Оглавление
Группа авторов. An Introduction to Molecular Biotechnology
Table of Contents
List of Tables
List of Illustrations
Guide
Pages
An Introduction toMolecular Biotechnology. Fundamentals, Methods and Applications
Copyright
Abbreviations
1 The Cell as the Basic Unit of Life
References
Further Reading
2 Structure and Function of Cellular Macromolecules
2.1 Structure and Function of Sugars
2.2 Structure of Membrane Lipids
2.3 Structure and Function of Proteins
2.4 Structure of Nucleotides and Nucleic Acids (DNA and RNA)
References
Further Reading
3 Structure and Functions of a Cell
3.1 Structure of a Eukaryotic Cell
3.1.1 Structure and Function of the Cytoplasmic Membrane
3.1.1.1 Membrane Permeability
3.1.1.2 Transport Processes Across Biomembranes
3.1.1.3 Receptors and Signal Transduction at Biomembranes
3.1.2 Endomembrane System in a Eukaryotic Cell
3.1.3 Mitochondria and Chloroplasts
3.1.4 Cytoplasm
3.1.5 Cytoskeleton
3.1.6 Cell Walls
3.2 Structure of Bacteria
3.3 Structure of Viruses
3.4 Differentiation of Cells
3.5 Cell Death
References
Further Reading
4 Biosynthesis and Function of Macromolecules (DNA, RNA, and Proteins)
4.1 Genomes, Chromosomes, and Replication
4.1.1 Genome Size
4.1.2 Composition and Function of Chromosomes
4.1.3 Mitosis and Meiosis
4.1.4 Replication
4.1.5 Mutations and Repair Mechanisms
4.2 Transcription: From Gene to Protein
4.3 Protein Biosynthesis (Translation)
Further Reading
5 Distributing Proteins in the Cell (Protein Sorting)
5.1 Import and Export of Proteins via the Nuclear Pore
5.2 Import of Proteins in Mitochondria, Chloroplasts, and Peroxisomes
5.3 Protein Transport into the Endoplasmic Reticulum
5.4 Vesicle Transport from the ER via the Golgi Apparatus to the Cytoplasmic Membrane
References
Further Reading
6 Evolution and Diversity of Organisms
6.1 Prokaryotes
6.2 Eukaryotes
References
Further Reading
7 Isolation and Purification of Proteins
7.1 Introduction
7.2 Producing a Protein Extract
7.3 Gel Electrophoretic Separation Methods. 7.3.1 Principles of Electrophoresis
7.3.2 Native Gel Electrophoresis
7.3.3 Discontinuous Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS‐PAGE)
7.3.4 Two‐Dimensional (2D) Gel Electrophoresis and Isoelectric Focusing (IEF)
7.3.5 Detecting Proteins in Gels
7.4 Methods of Protein Precipitation
7.5 Column Chromatography Methods. 7.5.1 General Principles of Separation
7.5.1.1 Size Exclusion Chromatography (Gel Filtration)
7.5.1.2 Hydrophobic Interaction Chromatography
7.5.1.3 Ion Exchange Chromatography
7.5.1.4 Hydroxyapatite Chromatography
7.5.2 Group‐Specific Separation Techniques
7.5.2.1 Chromatography on Protein A or Protein G
7.5.2.2 Chromatography on Cibacron Blue (Blue Gel)
7.5.2.3 Chromatography on Lectins
7.5.2.4 Chromatography on Heparin
7.5.3 Purification of Recombinant Fusion Proteins
7.5.3.1 Chromatography on Chelating Agents
7.5.3.2 Chromatography on Glutathione Matrices
7.6 Examples
7.6.1 Example 1: Purification of Nucleoside Diphosphate Kinase from the Cytosol of Bovine Retina Rod Cells
7.6.2 Example 2: Purification of Recombinant His6‐RGS16 After Expression in E. coli
Further Reading
8 Mass Spectrometry and Applications in Proteomics and Microbial Identification
8.1 Principles of ESI and MALDI Mass Spectrometry
8.2 Instrumental Setup
8.3 Intact Protein Analysis
8.3.1 Protein Digestion
8.3.2 Peptide Fragmentation
8.3.3 Protein Identification with MS/MS Spectra
8.4 Protein and Proteome Quantification. 8.4.1 Label‐Free Quantification
