Proteins E-Book

Gary Walsh

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 1012

Veröffentlichungsjahr: 2014

Beschreibung

Proteins Biochemistry and Biotechnology 2e is a definitive source of information for all those interested in protein science, and particularly the commercial production and isolation of specific proteins, and their subsequent utilization for applied purposes in industry and medicine.

Fully updated throughout with  new or fundamentally revised sections  on proteomics as, bioinformatics, protein glycosylation and  engineering,  well as sections detailing advances in upstream processing and  newer protein  applications such as enzyme-based biofuel production  this new edition has an increased focus on biochemistry to ensure the balance between biochemisty and biotechnology, enhanced with numerous case studies.

This second edition is an invaluable text for undergraduates of biochemistry and biotechnology but will also be relevant to students of microbiology, molecular biology, bioinformatics and any branch of the biomedical sciences who require a broad overview of the various medical, diagnostic and industrial uses of proteins.

• Provides a comprehensive overview of all aspects of protein biochemisty and protein biotechnology
• Includes numerous case studies
• Increased focus on protein biochemistry to ensure balance between biochemisty and biotechnology
• Includes new section focusing on proteomics as well as sections detailing protein function and enzyme-based biofuel production

"With the potential of a standard reference source on the topic, any molecular biotechnologist will profit greatly from having this excellent book. " (Engineering in Life Sciences, 2004; Vol 5; No. 5)

“Few texts would be considered competitors, and none compare favorably." (Biochemistry and Molecular Education, July/August 2002)

"...The book is well written, making it informative and easy to read..." (The Biochemist, June 2002)

Leseprobe

CONTENTS

Cover

Title page

Copyright page

Dedication

Preface

Chapter 1: Proteins and proteomics

1.1 Proteins, an introduction

1.2 Genes, genomics and proteomics

1.3 Bioinformatics

1.4 Proteomics: goals and applications

Further reading

Chapter 2: Protein structure and engineering

2.1 Primary structure

2.2 Higher-level structure

2.3 Protein classification on the basis of structure

2.4 Protein structural stability

2.5 Higher-order structure prediction

2.6 Protein folding

2.7 Intrinsically disordered proteins

2.8 Protein engineering

2.9 Protein post-translational modification

Further reading

Chapter 3: Protein sources

3.1 Recombinant versus non-recombinant production

3.2 Approaches to recombinant protein production

3.3 Heterologous protein production in E. coli

3.4 Heterologous production in bacteria other than E. coli

3.5 Heterologous protein production in yeast

3.6 Heterologous protein production in fungi

3.7 Proteins from plants

3.8 Animal tissue as a protein source

3.9 Heterologous protein production in transgenic animals

3.10 Heterologous protein production using animal cell culture

3.11 Insect cell culture systems

Further reading

Chapter 4: Protein purification and characterization

4.1 Protein detection and quantification

4.2 Initial recovery of protein

4.3 Removal of whole cells and cell debris

4.4 Concentration

4.5 Chromatographic purification

4.6 Protein inactivation and stabilization

4.7 Protein characterization

Further reading

Chapter 5: Large-scale protein production

5.1 Upstream processing

5.2 Downstream processing

5.3 Therapeutic protein production: some special issues

5.4 Range and medical significance of impurities potentially present in protein-based therapeutic products

Further reading

Chapter 6: Therapeutic proteins

6.1 Blood products

6.2 Anticoagulants

6.3 Thrombolytic agents

6.4 Additional blood-related products

6.5 Vaccine technology

6.6 Therapeutic enzymes

Further reading

Chapter 7: Therapeutic antibodies

7.1 Antibodies

7.2 IgG structure and activity

7.3 Antibody therapeutics: polyclonal antibody preparations

7.4 Antibody therapeutics: monoclonal antibodies

7.5 Therapeutic applications of monoclonal antibodies

7.6 Antibody conjugates

7.7 Bispecific antibodies

7.8 Antibody fragments

7.9 Engineering the antibody glycocomponent

7.10 Fc fusion proteins

Further reading

Chapter 8: Hormones and growth factors used therapeutically

8.1 Insulin

8.2 Glucagon

8.3 Gonadotrophins

8.4 Growth hormone

8.5 Erythropoietin

8.6 Other hormones

8.7 Growth factors

Further reading

Chapter 9: Interferons, interleukins and tumour necrosis factors

9.1 Regulatory factors: cytokines versus hormones

9.2 Interferons

9.3 Interleukins

9.4 Tumour necrosis factors

Further reading

Chapter 10: Proteins used for analytical purposes

10.1 The IVD sector

10.2 The basis of analyte detection and quantification

10.3 Enzymes as diagnostic/analytical reagents

10.4 Biosensors

10.5 Antibodies as analytical reagents

Further reading

Chapter 11: Industrial enzymes: an introduction

11.1 Sales value and manufacturers

11.2 Sources and engineering

11.3 Environmental benefits

11.4 Enzyme detection and quantification

11.5 Immobilized enzymes

11.6 Extremophiles

11.7 Enzymes in organic solvents

11.8 Industrial enzymes: the future

Further reading

Chapter 12: Industrial enzymes

12.1 Proteolytic enzymes

12.2 Carbohydrases

Further reading

Chapter 13: Additional industrial enzymes

13.1 Lipases

13.2 Penicillin acylase

13.3 Amino acylase and amino acid production

13.4 Cyclodextrins and cyclodextrin glycosyltransferase

13.5 Enzymes and animal nutrition

13.6 Enzymes in molecular biology

Further reading

Chapter 14: Non-catalytic industrial proteins

14.1 Functional properties of proteins

14.2 Milk and milk proteins

14.3 Animal-derived proteins

14.4 Plant-derived proteins

14.5 Sweet and taste-modifying proteins

Further reading

Index

About the companion website

Eula

List of Tables

Chapter 01

Table 1.1 Selected examples of proteins. The number of polypeptide chains and amino acid residues constituting the protein are listed, along with its molecular mass and biological function.

Table 1.2 The three main global sequence databases, their host organizations and web addresses. Refer to text for details.

Chapter 02

Table 2.1 The 20 commonly occurring amino acids. They may be subdivided into five groups on the basis of side-chain structure. Their three- and one-letter abbreviations are also listed (one-letter abbreviations are generally used only when compiling extended sequence data, mainly to minimize writing space and effort). In addition to their individual molecular masses, the per cent occurrence of each amino acid in an ‘average’ protein is also presented. This data was generated from sequence analysis of over 1000 different proteins.

Table 2.2 Top matches obtained from a BLAST search using the human erythropoietin (EPO) amino acid sequence as a query sequence against the 42 million sequence entries present in the UniProtKB database (Box 2.2). A total of 121 hits were obtained, the top 26 of which are presented here. Unsurprisingly, the highest matches were to the human EPO sequence entries already present in the database. Many of the additional hits are EPO sequences from other species. An outline of how similarity is graded is presented in the main text.

Table 2.3 Approximate bond energies associated with various (non-covalent) electrostatic interactions, as compared with a carbon–carbon single bond.

Table 2.4 Some secondary structure predictive methods.

