 P. pastoris growth and protein expression 37
2.2.8 Protein extraction 37
2.2.8.1 Periplasmic protein extraction from E. coli 37
2.2.8.2 Protein extraction from P. pastoris 38
2.2.9 Protein purification and cleanup 38
2.2.9.1 Affinity chromatography using immobilised nickel 39
2.2.9.2 Affinity chromatography using cobalt resin 39
2.2.10 Protein processing and analysis 40
2.2.10.1 SDS-PAGE 40
2.2.10.2 Protein staining 40
2.2.10.3 Western blotting 40
2.2.10.4 Glycan detection/staining 40
2.2.10.5 Lectin blotting 41
2.2.10.6 Protein quantification 42
2.2.10.7 Glycoprotein oxidation 42
2.2.11 Enzyme-Linked Immunosorbent Assay (ELISA) 42
2.2.11.1 Indirect ELISA 42
2.2.11.2 Inhibition ELISA 43
ii
2.2.12 Protein immobilisation 43
2.2.12.1 Adsorption to microplates 43
2.2.12.2 Covalent attachment to microplates 44
2.2.12.3 Immobilisation on TiO -coated 316L stainless steel 44
2
2.2.12.4 Disk washing, antigen incubation, and imaging 44
2.2.12.5 Densitometry analysis of immobilisations 45
2.2.13 Oligosaccharide extension 45
2.2.14 Lectin array 45
2.2.15 Molecular techniques – DNA 46
2.2.15.1 Plasmid isolation and purification 46
2.2.15.2 Restriction enzyme digestion 46
2.2.15.3 Dephosphorylation and ligation 46
2.2.15.4 Polymerase chain reaction 47
2.2.15.5 Agarose gel electrophoresis 47
2.2.15.6 DNA purification and concentration 47
2.2.15.7 Mutagenesis 47
2.2.15.8 Sequencing 48
2.2.16 Molecular techniques – RNA 48
2.2.16.1 RNA isolation from Pichia pastoris 48
2.2.16.2 cDNA synthesis 48
2.2.17 Computational analyses 49
2.2.17.1 Codon usage analysis 49
2.2.17.2 Sequence alignment and secondary structure
prediction 49
Chapter 3: Glycoprotein expression in E. coli and engineering of the
glycan chain 50
3.1 Introduction 51
3.2 Production of glycosylated scFvs in E. coli 56
3.2.1 Selection of scFv for glycoengineering 56
iii
3.2.2 Optimisation of glycoprotein production and recovery 57
3.3 Generation of alternative pgl constructs 60
3.3.1 Effect on glycan length of site-directed mutagenesis of pglJ 60
3.3.2 Investigation of pgl locus redundancy by truncation
mutagenesis 62
3.3.3 Western blot-based characterisation of glycoprofiles 64
3.3.3.1 DIG-specific analysis of glycoproteins 64
3.4 Functionality of glycoproteins 66
3.4.1 Antigen binding 66
3.4.2 Immobilisation of scFvs using adsorption 67
3.4.2.1 Adsorption - antigen-binding 71
3.4.3 Covalent immobilisation 73
3.4.3.1 Time course of covalent immobilisation of scFvs 75
3.4.3.2 Antigen-binding by covalently immobilised scFvs 78
3.4.4 Analysis of antigen binding by oxidised scFvs 81
3.5 Discussion 83
Chapter 4: Investigation into extension of the established Campylobacter
jejuni N-linked glycan in Escherichia coli and additional
characterisations of glycosylated scFvs 89
4.1 Introduction 90
4.2 Investigating protein sialylation in E. coli 94
4.3 CgtB-mediated extension and expression in E. coli 96
4.4 cgtB expression in P. pastoris 97
4.5 CgtB purification and oligosaccharide extension 100
4.6 Alternate characterisation of glycoproteins 101
4.6.1 Lectin array analysis 101
4.6.2 Lectin blot analysis of glycoproteins 111
4.7 Glycan-mediated immobilisation of scFvs 113
4.8 Discussion 119
iv
Chapter 5: Discussion 127
5.1 Discussion 128
5.2 Conclusion 138
References 140
Appendices 174
Appendix i 175
Appendix ii 177
Appendix iii 178

