An Introduction to Molecular Biotechnology- Fundamentals, Methods and Applications

637 Pages·2011·135.531 MB·English

by  Wink, Michael

AnIntroduction to
Molecular Biotechnology
Editedby
MichaelWink
AnIntroductiontoMolecularBiotechnology,2ndEdition.
EditedbyMichaelWink
Copyright©2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim
ISBN:978-3-527-32637-2
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An Introduction to
Molecular Biotechnology
Fundamentals, Methods, and Applications
Edited by
Michael Wink
Second, Updated Edition
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MolecularBiotechnology completenessofthecontentsofthisbookand
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ISBN 978-3-527-32637-2
V
Contents
Preface XIX
List of Contributors XXI
Abbreviations XXV
Part I Fundamentals of Cellular and Molecular Biology 1
1 The Cell as the Basic Unit of Life 3
M.Wink
2 Structureand Function of CellularMacromolecules 7
M.Wink
2.1 Structure and Function ofSugars 8
2.2 Structure ofMembraneLipids 10
2.3 Structure and Function ofProteins 14
2.4 Structure ofNucleotidesand Nucleic Acids(DNA and RNA) 21
2.5 References 27
3 Structureand Functions of a Cell 29
M.Wink
3.1 Structure ofa Eukaryotic Cell 29
3.1.1 Structure and Function ofthe Cytoplasmic Membrane 29
3.1.1.1 MembranePermeability 30
3.1.1.2 Transport ProcessesacrossBiomembranes 31
3.1.1.3 Receptorsand Signal Transductionat Biomembranes 33
3.1.2 EndomembraneSystem in a Eukaryotic Cell 38
3.1.3 Mitochondriaand Chloroplasts 40
3.1.4 Cytoplasm 45
3.1.5 Cytoskeleton 47
3.1.6 Cell Walls 49
3.2 Structure ofBacteria 50
3.3 Structure ofViruses 51
3.4 Differentiationof Cells 52
4 Biosynthesis and Function of Macromolecules
(DNA, RNA, and Proteins) 57
M.Wink
4.1 Genomes,Chromosomes,and Replication 57
4.1.1 Genome Size 57
4.1.2 Compositionand Function ofChromosomes 62
4.1.3 Mitosisand Meiosis 64
4.1.4 Replication 66
4.1.5 Mutations and Repair Mechanisms 66
AnIntroductiontoMolecularBiotechnology,2ndEdition.
EditedbyMichaelWink
Copyright©2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim
ISBN:978-3-527-32637-2
VI Contents
4.2 Transcription:From Geneto Protein 71
4.3 Protein Biosynthesis(Translation) 76
5 Distributing Proteins in the Cell (Protein Sorting) 81
M.Wink
5.1 Import and Export ofProteinsvia the Nuclear Pore 82
5.2 Import ofProteinsin Mitochondriaand Chloroplasts 83
5.3 Protein Transport into the Endoplasmic Reticulum 85
5.4 VesicleTransport fromthe ER via the Golgi Apparatus to the Cyto-
plasmic Membrane 86
6 Evolution and Diversity of Organisms 91
M.Wink
6.1 Prokaryotes 91
6.2 Eukaryotes 91
Part II Standard Methods in Molecular Biotechnology 99
7 Isolation and Purification of Proteins 101
T.Wieland,M.Lutz
7.1 Introduction 101
7.2 Producing a Protein Extract 102
7.3 Gel ElectrophoreticSeparation Methods 103
7.3.1 Principles ofElectrophoresis 103
7.3.2 Native Gel Electrophoresis 104
7.3.3 DiscontinuousSodium DodecylSulfatePolyacrylamide
Gel Electrophoresis(SDS-PAGE) 104
7.3.4 Two-Dimensional(2D) Gel Electrophoresis,Isoelectric
Focusing (IEF) 105
7.3.5 Detecting Proteinsin Gels 105
7.4 Methods ofProtein Precipitation 106
7.5 Column Chromatography Methods 107
7.5.1 General Principles ofSeparation 107
7.5.1.1 Size ExclusionChromatography (Gel Filtration) 107
7.5.1.2 HydrophobicInteraction Chromatography 108
7.5.1.3 Ion Exchange Chromatography 109
7.5.1.4 Hydroxyapatite Chromatography 110
7.5.2 Group-specific Separation Techniques 110
7.5.2.1 Chromatography on Protein A orProtein G 110
7.5.2.2 Chromatography on CibacronBlue(Blue Gel) 111
7.5.2.3 Chromatography on Lectins 111
7.5.2.4 Chromatography on Heparin 111
7.5.3 Purification ofRecombinant FusionProteins 112
7.5.3.1 Chromatography on Chelating Agents 112
7.5.3.2 Chromatography on Glutathione Matrices 112
7.6 Examples 113
7.6.1 Example 1: Purification of NucleosideDiphosphate Kinase
fromthe Cytosolof BovineRetina Rod Cells 113
7.6.2 Example 2: Purification of Recombinant His -RGS16
6
after Expressionin E. coli 114
8 Peptide and Protein Analysis with Electrospray