8.4.2 Chemical Stable Isotope Labeling
8.4.3 Metabolic Stable Isotope Labeling
8.5 Protein–Protein Interaction Analysis
8.6 Analysis of Posttranslational Modifications
8.7 Microbial Identification and Resistance Detection
References
9 Isolation of DNA and RNA
9.1 Introduction
9.2 DNA Isolation
9.3 RNA Isolation
9.3.1 Enrichment of mRNA
Reference
10 Chromatography and Electrophoresis of Nucleic Acids
10.1 Introduction
10.2 Chromatographic Separation of Nucleic Acids
10.3 Electrophoresis
10.3.1 Agarose Gel Electrophoresis: Submarine Electrophoresis
10.3.2 Pulsed‐Field Agarose Gel Electrophoresis
10.3.3 Polyacrylamide Gel Electrophoresis (PAGE)
Further Reading
11 Hybridization of Nucleic Acids
11.1 Significance of Base Pairing
11.2 Experimental Hybridization: Kinetic and Thermodynamic Control
11.3 Analytical Techniques
11.3.1 Clone Detection, Southern Blotting, Northern Blotting, and Gene Diagnosis
11.3.2 Systematic Gene Diagnosis and Expression Screening Based on Gene Arrays
11.3.3 In Situ Hybridization
References
Further Reading
12 Use of Enzymes in the Modification of Nucleic Acids
12.1 Restriction Enzymes (Restriction Endonucleases)
12.2 Ligases
12.3 Methyltransferases
12.4 DNA Polymerases
12.5 RNA Polymerases and Reverse Transcriptase
12.6 Nucleases
12.7 T4 Polynucleotide Kinase
12.8 Phosphatases
Further Reading
13 Polymerase Chain Reaction
13.1 Introduction
13.2 PCR Methods. 13.2.1 Basic Principle
13.2.2 Primer Design and Hot Start PCR
13.2.3 Multiplex PCR
13.2.4 RT‐PCR
13.2.5 Qualitative Analysis of the PCR Products
13.3 PCR as a Quantitative Method. 13.3.1 PCR Phases and PCR Efficiency
13.3.2 Quantitative Real‐Time PCR
13.3.3 Digital PCR
13.4 Areas of Application. 13.4.1 Genome Analysis
13.4.2 Cloning Techniques
13.4.3 Gene Expression Studies
Further Reading
14 DNA Sequencing
14.1 Introduction
14.2 The Sanger Method
14.3 Pyrosequencing
14.4 Second‐Generation Sequencing: Illumina and Ion Torrent. 14.4.1 Overview
14.4.2 The Illumina Sequencing System
14.4.3 The Ion Torrent Sequencing System
14.5 Third‐Generation Sequencing Techniques. 14.5.1 Overview
14.5.2 SMRT Sequencing
14.5.3 Nanopore Sequencing
14.6 The Impact of the DNA Sequencing Technology
References
Further Reading
Websites
15 Cloning Procedures
15.1 Introduction
15.2 Construction of Recombinant Vectors
15.2.1 Insert
15.2.2 Vector
15.2.3 Essential Components of Vectors. 15.2.3.1 Bacterial Origin of Replication (ori)
15.2.3.2 Antibiotic Resistance
15.2.3.3 Polylinkers
15.2.4 Cloning Using Recombination Systems
15.2.5 Further Components of Vectors for Prokaryotic Expression Systems
15.2.5.1 Promoter
15.2.5.2 Ribosome‐Binding Site
15.2.5.3 Termination Sequence
15.2.5.4 Fusion Sequence
15.2.6 Further Components of Eukaryotic Expression Vectors
15.2.6.1 Eukaryotic Expression Vectors: Yeast
15.2.6.2 Eukaryotic Expression Vectors for Mammal Cells
Promoters in Eukaryotic Expression Vectors for Mammalian Cells
Termination Sequences in Eukaryotic Expression Vectors for Mammal Cells
Sequences for the Replication of Eukaryotic Expression Vectors in Mammal Cells
Genes for the Selection of Stably Transfected Cell Clones
Fusion Sequences in Eukaryotic Expression Vectors for Mammalian Cells
15.2.6.3 Viral Expression Systems for Mammalian Cells
Adenoviral Expression Systems
Retroviral Expression Systems
15.2.7 Nonviral Introduction of Heterologous DNA to Host Organisms (Transformation, Transfection) 15.2.7.1 Transformation of Prokaryotes