v
List of abbreviations

Abbreviations

(NH ) SO Ammonium sulfate
4 2 4
* Stop codon
2H12 Anti-domoic acid scFv
4M5.3 Anti-fluorescein scFv
AFM Atomic force microscopy
Amp Ampicillin
AOX1 Alcohol oxidase 1 promoter
APTES 3-(aminopropyl) triethoxysilane
Asn Asparagine
AURIF Auristatin F
Bac Bacillosamine
BMGY Buffered glycerol-complex medium
BMMY Buffered methanol-complex medium
BSA Bovine serum albumin
Cam Chloramphenicol
CDR Complementarity-determining region
C Heavy chain constant domain
H
CHO Chinese hamster ovary cell
C Light chain constant domain
L
C-terminal Carboxyl terminal
Cy5 Cyanine5 NHS ester
dH O Deionised water
2
DHB 2,5-dihydroxybenzoic acid
DIG Digoxigenin
DNA Deoxyribonucleic acid
DsbA/C Disulfide bond isomerase A/C
dsFv Disulfide-stabilised Fv antibody fragment
EDA Ethylenediamine
EDTA Ethylenediamine tetraacetic acid
ELISA Enzyme-linked immunosorbent assay
Endo H/F Endoglycosidase H/F
Fab Antigen-binding fragment
Gal Galactose
GalNAc N-Acetylgalactosamine
Glc Glucose
GlcNAc N-Acetylglucosamine
GST Glutathione S-transferase
H O Water
2
H SO Sulphuric acid
2 4
HAMA Human anti-mouse antibody
HER2 Human epidermal growth factor receptor 2
His tag Hexahistidine tag
Hrp Horseradish peroxidase
Hsp Heat shock protein
IC Half maximal inhibitory concentration
50
vi
Ig Immunoglobulin
IgG Immunoglobulin G
IMAC Immobilised metal ion affinity chromatography
IPTG Isopropyl β-D-1-thiogalactopyranoside
KH PO Monopotassium phosphate
2 4
LB Luria-Bertani broth
LOS Lipooligosaccharide
LPS Lipopolysaccharide
mAb Monoclonal antibody
MALDI-TOF Matrix assisted laser desorption/ionisation time-of-
flight
Man Mannose
MBP Maltose-binding protein
MgCl Magnesium chloride
2
MgSO Magnesium Sulphate
4
MS Mass spectrometry
MWCO Molecular weight cut off
Na HPO .2H O Di-sodium hydrogen phosphate dihydrate
2 4 2
NeuAc Neuraminic acid (Sialic acid)
NH Primary amine
2
N-linked/glycosylated Asparagine-linked/glycosylated
NMR Nuclear magnetic spectroscopy
N-terminal Amino terminal
OD Optical density
O-linked/glycosylated Serine/threonine-linked/glycosylated
Omp Outer membrane protein
OST Oligosaccharyltransferase
pAb Polyclonal antibody
PBS Phosphate buffered saline
PelB Pectate lyase B
pgl C. jejuni protein glycosylation gene family
PNGase F Peptide-N-Glycosidase F
PTM Post-translational modification
QCM-D Quartz crystal microbalance with dissipation
monitoring
R2 Coefficient of determination
RNA Ribonucleic acid
RPP Recombinant protein production
SBA Soybean lectin from Glycine Max
scFv Single chain variable fragment
SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis
Strep tag Streptavidin tag
SUMO Small ubiquitin-like modifier protein
TBS Tris buffered saline
TEV Tobacco etch virus
V Heavy chain variable domain
H
V H Single domain antibodies
H
V Light chain variable domain
L
YPDS Yeast extract peptone dextrose medium
vii