Table 2.5 Conformational preferences and assignments of amino acid residues with regard to stretches of α-helix and β structure.

Table 2.6 Representative engineered proteins which are now used commercially. These and other examples are considered in later chapters of this book.

Table 2.7 The more common forms of post-translational modifications that polypeptides may undergo. Refer to text for additional details.

Table 2.8 Representative examples of some acylated proteins. The fatty acid moiety attached and the location of eukaryotic-derived acylated proteins are also listed.

Table 2.9 Some post-translational modifications (PTMs) associated with proteins for therapeutic use. The vast majority of such proteins are (recombinant forms of) native human extracellular proteins, and as such their PTM profile is biased towards PTMs characteristic of extracellular proteins derived from higher eukaryotes. The list is representative only, with specific examples being found throughout Chapters 6–9.

Chapter 03

Table 3.1 Some of the proteins and protein families produced by recombinant means that have found commercial application. In some instances the commercial products are produced exclusively via recombinant technology. In other cases (e.g. insulin) both non-recombinant and recombinant versions of the product are commercially available. The chapters in which the various proteins are discussed are also indicated.

Table 3.2 Some proteins obtained commercially from non-genetically engineered microorganisms. Sources include a range of bacteria, fungi and yeast, and the proteins have found medical, analytical and industrial uses.

Table 3.3 Illustrative examples of proteins manufactured commercially via heterologous expression in E. coli. These and other examples are considered in later chapters of this book.

Table 3.4 Some promoters that have been used to control expression of recombinant proteins in engineered E. coli cells. For example, the widely used pET expression vectors use a T7-based promoter.

Table 3.5 Some heterologous proteins produced in E. coli, and the levels of expression achieved.

Table 3.6 Some advantages and disadvantages of E. coli used for the production of recombinant proteins.

Table 3.7 Selected proteins of therapeutic importance which have been produced (at a research level at least) by recombinant means in yeast.

Table 3.8 Some advantages and disadvantages of heterologous protein production in yeast.

Table 3.9 Some proteins of industrial significance which have been expressed in recombinant fungal systems.

Table 3.10 Some proteins of industrial/medical interest which have been produced by recombinant means in plants.

Table 3.11 Major advantages and disadvantages of recombinant protein production in transgenic plants.

Table 3.12 Some proteins of industrial/medical significance which have been traditionally obtained from animal sources. Many of the listed examples may now also be obtained via recombinant production.

Table 3.13 Some proteins of therapeutic use which have been expressed in the milk of transgenic animals.

Table 3.14 Some recombinant pharmaceutical proteins approved for general medical use which are produced commercially via animal cell culture.

Chapter 04

Table 4.1 Chromatographic techniques most commonly applied to protein purification protocols. The basis of separation is listed in each case.

Table 4.2 Typical quantities of any given protein required to meet the indicated academic/applied purposes.

Table 4.3 The various methods most commonly used to detect and quantify protein levels in a biological sample. Notable characteristics of each method are also listed.

Table 4.4 Example bioassays designed to detect/quantify the indicated protein.

Table 4.5 Some chemical, physical and enzyme-based techniques that may be employed to achieve microbial cell disruption.

Table 4.6 Various methods/techniques that may be used to precipitate proteins from solution.

Table 4.7 Functional groups commonly attached to chromatographic beads in order to generate cation or anion exchangers.

Table 4.8 Some lectins commonly used in immobilized format for the purification of glycoproteins. The sugar specificity is listed, as are the free sugars used to elute the bound glycoprotein.

Table 4.9 Various chemical, biological and physical influences that can lead to protein inactivation. The negative impact of many such influences is time and concentration dependent. Refer to text for details.

Table 4.10 Buffers commonly used to maintain the pH of a protein solution at a pre-specified value.

Table 4.11 Substances often added to protein solutions in order to enhance protein stability.

Table 4.12 Major studies typically undertaken in order to characterize a protein, along with the associated analytical techniques/approaches by which these are generally achieved.

Chapter 05

Table 5.1 Quantitative composition of the various constituents in a complex media (Terrific broth, TB), a fully defined media (Minimal media 9, M9) and a semi-defined media (M9-yeast extract, M9/YE).

Table 5.2 Some advantages and disadvantages associated with the lyophilization (freeze-drying) of proteins.

Table 5.3 The range of substances that cleaning, decontamination and sanitation of therapeutic protein production equipment aims to remove.

Table 5.4 The range and medical significance of potential impurities present in therapeutic protein products.

Table 5.5 Molecular mass of some therapeutic proteins. Many are glycosylated, thereby exhibiting a range of molecular masses due to differential glycosylation.

Chapter 06

Table 6.1 Blood clotting factors.

Table 6.2 Recombinant blood clotting factors that have gained approval for general medical use in the EU and/or the USA. Most are also approved for use in various other world regions.

Table 6.3 Anticoagulants which are used therapeutically.

Table 6.4 Recombinant tPA-based products that have gained approval for general medical use in the EU and/or the USA. Most are also approved for use in various other world regions.

Table 6.5 The five structural domains of native tPA and the biological function of each domain.

Table 6.6 Recombinant subunit vaccines that have gained approval for general medical use in the EU and/or the USA. Many are also approved for use in various other world regions.

Table 6.7 Traditional (non-recombinant) enzymes used medically. Refer to text for details.

Table 6.8 Recombinant therapeutic enzymes now in medical use.

Chapter 07

Table 7.1 Polyclonal antibody preparations most commonly used to induce passive immunity.

Table 7.2 First-generation murine therapeutic monoclonal antibodies (Mabs) produced via hybridoma technology.

Table 7.3 Chimeric and humanized monoclonal antibodies (Mabs) that have gained approval for general medical use in the EU and/or the USA. Many are also marketed in other world regions.

Table 7.4 Fully human monoclonal antibodies (Mabs) that have gained approval for general medical use in the EU and/or the USA. Many are also marketed in other world regions.

Table 7.5 Some approved therapeutic monoclonal antibodies that bring about their effect by neutralizing/blocking the biological activity of a target signalling ligand.

Table 7.6 Some monoclonal antibody-based products approved for the treatment of cancer that bring about their effect via triggering of various antibody-mediated immune effector functions on binding to the cancer cell surface.

Table 7.7 Antibody fragments approved for general medical use. For various commercial and technical reasons many of these products no longer remain on the market. Refer to text for details.

Table 7.8 Fc-based fusion products approved for general medical use in the EU and/or the USA. Many are also available in several other world regions.

Chapter 08

Table 8.1 Major polypeptide hormones and growth factors used therapeutically.

Table 8.2 Some human insulin preparations produced via recombinant DNA technology that have been approved for general medical use.

Table 8.3 Some therapeutic characteristics of short- and longer-acting insulin preparations.

Table 8.4 Engineered rapid and long-acting insulin products now used therapeutically.

Table 8.5 Recombinant gonadotrophins now used to treat various fertility-related conditions. All the products are expressed in CHO cell lines, which facilitate appropriate product glycosylation.

Table 8.6 Representative recombinant human growth hormone (rhGH)-based products now approved for general medical use.

Table 8.7 Representative recombinant human erythropoietin (rhEPO)-based products now approved for general medical use.