Electroporation
Chemical Transformation
15.2.7.2 Transformation of Yeast Cells
15.2.7.3 Transfection of Mammal Cells
Calcium Phosphate‐Mediated Transfection
Liposomal Transfection
Electroporation
Further Reading
16 Expression of Recombinant Proteins
16.1 Introduction
16.2 Expression of Recombinant Proteins in Host Organisms
16.2.1 Expression in E. coli
16.2.2 Expression in Yeasts
16.2.3 Expression in Insect Cells
16.2.3.1 Expression Based on Recombinant Baculoviruses
16.2.3.2 Expression of Proteins in Stably Transfected Insect Cells
16.2.4 Expression of Proteins in Mammalian Cells
16.3 Expression in Cell‐Free Systems
16.3.1 Expression of Proteins in Reticulocyte Lysates
16.3.2 Protein Expression Using E. coli Extracts
Further Reading
17 Patch Clamp Method
17.1 Ion Channels
17.2 Technical Requirements of the Patch Clamp Method
17.3 Patch Clamp Configurations
17.4 Applications of the Patch Clamp Method
Reference
Further Reading
18 Cell Cycle Analysis
18.1 Introduction
18.2 Analyzing the Cell Cycle
18.3 Experimental Analysis of the Cell Cycle
18.3.1 Preparing Synchronized Cell Cultures of S. cerevisiae
18.3.1.1 Centrifugal Elutriation
18.3.1.2 Cell Cycle Arrest Using α‐Factor
18.3.2 Identification of Cell Cycle Stages
18.3.2.1 Budding Index
18.3.2.2 Fluorescent Staining of the Nucleus
18.3.2.3 Detection of Cell Cycle Phases Using Fluorescent Proteins as Reporters
Acknowledgments
Further Reading
19 Microscopic Techniques
19.1 Introduction
19.2 Electron Microscopy
19.2.1 Cryo‐electron Microscopy
19.2.2 Electron Tomography
19.3 Atomic or Scanning Force Microscopy
19.3.1 Force Spectroscopy
19.3.2 Advantages and Disadvantages
19.4 Light Microscopy
19.4.1 Deconvolution
19.4.2 Confocal Microscopy
19.4.3 Why Fluorescence?
19.4.4 Nanoscopy
19.5 Microscopy in the Living Cell
19.5.1 Analysis of Fluorescently Labeled Proteins In Vivo
19.5.2 Fluorescence Recovery After Photobleaching
19.5.3 Fluorescence Correlation Spectroscopy
19.5.4 Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscopy
19.5.5 Single‐Molecule Fluorescence
Further Reading
20 Laser Applications
20.1 Laser Development: A Historical Perspective
20.2 Types of Lasers and Setups
20.3 Properties of Laser Radiation
20.4 Applications
20.4.1 Laser Scanning Microscopy
20.4.2 Optical Tweezers
20.4.3 Laser Microdissection and Laser Therapy
20.4.4 Manufacturing of Products in Medical Technology and Biotechnology Products
Further Reading
21 Sequencing the Universe of Life
21.1 What to Sequence?
21.1.1 Whole‐Genome Sequencing
21.1.2 Exome Sequencing
21.1.3 (Gene) Panel Sequencing
21.1.4 RNA Sequencing. 21.1.4.1 Tag‐ vs. Full‐Length Sequencing
21.1.4.2 Sequencing of RNA Species and Modifications
21.1.4.3 Sequencing of Single Cells
21.1.4.4 In Situ Sequencing
21.1.5 (Whole‐Genome) Bisulfite Sequencing of DNA
21.1.6 Sequencing to Characterize Chromatin Structure and Beyond
21.2 Sequencing Projects: Human. 21.2.1 Initial Sequencing of the Human Genome
21.2.2 The 1000 Genomes Project: Assessing Natural Variation
21.2.3 Screening for Genetic Disease
21.2.4 Sequencing of Populations
21.2.5 TCGA and ICGC: Screening for Cancer Driver Mutations
21.3 Sequencing Other Species, Environments, …
21.4 Sequencing in the Clinics: Personalizing Oncology
21.5 Sequencing in the Private Sector: Direct to Consumer Testing (DTC)
21.6 The Information Content of a Genome Sequence and Ethical Consequences