Tandem Mass Spectrometry 115
A.Schlosser, W.D. Lehmann
8.1 Introduction 115
8.2 Principles ofMassSpectrometry 115
8.3 MassPrecision,Resolution,and IsotopeDistribution 116
Contents VII
8.4 Principles ofESI 116
8.5 Tandem MassSpectrometers 117
8.5.1 MassAnalyzers 117
8.5.2 Triple Quadrupole 118
8.5.3 LinearTrap Quadrupole (LTQ) and LTQ Orbitrap 118
8.5.4 Q-TOF 119
8.5.5 Q-FT-ICR 119
8.6 Peptide Sequencing with MS/MS 119
8.7 Identifying Proteinswith MS/MSData and Protein Databases 120
8.7.1 DatabaseSearch with MS/MSRaw Data 120
8.8 Determining Protein MolecularMass 121
8.9 Analysis ofCovalent Protein Modification 122
8.10 Relative and AbsoluteQuantification 123
9 Isolation of DNAand RNA 125
H. Weiher, R. Zwacka, I. Herr
9.1 Introduction 125
9.2 DNA Isolation 125
9.3 RNA Isolation 127
9.3.1 Enrichment ofmRNA 127
10 Chromatography and Electrophoresis of Nucleic Acids 129
H. Weiher, R. Zwacka, I. Herr
10.1 Introduction 129
10.2 Chromatographic Separation ofNucleic Acids 129
10.3 Electrophoresis 130
10.3.1 AgaroseGel Electrophoresis:Submarine Electrophoresis 130
10.3.2 Pulsed Field AgaroseGel Electrophoresis 131
10.3.3 Polyacrylamide Gel Electrophoresis(PAGE) 131
11 Hybridization of Nucleic Acids 133
H. Weiher, R. Zwacka, I. Herr
11.1 Significance ofBase Pairing 133
11.2 Experimental Hybridization:Kinetic
and Thermodynamic Control 133
11.3 Analytical Techniques 134
11.3.1 CloneDetection,Southern Blotting, Northern Blotting,
and GeneDiagnosis 134
11.3.2 Systematic GeneDiagnosis and ExpressionScreening
basedon Gene Arrays 135
11.3.3 In Situ Hybridization 135
12 Use of Enzymes in the Modification of Nucleic Acids 137
A.Groth, R.Zwacka, H.Weiher, I.Herr
12.1 RestrictionEnzymes (RestrictionEndonucleases) 137
12.2 Ligases 139
12.3 Methyltransferases 139
12.4 DNA Polymerases 140
12.5 RNA Polymerasesand ReverseTranscriptase 141
12.6 Nucleases 141
12.7 T4 Polynucleotide Kinase 141
12.8 Phosphatases 142
13 Polymerase Chain Reaction 143
A.Mohr, H.Weiher, I.Herr, R.Zwacka
13.1 Introduction 143
13.2 Techniques 143
VIII Contents
13.2.1 Standard PCR 143
13.2.2 RT-PCR 144
13.2.3 Quantitative/Real-Time PCR 145
13.2.4 Rapid Amplification of cDNA Ends (RACE) 146
13.3 Areasof Application 146
13.3.1 Genome Analysis 146
13.3.2 Cloning Techniques 147
13.3.3 ExpressionStudies 147
14 DNA Sequencing 149
R.Zwacka, A.Mohr, I.Herr, H.Weiher
14.1 Introduction 149
14.2 DNA Sequencing Methods 149
14.2.1 Chemical Sequencing Method (MaxamGilbertMethod) 150
14.2.2 Enzymatic Sequencing (SangerCoulson Method) 150
14.2.3 Pyrosequencing 151
14.3 Strategies forSequencing the Human Genome 151
14.4 Practical SignificanceofDNA 152
15 Cloning Procedures 153
T.Wieland,S.Lutz
15.1 Introduction 153
15.2 ConstructionofRecombinant Vectors 153
15.2.1 Insert 154
15.2.2 Vector 156
15.2.3 Essential Components ofVectors 156
15.2.3.1 Bacterial Origin of Replication (ori) 156
15.2.3.2 Antibiotic Resistance 156
15.2.3.3 Polylinkers 157
15.2.4 Cloning Using RecombinationSystems 157
15.2.5 Further ComponentsofVectorsfor Prokaryotic
ExpressionSystems 158
15.2.5.1 Promoter 158
15.2.5.2 Ribosome-BindingSite 159
15.2.5.3 Termination Sequence 159
15.2.5.4 FusionSequence 159
15.2.6 Further ComponentsofEukaryotic ExpressionVectors 159
15.2.6.1 Eukaryotic ExpressionVectors:Yeast 160
15.2.6.2 Eukaryotic ExpressionVectorsforMammal Cells 161
15.2.6.3 Viral ExpressionSystems forMammalian Cells 163
15.2.7 Nonviral Introduction of HeterologousDNAto HostOrganisms
(Transformation,Transfection) 165
15.2.7.1 TransformationofProkaryotes 165
15.2.7.2 TransformationofYeastCells 166
15.2.7.3 TransfectionofMammal Cells 166
16 Expression of Recombinant Proteins 169
T.Wieland,S.Lutz
16.1 Introduction 169
16.2 ExpressionofRecombinant Proteinsin HostOrganisms 170
16.2.1 Expressionin E. coli 173
16.2.2 Expressionin Yeasts 174
16.2.3 Expressionin Insect Cells 176
16.2.3.1 ExpressionBasedon RecombinantBaculoviruses 176
16.2.3.2 ExpressionofProteinsin Stably TransfectedInsect Cells 177
16.2.4 ExpressionofProteinsin Mammalian Cells 177
16.3 Expressionin Cell-FreeSystems 178