Table 8.8 Some polypeptide growth factors. Many can be grouped into families on the basis of amino acid sequence homology, or the cell types affected. Most growth factors are produced by more than one cell type and display endocrine, paracrine or autocrine effects on target cells by interacting with specific cell-surface receptors.

Table 8.9 Human colony-stimulating factors (CSFs) and their sources.

Table 8.10 Colony-stimulating factors (CSFs) approved for general medical use.

Table 8.11 Some additional recombinant growth factors approved for general medical use.

Chapter 09

Table 9.1 The major polypeptides/polypeptide families that constitute the cytokine group of regulatory molecules.

Table 9.2 Human interferons and the cellular sources from which they were initially isolated.

Table 9.3 Summary of the biological characteristics of the interferons. Details provided relate to human interferons, except in the case of IFN-τ, which is only produced by ruminants.

Table 9.4 Interferon preparations approved for general medical use. In each case the original indication for which the product was approved is listed. Additional indications have subsequently been approved for some such products.

Table 9.5 Some human interleukins: summary of size, sources and major biological activities.

Table 9.6 Interleukins and interleukin antagonists approved for general medical use.

Table 9.7 Summary of the physicochemical characteristics of human TNF-α and TNF-β.

Chapter 10

Table 10.1 Some analytes routinely measured in clinical chemistry laboratories, along with their diagnostic significance.

Table 10.2 Some enzymes used directly or indirectly as diagnostic reagents. The original enzyme source and likely applications are also listed. Many are now produced via recombinant means.

Table 10.3 Approximate content of triglycerides and cholesterol in various lipoproteins.

Table 10.4 Some analytes of diagnostic importance for which specific quantitative enzyme electrodes have been developed. The enzymes used in these test systems are also listed.

Table 10.5 Some analytes of importance in the food industry for which biosensors have been developed (although not all have been commercialized).

Table 10.6 Some specific examples of analytes of diagnostic importance for which immunoassays are commercially available.

Table 10.7 Labels usually used in immunoassay systems. Refer to text for additional details.

Chapter 11

Table 11.1 Selected attributes of industrial enzymes compared with those of proteins used for healthcare purposes.

Table 11.2 Some industrially important enzymes, their traditional sources and sample industrial applications.

Table 11.3 Some enzyme-producing companies, as well as technical associations representing enzyme producers.

Table 11.4 Some industrial enzymes produced by recombinant DNA technology. Several of these are considered in more details in subsequent chapters.

Table 11.5 Selected examples of commercialized detergent enzymes whose amino acid sequence was logically altered by site-directed mutagenesis in order to enhance their industrial utility by rendering them oxidation-resistant.

Table 11.6 Some databases providing specific information relating to enzymes.

Table 11.7 Some enzymes currently used in an immobilized format for industrial purposes, the reactions catalysed and the industrial processes in which they are used.

Table 11.8 Some amino acid residues normally involved in the formation of covalent linkages in enzyme immobilization.

Table 11.9 Various categories of extremophiles and the habitats in which they live.

Table 11.10 (Hyper)thermophilic Archaea and their respective maximum growth temperatures.

Table 11.11 Temperature optimum and stability of selected enzymes isolated from various hyperthermophiles which may have potential industrial application.

Table 11.12 Some thermoacidophiles and the temperature and pH values at which they grow optimally.

Table 11.13 Some enzymes derived from thermoacidophiles which may have potential industrial application.

Table 11.14 Comparison of the thermal stability of selected enzymes in aqueous versus non-aqueous media.

Chapter 12

Table 12.1 Overview of the various industrial uses of proteolytic enzymes.

Table 12.2 Peptide bonds hydrolysed by various types of exopeptidases.

Table 12.3 The major classes of proteolytic enzymes (classified on the basis of their mechanism of catalytic conversion). Some better-known examples of each class are also presented.

Table 12.4 Principal ingredients of detergent preparations.

Table 12.5 The major commercialized subtilisin proteases, their sources, properties and manufacturers.

Table 12.6 Some proteolytic preparations (recombinant and non-recombinant) used to promote curd formation in cheesemaking.

Table 12.7 Some microorganisms that produce keratinases and selected biochemical properties of the keratinases they produce.

Table 12.8 Major industrial applications of amylolytic enzymes.

Table 12.9 The relative percentage content of amylose and amylopectin in the starch derived from various plant sources.

Table 12.10 Sources and characteristics of some industrially significant amylolytic enzymes.

Table 12.11 Optimum temperature and pH for glucoamylases isolated from the indicated sources.

Table 12.12 The relative sweetness of a number of commonly occurring sugars of industrial importance. For comparative purposes, sucrose has been assigned a relative sweetness of 100%.

Table 12.13 Various substances used in textile sizing. Refer to text for details.

Table 12.14 Some well-characterized microbial sources of cellulolytic enzymes.

Table 12.15 Sources, characteristics and industrial producers of some detergent cellulases.

Chapter 13

Table 13.1 Major detergent lipase preparations now commercially available, their sources and manufacturers.

Table 13.2 Lipid-based stains commonly found on soiled clothing/laundry.

Table 13.3 Various microorganisms that produce penicillin acylase.

Table 13.4 Some bacterial producers of cyclodextrin glycosyltransferase.

Table 13.5 Effect of supplementing barley-based diets with β-glucanase on performance of poultry. The wheat-based diets serves as a control. Bird performance may be assessed by the feed to weight gain ratio. The lower this value, the more efficiently the bird has utilized the feed provided.

Table 13.6 Typical effects of phytase on phosphorus balance in pigs. Phytase diet was similar to control diet but was supplemented with phytase. Both phosphorus intake and absorption were monitored. Inclusion of phytase resulted in increases in absorbed and retained phosphorus. All differences recorded were found to be statistically significant.

Table 13.7 Enzymes most often added to animal feed and the natural substrates that are most often used in their assay.

Table 13.8 Synthetic chromogenic substrates that may be used to assay the enzymes indicated.

Table 13.9 Some commercially available restriction endonucleases, their sources, DNA recognition sites and cleavage points.

Table 13.10 Some DNA polymerases available commercially.

Chapter 14

Table 14.1 Principal non-catalytic proteins that find bulk industrial application. Although most are used as, or added to, human food/animal feed, some also have non-food applications in the pharmaceutical and cosmetics sectors for example.

Table 14.2 Functional properties exhibited by some proteins which renders attractive their addition to selected foods.

Table 14.3 Average composition (% w/v) of the milk of humans and various agriculturally important species.

Table 14.4 The major dairy-based products, how they are produced and their uses.

Table 14.5 Methods by which micellar caseins may be extracted from milk on a laboratory scale.

Table 14.6 Major applications of milk-derived proteins in the food industry.

Table 14.7 Composition (%) of rennin-derived whey.

Table 14.8 Some physicochemical characteristics of the major (bovine) whey proteins.

Table 14.9 Amino acid composition (%) of gelatin.

Table 14.10 Major food and non-food applications of collagen-derived gelatin.

Table 14.11 Major protein types found in egg white. Immunoglobulins are found in egg white and yolk. Refer to text for further details.