References
22 Cellular Systems Biology
22.1 Introduction
22.2 Analysis of Cellular Networks by Top‐Down Approaches
22.2.1 Motivation
22.2.2 Definitions and Construction of the Networks
22.2.3 Gene Set Enrichment Tests
22.2.4 Inferring Gene Regulators Employing Gene Regulatory Models
22.2.5 Network Descriptors
22.2.5.1 Scale‐Free Networks
22.2.5.2 Centrality
22.2.5.3 The Clustering Coefficient
22.2.6 Detecting Essential Enzymes with a Machine Learning Approach
22.2.7 Elementary Flux Modes
22.3 Overview over Bottom‐Up Modeling of Biochemical Networks
22.3.1 Motivation
22.3.2 Choosing Model Complexity and Model Building
22.3.3 Model Simulation
22.3.4 Model Calibration
22.3.5 Model Verification and Analysis
22.3.6 Examples
Further Reading
References
23 Protein–Protein and Protein–DNA Interactions
23.1 Protein–Protein Interactions
23.1.1 Classification and Specificity: Protein Domains
23.1.2 Protein Networks and Complexes
23.1.3 Structural Properties of Interacting Proteins
23.1.4 Which Forces Mediate Protein–Protein Interactions?
23.1.4.1 Thermodynamics
23.1.4.2 Energetics
23.1.5 Methods to Examine Protein–Protein Interactions
23.1.6 Theoretical Prediction of Protein–Protein Interactions
23.1.7 Regulation of Protein–Protein Interactions
23.1.8 Biotechnological and Medical Applications of Protein–Protein Interactions
23.2 Protein–DNA Interactions
23.2.1 Specific Protein–DNA Interaction
23.2.2 Thermodynamic Consideration
23.2.3 Methods to Study Protein–DNA Interactions
23.2.3.1 Structural Classification of Protein–DNA Complexes
23.2.4 Regulatory Networks and System Biology
23.2.5 Medical Importance of Protein–DNA Interactions
23.2.6 Biotechnological Applications
References
Further Reading
24 Bioinformatics
24.1 Introduction
24.2 Data Sources
24.2.1 Primary Databases: EMBL/GenBank/DDBJ, PIR, and Swiss‐Prot
24.2.2 Genome Databases: Ensembl and GoldenPath
24.2.3 Motif Databases: BLOCKS, PROSITE, Pfam, ProDom, and SMART
24.2.4 Molecular Structure Databases: PDB and SCOP
24.2.5 Transcriptome Databases: SAGE, ArrayExpress, and GEO
24.2.6 Reference Databases: PubMed, OMIM, and GeneCards
24.2.7 Pathway Databases and Gene Ontology
24.3 Sequence Analysis
24.3.1 Kyte–Doolittle Plot, Helical Wheel Analysis, and Signal Sequence Analysis
24.3.2 Pairwise Alignment
24.3.2.1 Local/Global
24.3.2.2 Optimal/Heuristic
24.3.3 Alignment Statistics
24.3.4 Multiple Alignment
24.4 Evolutionary Bioinformatics
24.4.1 Statistical Models of Evolution
24.4.2 Relation to Score Matrices
24.4.3 Phylogenetic Analysis
24.5 Gene Prediction
24.5.1 Neural Networks or HMMs Based on Hexanucleotide Composition
24.5.2 Comparison with Expressed Sequence Tags or Other Genomes (Fugu, Mouse)
24.6 Bioinformatics in Transcriptome and Proteome Analysis
24.6.1 Preprocessing and Normalization
24.6.2 Feature Selection
24.6.3 Similarity Measures: Euclidean Distance, Correlation, Manhattan Distance, Mahalanobis Distance, and Entropy Measures
24.6.4 Unsupervised Learning Procedures: Clustering, Principal Component Analysis, Multidimensional Scaling, and Correspondence Analysis
24.6.5 Supervised Learning Procedures: Linear Discriminant Analysis, Decision Trees, Support Vector Machines, and ANNs
24.6.6 Analysis of Overrepresentation of Functional Categories
24.7 Analysis of Ultraparallel Sequencing Data