Table 14.12 The major globulin-based seed proteins found in soybean.

Table 14.13 Some sweet/taste-modifying proteins and their sources.

List of Illustrations

Chapter 01

Figure 1.1 Differential splicing of mRNA can yield different polypeptide products. Transcription of a gene sequence yields a ‘primary transcript’ RNA. This contains coding regions (exons) and non-coding regions (introns). A major feature of the subsequent processing of the primary transcript is ‘splicing’, the process by which introns are removed, leaving the exons in a contiguous sequence. Although most eukaryotic primary transcripts produce only one mature mRNA (and hence code for a single polypeptide), some can be differentially spliced, yielding two or more mature mRNAs. The latter can therefore code for two or more polypeptides. E, exon; I, intron.

Figure 1.2 Evolution of the various approaches used to study proteins. Refer to text for details.

Figure 1.3 Global proteomics approach. While target proteins may be obtained from native (i.e. naturally producing) source material, they are most commonly obtained by recombinant means via the construction of gene/cDNA libraries. In the case of a prokaryotic cell source, a collection of individual genes can be isolated and cloned by standard molecular biology techniques, forming a genomic library (consisting of just three genes in the simplified example portrayed here). Eukaryotic genes generally consist of coding sequences (exons) interrupted by non-coding sequences (introns), while processed mRNA transcripts derived from those genes reflect the coding sequence for the final polypeptide product only. Isolation of total cellular mRNA followed by incubation with a reverse transcriptase enzyme yields complementary double-stranded DNA (cDNA) sequences, directly encoding the polypeptide sequences of the complement of expressed genes, thereby generating a cDNA library. Again by using standard molecular biology techniques the gene/cDNA library products can be expressed, yielding the recombinant protein products. The proteins, in turn, can be purified and characterized via techniques considered in subsequent sections of this chapter, as well as in Chapters 4 and 5.

Figure 1.4 Separation of proteins by SDS-PAGE. Protein samples are incubated with SDS (as well as reducing agents, which disrupt disulfide linkages). The electric field is applied across the gel after the protein samples to be analysed are loaded into the gel wells. The rate of protein migration towards the anode depends on protein size. After electrophoresis is complete individual protein bands may be visualized by staining with a protein-binding dye.

Figure 1.5 Proteins are amphoteric molecules, displaying a positive, negative or zero overall net charge depending on the pH of the solution in which are they dissolved. Contributing to the overall charge of a protein are all the positive and negative charges of its amino acid side chains as well as the free amino and carboxyl groups present at its amino and carboxyl termini, respectively. The state of ionization of these groups is pH dependent. The pH at which the net number of positive charges equal the net number of negative charges (i.e. the protein has an overall net electric charge of zero, and hence will not move under the influence of an electric field) is known as its isoelectric point (pI).

Figure 1.6 Principle of two-dimensional gel electrophoresis. The protein sample is applied to the polyacrylamide gel and first subjected to isoelectric focusing (IEF). After this is complete the protein bands are subjected to SDS-PAGE in the perpendicular direction (a). This combination has greater resolving power than either technique alone. (b) Resolution of two proteins with equal pI values but different molecular masses. (c) Resolution of two proteins of equal molecular mass but differing pI values. (d) Example of a two-dimensional gel in which a microbial proteome has been resolved.

Figure 1.7 (a) Schematic representation of capillary electrophoresis. After sample application, a high voltage is applied and the proteins migrate under the influence of the resultant electric field. Visualization of proteins eluting is achieved using an in-line UV/visible, fluorescence or other appropriate detector. (b) Separation of individual constituents of a protein mixture, with the molecular mass of individual proteins (kDa) indicated above each peak.

Figure 1.8 HPLC-based chromatographic separation generally gives rise to better-resolved protein peaks (a) than do low pressure-based systems (b).

Figure 1.9 Basic principle of mass spectrometry. The system is composed of three essential components: an ion source which generates gas phase-ionized analytes; a mass analyser, which sorts the ions by mass via the application of, for example, electric or magnetic fields; and a detector, which detects and quantifies the ions. Data from the detector thus provides the mass and abundance of each ion present. Refer to text for further detail.

Figure 1.10 Schematic representation of a common approach to protein identification via MS-based peptide mass fingerprinting. Refer to text for detail.

Figure 1.11 Diagrammatic representation of the quantitative proteomic approach as illustrated by two-dimensional gel electrophoretic-based analysis. In this simplified illustrative example, the ‘proteome’ consists of just five proteins derived from a biological source material under investigation (e.g. a specific cell type exposed to two different stimuli). It is clear that, relative to stimulus (a), stimulus (b) results in an increase in the concentration of proteins 1 and 2, a decrease in the concentration of protein 3, while making no difference to the concentration of proteins 4 and 5. In reality, proteomic samples analysed would generally contain hundreds or thousands of different proteins.

Figure 1.12 Approach to interactome studies using a bait protein immobilized on a chromatographic bead. Beads can be incubated with the target proteome (which in this simplified example contains only three proteins). Only proteins interacting with the bait molecule in a biospecific manner will be retained on the column. After washing away the additional (non-binding) proteins, the captured (prey) protein(s) can be eluted from the bead and analysed in order to establish prey protein identity.

Figure 1.13 The basis on which the yeast two-hybrid (Y2H) system detects protein–protein interactions. Plasmid 1 (in yeast 1) contains a fusion construct housing a nucleotide sequence coding for a transcription activator DNA-binding domain (DBD) fused to a nucleotide sequence coding for the bait protein (X). Plasmid 2 (in yeast 2) contains a fusion construct housing a nucleotide sequence coding for a transcription activator domain (AD) fused to a nucleotide sequence coding for a possible prey protein (Y). The yeast are allowed to mate (or are transformed), bringing both plasmids into the one cell. If the bait and prey proteins (X–Y) actually do interact, they bring the transcription factor DBD and AD domains together in the one complex, which in turn specifically activates the downstream reporter gene. Refer to text for exact detail.

Figure 1.14 Generation of a protein array. (a) The genes/cDNAs coding for the proteome of interest (only six proteins in this simplified example) are expressed in a recombinant microbial library (i.e. individual genes/cDNAs are inserted into individual microbial cells which, when grown individually, will produce the recombinant protein product). This molecular biology approach also allows the attachment of affinity tags at the end of each protein, facilitating affinity-based protein purification subsequent to protein expression. The tags also allow the docking (attachment) of the proteins to a solid support (e.g. a glass slide) if a docking ligand for the affinity tag is first immobilized on that support (b). Refer to text for further details.

Figure 1.15 Some approaches that facilitate the detection of protein interactions. (a) The use of a substrate molecule which generates a coloured/fluorescent product if the array is designed to interact with a specific enzyme. (b) The use of a labelled (tagged) antibody capable of binding to human antibodies if the array is designed to interact with human antibodies. (c) Interaction detection via the use of a sample in which all analytes are pre-tagged. (d) A (simplified) array image in which molecules from a sample analysed have bound to one specific array protein. Refer to text for further detail.

Figure 1.16 Self-assembling protein arrays. The nucleotide sequences (e.g. cDNA) coding for individual tagged proteins are immobilized on the array solid support surface, as are tag docking ligands. Only a single illustrative sample is shown here (a). A commercial cell-free expression system is then incubated on the array surface. Cell-free expression systems contain a cocktail of the components necessary to transcribe and translate a coding sequence (RNA polymerase, ribosomes, tRNA and ribonucleotides), thus allowing protein synthesis to occur in vitro. The result therefore is synthesis of the tagged array proteins, which then spontaneously immobilize on the solid support surface via binding to the docking ligands (b).

Chapter 02

Figure 2.1 The chemical structure of the 20 amino acids commonly found in proteins.

Figure 2.2 The formation of cystine via disulfide bond formation between two cysteines.

Figure 2.3 Structure of some modified amino acids.

Figure 2.4 (a) Peptide bond formation. (b) Polypeptides consist of a linear chain of amino acids successively linked via peptide bonds. (c) The peptide bond displays partial double-bonded character.

Figure 2.5 Fragment of polypeptide chain backbone illustrating rigid peptide bonds and the intervening N─Cα and Cα─C backbone linkages, which are free to rotate.

Figure 2.6 A pairwise sequence alignment between the amino acid sequence of human erythropoietin (EPO, top line of each twin sequence) and canine EPO (bottom line of each twin sequence) (a). The sequence alignment was undertaken via the UniProt website. Asterisks are automatically placed underneath sequence positions housing identical amino acid residues while double or single dots (i.e. a colon or a period) appear underneath residue positions which display strongly or weakly similar properties, respectively. Thus, human and canine EPOs contain identical residues at 155 positions (i.e. they display approximately 75% identity) and similar residues at a further 24 positions. The software also facilitates the generation of additional information such as the positioning of amino acid residues with particular properties.

Figure 2.7 Ball and stick and ribbon representation of an α-helix. Reproduced from Current Protocols in Protein Science by kind permission of the publisher, John Wiley & Sons, Ltd.

Figure 2.8 The β sheet. (a) Two segments of β strands (antiparallel) forming a β sheet via hydrogen bonding. The β strand is drawn schematically as a thick arrow. By convention the arrowhead points in the direction of the polypeptide’s C-terminus. (b) Schematic of a two-strand β sheet in parallel and antiparallel modes.

Figure 2.9 (a) The β bend or β turn is often found between two stretches of antiparallel β strands. (b) It is stabilized in part by hydrogen bonding between the C═O bond and the NH groups of the peptide bonds at the neck of the turn.

Figure 2.10 Schematic representation of the domain structure of human tissue plasminogen activator, a 526 amino acid protein. A number of these domains have been independently produced via recombinant DNA technology and their three-dimensional structure resolved. These are also included (protein databank entries 1TPN, 1PK2 and 1RTF; www.pdb.org).

Figure 2.11 Some structural motifs commonly associated with (globular) polypeptides: (a) a four-helix bundle (b) a hairpin structure (c) a β sheet with a Greek key topology (d) a jelly roll motif (e) a β sandwich (f) a β barrel (g) an α/β barrel. Refer to text for further details. Reproduced from Current Protocols in Protein Science by kind permission of the publisher, John Wiley & Sons, Ltd.

Figure 2.12 Overview of the principles of X-ray diffraction. Refer to text for details.

Figure 2.13 Growth of protein crystals by the vapour diffusion (hanging drop) method. A small (20-μL) drop of a concentrated purified protein solution containing a suitable precipitant (e.g. polyethylene glycol or ammonium sulfate) is placed on a glass surface. This is subsequently inverted and sealed (e.g. with vacuum grease) to the top of a chamber containing a reservoir of the precipitant. The apparatus is then incubated at a temperature of the order of 22°C, resulting in slow evaporation of water from the protein-containing hanging drop. A supersaturated solution is slowly generated, which is conducive to crystal growth.

Figure 2.14 Three-dimensional structure of human erythropoietin (EPO) as deposited in the PDB (entry 1BUY). As well as the actual three-dimensional structure (a), entries contain additional information about proteins, including details of primary and secondary structure (b).

Figure 2.15 Overview of the protein folding pathway. Refer to text for details.

Figure 2.16 Cis versus trans forms of a peptide bond.

Figure 2.17 (a) N-linked versus (b) O-linked glycosylation. ‘Sugar’ represents an oligosaccharide chain, an example of which is provided in Figure 2.18.

Figure 2.18 Structure of two sample oligosaccharide side chains (one N-linked the other O-linked) found in glycoproteins. Man, mannose; Gal, galactose; SA, sialic acid; GlcNAc, N-acetylglucosamine.

Figure 2.19 (a) Reversible phosphorylation of a protein substrate via a kinase/phosphatase mechanism. (b) Phosphate groups are usually attached to the protein via the hydroxyl groups of serine, threonine or tyrosine residues.

Figure 2.20 Acetylation of a polypeptide N-terminal amino group as catalysed by an N-acetyltransferase enzyme. The acetyl group donor is usually acetyl-CoA.

Figure 2.21 Linkage of (a) palmitic acid via an ester bond to a serine residue, (b) palmitic acid via a thioester bond to a cysteine residue, and (c) myristic acid to an N-terminal glycine reside via an amide bond.

Chapter 03

Figure 3.1 (a) Overview of the molecular biology process by which a recombinant protein production system is developed. (b) A more detailed overview of common strategies adopted during this process. Refer to text for specific detail.

Figure 3.2 Affinity-based purification of mRNA. The unpurified mRNA-containing solution is percolated through a column packed with cellulose beads (a), to which a short chain of deoxythymidylate (an oligo dT chain) has been attached. Any mRNA present is retained in the column due to complementary base pairing between its 3′ poly-A tail and the immobilized oligo dT (b). Non-bound material can then be washed out of the column, with subsequent desorption of the mRNA by passing a low salt buffer through the column. The mRNA collected may then be precipitated out of solution using ethanol, followed by collection via centrifugation. An alternative and now more commonly used variation entails the direct addition of oligo (dT)-bound magnetic beads directly into the cell lysate and ‘pulling out’ the mRNA using a magnet. The method is rapid, thus minimizing contact time of the mRNA with degradative ribonucleases present naturally in the cytoplasm.

Figure 3.3 The plasmid pUC18, which is often used for cloning purposes. The plasmid contains three genes: the ampicillin resistance gene (ampR); the lacZ gene, which codes for the enzyme β-galactosidase; and the lacI gene, which codes for a factor that controls the transcription of lacZ. Also present is an origin of replication (ori) which is essential for plasmid replication within the cell. Note the presence of a short stretch of DNA called the polylinker region located within the lacZ gene. The polylinker (also called a multiple cloning site) contains cleavage sites for 13 different restriction endonucleases. This allows genetic engineers great flexibility to insert a DNA fragment for cloning into this area. The polylinker has been designed and positioned within the lacZ gene in a manner so as not to prevent the expression of functional β-galactosidase. However, if a piece of DNA for cloning is introduced into the polylinker region, the increased length does block β-galactosidase expression. The full sequence of the 2.69 kb plasmid is known and sequence analysis confirms the presence of multiple additional restriction endonuclease sites outside the polylinker region. There are at least six target sites for commonly used restriction enzymes within the ampR gene.