24.7.1 Mapping of Ultraparallel Sequencing Data
24.7.2 Genome (Re‐)sequencing
24.7.3 Transcriptome Sequencing
24.7.4 ChIP‐seq
24.7.5 Epigenetic Analysis
24.7.6 Single‐Cell Analysis
24.7.7 Bioethics of Human Sequencing Data
24.8 Bioinformatic Software
Further Reading
25 Drug Research
25.1 Introduction
25.2 Active Compounds and Their Targets
25.2.1 Identification of Potential Targets in the Human Genome
Box 25.1 Sequence Similarities
25.2.2 Comparative Genome Analysis
25.2.3 Experimental Target Identification: In Vitro Methods
Box 25.2 Reporter Gene Assays
25.2.4 Experimental Identification of Targets: Model Organisms
25.2.5 Experimental Target Identification in Humans
25.2.6 Difference Between Target Candidates and Genuine Targets
Box 25.3 The Low‐Density Lipoprotein (LDL) Receptor: Promising at First Sight, Yet Unsuitable as a Direct Target
25.2.7 Biologicals
25.2.8 DNA and RNA in New Therapeutic Approaches
25.2.9 Patent Protection for Targets
25.2.10 Compound Libraries as a Source of Drug Discovery
Box 25.4 Combinatorial Chemistry
Box 25.5 Preclinical Steps in Drug Development
25.2.11 High‐Throughput Screening
25.2.12 High‐Quality Paramounts in Screening Assays
25.2.13 Virtual Ligand Screening
25.2.14 Activity of Drugs Described in Terms of Efficacy and Potency
25.2.15 Chemical Optimization of Lead Structures
25.3 Preclinical Pharmacology and Toxicology
25.4 Clinical Development
25.5 Clinical Testing
Box 25.6 Regulatory Authorities
Further Reading
26 Drug Targeting and Prodrugs
26.1 Drug Targeting
26.1.1 Passive Targeting by Exploiting Special Physiological Properties of the Target Tissue
26.1.2 Physical Targeting
26.1.3 Active Targeting
26.1.4 Cellular Carrier Systems
26.2 Prodrugs
26.2.1 Prodrugs to Improve Drug Solubility
26.2.2 Prodrugs to Increase Stability
26.3 Penetration of Drugs Through Biological Membranes
26.4 Prodrugs to Extend Duration of Effect
26.5 Prodrugs for the Targeted Release of a Drug
26.6 Prodrugs to Minimize Side Effects
References
27 Molecular Diagnostics in Medicine
27.1 Introduction
27.2 Uses of Molecular Diagnostics. 27.2.1 Introduction
27.2.2 Monogenic and Polygenic Diseases
27.2.3 Individual Variability in the Genome: Forensics
27.2.4 Individual Variability in the Genome: HLA Typing
27.2.5 Individual Variability in the Genome: Pharmacogenomics
27.2.6 Individual Variability in the Genome: Susceptibility to Infectious Diseases
27.2.7 Viral Diagnosis
27.2.8 Microbial Diagnosis and Resistance Diagnosis
27.3 Which Molecular Variations Should be Detected
27.3.1 Point Mutations
27.3.2 Insertions and Deletions
27.3.3 Nucleotide Repeats
27.3.4 Deletion or Duplication of Genes
27.3.5 Recombination Between Chromosomes
27.3.6 Epigenetic Changes
27.4 Molecular Diagnostic Methods
27.4.1 DNA/RNA Purification
27.4.2 Detection of Target Sequence and Known Sequence Variations. 27.4.2.1 Nucleic Acid Tests
27.4.2.2 Quantitative PCR
27.4.2.3 Multiplexing of Nucleic Acid Detection: Nucleic Acid Microarrays
27.4.2.4 Production and Manufacture of Microarrays
27.4.2.5 Applications of Fragment Length Analysis
Detection of Length Polymorphism by PCR Fragment Size
Restriction Fragment Length Polymorphism (RFLP)
Amplification‐Created Restriction Sites (ACRS)
Amplification Refractory Mutation System (ARMS)
Mutationally Separated (MS)‐PCR
27.4.2.6 Minisequencing
27.4.2.7 Determination of Unknown Mutations