Figure 3.4 Overview of the recovery of biologically active protein from inclusion bodies. Refer to text for further detail.

Figure 3.5 Schematic diagram of the type I secretion mechanism which achieves one-step protein export from Gram-negative bacterial cells. The cell wall is omitted for clarity. At the core of the system is a complex of three different proteins, HlyB, HlyD and TolC, which cooperate to export proteins via an ATP-dependent mechanism.

Figure 3.6 Generalized schematic diagram of the production of a recombinant protein in the milk of transgenic mice. Refer to text for details.

Figure 3.7 Overview of the industrial production of the IFN-ω product Vibragen Omega. Refer to text for details.

Chapter 04

Figure 4.1 Generalized approach used to extensively purify a protein.

Figure 4.2 Schematic diagram of the Potter homogenizer, commonly used to homogenize gram-level quantities of animal tissue such as liver and brain.

Figure 4.3 Diagrammatic representation of a cell homogenizer. This is one of a number of instruments routinely used to rupture microbial cells and, in some cases, animal/plant tissue.

Figure 4.4 Laboratory-scale centrifuges (a, b), a centrifuge rotor (c) and a schematic representation of the centrifugation process (d). Centrifugation of a cell homogenate allows collection of the cellular debris as a ‘pellet’ at the base of the sample container (the centrifuge tube), while the clarified liquid (‘supernatant’) contains the intracellular contents.

Figure 4.5 Molecular structure of (a) polyethylene glycol (n is usually between 4 and 200) and (b) a fragment of the dextran backbone.

Figure 4.6 Principle of an aqueous two-phase purification system as applied to separation of cellular debris from soluble proteins. PEG, polyethylene glycol.

Figure 4.7 Molecular structure of the repeat unit of polyethylenimine.

Figure 4.8 Effect of salt concentration on protein solubility. Increases in salt concentration from low initial values often increase protein solubility (salting in). Further increases above an optimal value will result in destabilization of the protein, and eventually its precipitation from solution (salting out).

Figure 4.9 Structure of the organic solvents most commonly used as protein precipitants.

Figure 4.10 Ultrafiltration separates molecules on the basis of size and shape. (a) Diagramatic representation of a typical laboratory-scale ultrafiltration system. The sample (e.g. crude protein solution) is placed in the ultrafiltration chamber, where it sits directly above the ultrafilter membrane. The membrane in turn sits on a macroporous support that provides it with mechanical strength. Pressure is then applied (usually in the form of an inert gas) as shown. Molecules larger than the pore diameter (e.g. large proteins) are retained on the upstream side of the ultrafilter membrane. However, smaller molecules, particularly water molecules, are easily forced through the pores, thus effectively concentrating the protein solution (b). Membranes can be manufactured that display different pore sizes, i.e. have different molecular mass cut-off points. (c) An ultrafilter membrane. (d) A laboratory stirred cell ultrafilter unit in operation.

Figure 4.11 (a) Typical sequence of events during an adsorption-based protein purification chromatographic step. Fractions collected during an initial column wash (to wash unbound material out of the column) and during the elution of bound protein (usually triggered by an increasing concentration of desorbing molecules included in the elution running buffer) are usually assayed for (i) total protein (by measuring absorbance at 280 nm) and (ii) target protein bioactivity. In the case illustrated (b), a large amount of unbound protein emerges from the column during the initial wash, while three different protein peaks are subsequently eluted from the column. Only the last peak contains the protein of interest.

Figure 4.12 (a) A basic laboratory-scale protein purification system and (b) two chromatography columns.

Figure 4.13 The application of gel filtration chromatography to separate proteins from molecules of much lower molecular mass. The mobile phase (the ‘running buffer’) will be devoid of the molecular species to be removed from the protein. Highly cross-linked porous beads are used, which exclude all protein molecules. However, the lower molecular mass substances can enter the beads and their progress down through the column will therefore be retarded (a). The earlier fractions collected will contain the proteins while the latter fractions will contain the low-molecular-mass contaminants (b). In practice this ‘group separation’ application of gel filtration chromatography is mainly used to separate proteins from salt (e.g. after an ammonium sulfate precipitation step) or for buffer exchange.

Figure 4.14 Structure of amino acids having overall net charges at pH 7.0. In proteins the charges associated with the α-amino and α-carboxyl groups in all but the terminal amino acids are not present, as these groups are directly involved in the formation of peptide bonds.

Figure 4.15 Principle of ion-exchange chromatography, in this case anion exchange chromatography. The chromatographic beads exhibit an overall positive charge. Proteins displaying a net negative charge at the pH selected for the chromatography will bind to the beads due to electrostatic interactions.

Figure 4.16 Structural formulae of the eight commonly occurring amino acids that display hydrophobic characteristics.

Figure 4.17 Chemical structure of (a) phenyl and (b) octyl Sepharose, widely used in hydrophobic interaction chromatography.

Figure 4.18 Schematic representation of the principle of biospecific affinity chromatography. The chosen affinity ligand is chemically attached to the support matrix (agarose bead) via a suitable spacer arm. Only those ligands in solution that exhibit biospecific affinity for the immobilized species will be retained.

Figure 4.19 Principle of immunoaffinity chromatography. Only antigen that is specifically recognized by the immobilized antibody will be retained on the column.

Figure 4.20 Some monochloro and dichloro triazine dyes commonly used as affinity ligands in dye affinity chromatography.

Figure 4.21 Schematic representation of the basic principles of metal chelate affinity chromatography. (a) Certain proteins are retained on the column via the formation of coordinate bonds with the immobilized metal ion. (b) The structure of the most commonly used metal chelator, iminodiacetic acid (IDA).

Figure 4.22 A typical laboratory HPLC system (a) and a selection of typical HPLC chromatographic columns (b).

Figure 4.23 Schematic diagram of an elution profile from an IMAC column onto which a His-tagged unpurified protein extract has been loaded and unbound protein subsequently washed through. In this case elution is promoted via application of an increasing gradient of imidazole concentration in the eluting buffer, and protein elution from the column is followed by monitoring absorbance of the eluate at 280 nm. Sometimes an initial elution peak is observed at low imidazole concentrations (peak 1). This usually represents non-tagged proteins, which weakly bind the column via, for example, histidine residues naturally present on their surface. His-tagged proteins bind more tightly to the column and are therefore eluted only at higher imadizole concentrations (peak 2).

Figure 4.24 Overview of membrane protein isolation and purification.

Figure 4.25 Eukaryotic cellular organelle separation via differential centrifugation (a). Individual fractions (pellets) may then be resuspended in a small volume of buffered solution and applied to a density gradient centrifugation step in order to further purify individual organelle fractions (b).

Figure 4.26 Typical protocol used to isolate the membrane fraction from E. coli cells.

Figure 4.27 Examples of non-ionic, ionic and zwitterionic detergents. The chemical detail of the polar head group is shown. The non-polar tail group is represented by ‘R’ in each case. Generally, non-ionic detergents disrupt protein–lipid interactions but not protein–protein interactions, in contrast to both ionic and zwitterionic detergents.

Figure 4.28 Transverse section of a lipid bilayer membrane, detergent-solubilized integral membrane protein and micelle as they would occur in an aqueous-based solution.

Figure 4.29 Molecular structure of Triton, sodium dodecyl sulfate (SDS), urea and guanidinium chloride.

Figure 4.30 Oxidation of (a) methionine and (b) cysteine side chains, as can occur on exposure to air or more potent oxidizing agents (e.g. peroxide, superoxide, hydroxyl radicals). Refer to text for details.

Figure 4.31 Molecular structure of some reducing agents commonly/potentially used in order to maintain a protein in a reducing environment.

Figure 4.32 Deamidation of asparagine and glutamine residues, yielding aspartic acid and glutamic acid, respectively.

Figure 4.33 Phase diagram of water, which describes the various phases in which water can exist as a function of pressure and temperature. Thus, at normal atmospheric pressure (1 atm, 101 kPa) it exists as a solid phase (ice) at temperatures below 0°C, as a gas above 100°C and as a liquid in between. However, at reduced atmospheric pressures (below 0.006 atm, 0.6 kPa) the water will sublime directly from solid to gaseous phase, without existing in an intervening liquid phase.

Figure 4.34 A laboratory-scale freeze drier.

Chapter 05

Figure 5.1 Flow diagram outlining the major steps constituting upstream and downstream processing of protein products.

Figure 5.2 Overview of the traditional approach to strain improvement classically pursued when developing microbial strains for the production of commercial proteins such as industrial enzymes. Refer to text for further detail.

Figure 5.3 Genome locations potentially altered by random mutagenesis and the possible consequences thereof. Refer to text for further detail.

Figure 5.4 The master cell bank/working cell bank system. For simplicity each bank contains only five ampoules. In reality, each bank would likely consist of several hundred ampoules. Working cell bank number 2 will be generated from master cell bank vial number 2 only when working cell bank number 1 is exhausted, and so on.

Figure 5.5 Outline of typical upstream processing stages involved in the production of a single batch of product. Initially, the contents of a single ampoule of the working cell bank (a) are used to inoculate a few hundred millilitres of media (b). After growth, this laboratory-scale starter culture is used to inoculate several litres to tens of litres of media present in a small bioreactor (c). This production-scale starter culture is used to inoculate the production-scale bioreactor (d). This process is equally applicable to prokaryotic or eukaryotic-based producer cell lines, although the bioreactor design, conditions of growth, etc. will differ in these two instances.

Figure 5.6 Schematic diagram of a generalized microbial fermenter.

Figure 5.7 Typical bioprocess-scale fermentation equipment. Photo of a 20 litre pilot scale/small bioprocess-scale fermentation unit (a). Photo of a process scale mammalian cell culture facility, housing a 1000 litre bioreactor (on the left) and a 200 litre scale bioreactor (on the right), (b). Photo (b) copyright FUJIFILM Diosynth Biotechnologies UK Ltd, www.fujifilmdiosynth.com

Figure 5.8 Conventional design of an animal cell bioreactor. The bioreactor displays several structural differences compared with microbial fermentation vessels (see Figure 5.6). Note in particular (i) the use of a marine-type impeller (some animal cell bioreactors – air lift fermenters – are devoid of impellers, and use sparging of air/gas as the only means of media agitation); (ii) the absence of baffles; and (iii) curved internal surfaces at the bioreactor base. These modifications aim to minimize damage to the fragile animal cells during culture. Many (mainly transformed) cells can grow in free submerged suspension in such vessels. However, other cell lines grow only when attached to a solid substratum. In such cases microcarrier beads (described later in the text) can be used.

Figure 5.9 A generalized outline of bulk protein production from microbial sources. Solid-state fermentation can be used to produce some extracellular proteins. Under such circumstances, the product is extracted from the solid culture media in an aqueous media, followed by its clarification (filtration or centrifugation). It is then concentrated if required and further processed as shown.

Figure 5.10 (a) A range of cartridge filters and (b) a range of filters and their stainless steel housings. In each case the Pleated filter is protected by an outer plastic supporting mesh. Photographs courtesy of Pall Corporation. Schematic (c) represents a cartridge filter in which filter sheets of three different pore sizes are housed.

Figure 5.11 Example of an ultrafiltration system used on an industrial scale. This system (SPM 180) comprises 16.7 m2 of spiral wound membrane. It can be used in ultrafiltration or diafiltration mode. Photograph courtesy of Amicon Ltd.

Figure 5.12 Schematic representation of the spray-drying process. The protein solution is atomized, forming tiny product droplets. Hot air/gas is fed into the system as shown, and travels concurrently with the atomized product down the length of the drying chamber. In this way the hottest driest air comes into contact with the wettest material. The product is dried by the air. The dried product collects at the base of the drying chamber, while the wet air/gas is removed as shown.

Figure 5.13 Schematic representation of the drum-drying process. The protein solution is fed into the system such that a thin layer coats the surface of the heated revolving drums. The heat evaporates off the solvent water, leaving a dried film of product which is scraped off by knives.

Figure 5.14 Generalized outline of the production of protein products destined for therapeutic or diagnostic application. Additional steps may be required to ensure complete removal of specific non-protein contaminants. Other chromatography types sometimes used include affinity chromatography or chromatofocusing. Final protein products are generally greater than 99% pure if destined for therapeutic application. Numerous quality control steps are also undertaken.

Figure 5.15 The drying chamber of an industrial-scale freeze-dryer. VirTis image is reproduced by permission of SP Scientific, www.SPScientific.com.

Figure 5.16 Generalized system by which potable water is purified in order to facilitate its use in biopharmaceutical processing. Water passed through cation and anion exchange columns is termed ‘purified’ or ‘deionized’ water. The generation of water for injection (WFI) entails a subsequent distillation or reverse osmosis step.

Figure 5.17 Schematic representation of HEPA-filtered air generation and flow through a clean room.

Figure 5.18 Structure of a generalized lipopolysaccharide (LPS) molecule. LPS consitutes the major structural component of the outer membrane of Gram-negative bacteria. Although LPS of different Gram-negative organisms differ in their chemical structure, each consists of a complex polysaccharide component linked to a lipid component. Refer to text for specific details.

Figure 5.19 Activation of clot formation by endotoxin. The presence of endotoxin causes stepwise sequential activation of various clotting factors present naturally within the amoebocytes of the American horseshoe crab. The net result is the generation of the polypeptide fragment coagulin, which polymerizes thus forming a gel or clot.

Chapter 06

Figure 6.1 Simplified version of the intrinsic and extrinsic blood coagulation pathways.

Figure 6.2 (a) Synthesis of factor VIII complex as occurs in healthy individuals. In the case of persons suffering from haemophilia A, synthesis of factor VIII:C is blocked (b), preventing constitution of an active factor VIII complex in plasma. Persons suffering from von Willebrand disease fail to synthesize vWF (c). Although they can synthesize VIII:C, this is rapidly degraded on entering the blood due to lack of its vWF stabilizing factor.

Figure 6.3 Overview of the likely mode of manufacture of ATryn, a recombinant antithrombin produced in the milk of transgenic goats.