27.5 Outlook
Further Reading. Historic Article: “News & Views”
Reviews
Web Link
Textbooks
28 Recombinant Antibodies and Phage Display
28.1 Introduction
28.2 Generation of Specific Recombinant Antibodies
28.2.1 Generation of Antibody Gene Libraries
28.2.2 Selection Systems for Recombinant Antibodies. 28.2.2.1 Transgenic Mice with Human IgG Genes
28.2.2.2 In Vitro Selection Systems
28.3 Production and Purification of Recombinant Antibodies
28.4 Features and Applications of Recombinant Antibodies. 28.4.1 Advantages of Recombinant Antibodies
28.4.2 Formats and Applications of Recombinant Antibodies
28.4.2.1 Camelid Antibodies and VH Domains
28.4.2.2 scFv and dsFv
28.4.2.3 scFv–Fc Fusions, Fc Engineering, and the Addition of Constant Domains
28.4.2.4 IgG, Fusion Proteins, and Derivatives for Therapy
28.4.2.5 Bispecific Antibodies
28.4.2.6 Chimeric Antigen Receptors (CARs)
28.4.3 The Future of Therapeutic Antibodies
28.4.4 Research and In Vitro Diagnostics
28.4.5 Intracellular and Cell‐Penetrating Antibodies
28.5 Outlook
Further Reading. Textbooks
References
29 Genetically Modified Mice and Their Impact in Medical Research
29.1 Overview
29.2 Transgenic Mice
29.2.1 Retroviral Infection
29.2.2 Pronuclear Injection
29.3 Homologous Recombination: Knockout (Knock‐In) Mice
29.4 Endonuclease‐Based Knockout Mice
29.5 Endonuclease‐Based Knock‐In Mice
29.6 Conditionally Regulated Gene Expression
29.7 Gene Transfer to Subpopulations of Cells
29.7.1 Electroporation of Mouse Embryos (Plasmid DNA)
29.7.2 Virus‐Mediated Gene Transfer (Lentivirus, rAAVs)
29.7.3 Virus‐Mediated Gene Deletion (Cre/lox)
29.7.4 Virus‐Mediated Gene Knockdown (shRNA, Antagomirs)
29.8 Impact of Genetically Modified Mice in Biomedicine
29.8.1 Alzheimer's Disease
29.8.2 Amyotrophic Lateral Sclerosis (ALS)
29.8.3 Psychological and Cognitive Disorders
29.8.4 Autism Spectrum Disorder (ASD)
29.8.5 Chemogenetics, Optogenetics, and Magnetogenetics
29.9 Outlook
Reference
Further Reading
30 Plant Biotechnology
30.1 Introduction. 30.1.1 Green Genetic Engineering: A New Method Toward Traditional Goals
30.1.2 Challenges in Plant Biotechnology
30.2 Gene Expression Control and Genome Editing
30.2.1 Gene Expression Control
30.2.2 Genome Editing
30.3 Production of Transgenic Plants
30.3.1 Transformation Systems
30.3.1.1 Agrobacterium as a Natural Transformation System
30.3.1.2 Biolistic Method: Gene Gun
30.3.1.3 Plastid Transformation
30.3.1.4 Viral Systems
30.4 Selection of Transformed Plant Cells
30.4.1 Requirements for an Optimal Selection Marker System
30.4.2 Negative Selection Marker Systems
30.4.3 Positive Selection Marker Systems
30.4.4 Selection Systems, Genetic Engineering Safety, and Marker‐Free Plants
30.5 Regeneration of Transgenic Plants. 30.5.1 Regeneration Procedures
30.5.2 Composition of Regeneration Media
30.6 Plant Analysis: Identification and Characterization of Genetically Engineered Plants. 30.6.1 DNA and RNA Verification
30.6.2 Protein Analysis
30.6.3 Genetic and Molecular Maps
30.6.4 Stability of Transgenic Plants
Further Reading
31 Biocatalysis in the Chemical Industry
31.1 Introduction
31.2 Bioconversion/Enzymatic Procedures
31.3 Development of an Enzyme for Industrial Biocatalysis
31.3.1 Identification of Novel Biocatalysts
31.3.2 Improvement of Biocatalysts
31.3.3 Production of Biocatalysts
31.3.4 Outlook
31.3.5 Case Study 1: Screening for New Nitrilases
31.3.6 Case Study 2: Use of Known Enzymes for New Reactions: Lipases for the Production of Optically Active Amines and Alcohols
31.3.7 Case Study 3: Enzyme Optimization with Rational and Evolutive Methods
31.4 Fermentative Procedures
31.4.1 Improvement of Fermentation Processes
31.4.2 Classical Strain Optimization
31.4.3 Metabolic Engineering
31.4.4 Case Study 4: Fermentative Production of n‐Butanol
31.4.5 Case Study 5: Production of Glutamic Acid with C. glutamicum
31.4.5.1 Molecular Mechanism of Glutamate Overproduction
31.4.6 Case Study 6: Production of Lysine with C. glutamicum
31.4.6.1 Molecular Mechanism of Lysine Biosynthesis
31.4.6.2 Deregulation of the Key Enzyme Aspartate Kinase
31.4.7 Genomic Research and Functional Genomics
31.4.8 Case Study 7: Fermentative Penicillin Production
31.4.9 Case Study 8: Vitamin B2 Production
31.4.9.1 Riboflavin Biosynthesis
31.4.9.2 Classical Strain Development
References
32 Industrial Application: Biotech Industry, Markets, and Opportunities
32.1 Historical Overview and Definitions of Concepts
32.2 Areas of Industrial Application of Molecular Biotechnology
32.2.1 Red Biotechnology
32.2.1.1 Biopharmaceutical Drug Development
32.2.1.2 Gene and Cell Therapy
32.2.1.3 Tissue Engineering/Regenerative Medicine
32.2.1.4 Pharmacogenomics and Personalized Medicine
32.2.1.5 Molecular Diagnostic Agents
32.2.1.6 Systems Biology
32.2.1.7 Synthetic Biology
32.2.2 Green Biotechnology
32.2.2.1 Transgenic Plants
32.2.2.2 Genomic Approaches in Green Biotechnology
32.2.2.3 Novel Food and Functional Food
32.2.2.4 Livestock Breeding
32.2.3 White Biotechnology
32.3 Status Quo of the Biotech Industry Worldwide
32.3.1 Global Overview
32.3.2 United States
32.3.3 Europe
33 Patents in the Molecular Biotechnology Industry: Legal and Ethical Issues
33.1 Patent Law
33.1.1 What is a Patent?
33.1.2 How Does One Obtain a Patent?
33.1.3 What is the Proper Subject Matter for a Patent?
33.1.4 Types of Patents in Pharmaceutical and Molecular Biotechnology
33.1.5 Patent Infringement
33.1.6 International Patent Law
33.2 Ethical and Policy Issues in Biotechnology Patents
33.2.1 No Patents on Nature
33.2.2 Threats to Human Dignity
33.2.3 Problems with Access to Technology
33.2.4 Benefit Sharing
33.3 Conclusions
Acknowledgments
34 Drug Approval in the European Union and United States
34.1 Introduction
34.2 Regulation Within the European Union. 34.2.1 The EU Regulatory Framework
34.2.2 The EMA and National Competent Authorities
34.2.3 New Drug Approval Routes
34.2.3.1 The Centralized Procedure
34.2.3.2 Decentralized Procedure and Mutual Recognition
34.3 Regulation in the United States
34.3.1 CDER and CBER
34.3.2 The Approvals Procedure
34.4 The Advent and Regulation of Biosimilars
34.5 International Regulatory Harmonization
References
35 Emergence of a Biotechnology Industry
Reference
Further Reading
36 The 101 of Founding a Biotech Company
36.1 First Steps Toward Your Own Company
Box 36.1 Venture Capital and Biotech Funding
36.2 Employees: Recruitment, Remuneration, and Participation
37 Marketing
37.1 Introduction
37.2 What Types of Deals Are Possible?
37.3 What Milestone or License Fees Are Effectively Paid in a Biotech/Pharma Cooperation?
37.4 PR and IR in Biotech Companies
Further Reading
Websites
Glossary
Index
WILEY END USER LICENSE AGREEMENT