Figure 6.4 Simplified representation of wound healing.

Figure 6.5 The fibrinolytic system, as triggered naturally by tissue plasminogen activator (tPA).

Figure 6.6 Overview of the production of Rapilysin, a modified tPA produced in recombinant E. coli cells. IB, inclusion body.

Figure 6.7 Overview of the manufacture of Cerevarix. The adjuvant used is MPL (monophosphoryl lipid A), a detoxified derivative of the lipopolysaccharide moiety of the Gram-negative bacterium Salmonella minnesota. Each dose of the final vaccine suspension contains 20 µg of both HPV type 16 and type 18 L1 capsid proteins.

Figure 6.8 Basic approach to developing recombinant vaccine vectors. The nucleotide sequence coding for most or all of a selected surface antigen on the target pathogen is identified and isolated. This is then introduced into the genome of a clinically safe virus (e.g. vaccinia). It is modified (e.g. by fusion with a viral gene coding for a viral surface protein) to ensure that the transferred gene product will be expressed on the viral cell surface.

Figure 6.9 The biosynthesis and degradation of L-asparagine.

Figure 6.10 Cleavage of DNA by DNase I. Hydrolysis generates two fragments, one with a free 3′ hydroxy terminus, the other with a free 5′ phosphate terminus.

Figure 6.11 The reaction catalysed by β-glucocerebrosidase.

Figure 6.12 Simplified schematic overview of the glycoengineering of β-glucocerebrosidase (a). Enzymatic remodelling of the sugar chain removes terminal sialic acids, i.e. derivatives of the monosaccharide neuraminic acid such as N-acetylneuraminic acid (Neu5Ac or NANA), as well as galactose (Gal) exposing mannose (Man) residues underneath. Macrophages are phagocytes responsible for engulfing bacteria (and other pathogens/cell debris, etc.), followed by lysosomal destruction of the phagocytosed material. Bacteria often contain mannose residues on their surface, hence it is unsurprising that macrophages have evolved to express a mannose receptor on their surface. The carbohydrate remodelling undertaken therefore exploits this pathway, allowing for uptake of the enzyme with subsequent lysosomal delivery (a). The actual carbohydrate remodelling is likely more extensive than described above, with a necessity in practice to remove not only terminal sialic acids but also Gal residues. A likely remodelling process is presented in (b). Fuc, fucose; GlcNAc, N-acetylglucose.

Chapter 07

Figure 7.1 IgG structure. Refer to text for details.

Figure 7.2 Prominent effector mechanisms by which antibody binding to an antigen on the surface of a target cell (e.g. a microbial cell, a cancer cell or a virally infected cell) can trigger destruction of that cell. In scenario (a) phagocytic cells such as macrophages and neutrophils, because they display antibody Fc receptors on their surface, can dock on the surface of an opsonized target cell (often a microorganism), and subsequently engulf (phagocytose) the target cell. Opsonization refers to the binding of antibodies or certain other immunological mediators to an antigen, marking the antigen for recognition by immune cells. In scenario (b) certain immune system cells, in particular natural killer (NK) cells, dock at the target cell surface, also via binding to the antibody Fc region, inducing the latter’s destruction via a process called antibody-dependent cell-mediated cytotoxicity (ADCC). This process often targets cancer cells and virally infected cells in the body. NK cells are cytotoxic lymphocytes that secrete proteins which insert themselves into the membrane of the target cell, forming a pore. This in turn lyses the cell. NK cells also secrete various serine proteases that are also capable of inducing cell death.

Figure 7.3 The proposed process of antibody recycling via endocytosis. Several aspects of the exact molecular mechanisms underpinning this overall process remain to be fully elucidated. Refer to text for further details.

Figure 7.4 Schematic overview of IgG–FcRn interaction. Refer to text for details.

Figure 7.5 Overview of a generalized production protocol used to enrich/purify polyclonal antibodies for therapeutic use.

Figure 7.6 Production of (a) chimeric and (b) humanized antibodies (via recombinant DNA technology). Chimeric antibodies consist of murine monoclonal VH and VL domains grafted onto the Fc region of a human antibody. Humanized antibody consists of murine CDR regions grafted into a human antibody.

Figure 7.7 Overview of the general approach undertaken to produce fully human monoclonal antibodies. Refer to text for further details.

Figure 7.8 Overview of the likely manufacturing scheme of the antibody product Herceptin.

Figure 7.9 The various antibody-mediated approaches that may be used to induce target cell destruction. Refer to text for details. TSA, tumour surface antigen.

Figure 7.10 Chemical structure of monomethyl auristatin E (MMAE), the antineoplastic agent conjugated to the CD30-directed monoclonal antibody Adcetris. Refer to text for further details.

Figure 7.11 Schematic of the bispecific antibody Removab, and the potential methods by which it may trigger the destruction of its tumour cell. One arm of the antibody binds the EpCAM antigen on the target cell surface, while the other binds the CD3 antigen found on the surface of T lymphocytes (cells which can mediate tumour destruction). Additionally, the Fc region of the antibody facilitates docking of various immune effector cells (e.g. phagocytes and NK cells) which can induce tumour cell destruction. Overall therefore the product may induce a twin tumoricidal effect.

Figure 7.12 Structure of two of the more common antibody fragments (ScFv and the ‘minibody’) which are produced by recombinant means. Refer to text for details.

Figure 7.13 Typical diantennary structure of the IgG glycocomponent, as attached to the antibody’s backbone at Asn297 of both heavy chains. The fucose (Fuc) residue discussed in the text is highlighted in bold.

Chapter 08

Figure 8.1 Synthesis of human insulin from preproinsulin. The initial proteolytic event involves removal of a 23 amino acid signal sequence from the amino-terminal end of preproinsulin, thus yielding proinsulin. Proinsulin is converted into insulin by additional proteolytic events, resulting in the generation of not only insulin but also a 30 amino acid sequence (termed C or connecting peptide) and two dipeptide moieties. Mature human insulin thus consists of two polypeptides, the A and B chains. The B chain contains 30 amino acid residues whereas the A chain contains 21 amino acids. The chains are covalently linked via two interchain disulfide linkages. One intrachain disulfide linkage is also present in the A chain.

Figure 8.2 A typical purification scheme for the production of traditional insulin preparations derived from the pancreatic tissue of slaughtered animals.

Figure 8.3 A likely purification scheme for human insulin produced via the ‘proinsulin’ route. Refer to text for further details.

Figure 8.4 Changes in plasma (a) FSH and (b) LH levels during the menstrual cycle of a healthy human female.

Figure 8.5 Overview of a typical scheme by which hCG may be purified from the urine of pregnant females.

Figure 8.6 Production of recombinant human growth hormone (rhGH) in E. coli. Subsequent to fermentation, the cells are collected by centrifugation or filtration. After homogenization (the product accumulates intracellularly), nucleic acids as well as some membrane constituents are precipitated by the addition of polyethylenimine. Ammonium sulfate precipitation of the supernatant concentrates the crude rhGH preparation. Chromatographic purification follows, as illustrated.

Figure 8.7 Major blood cell types/derivatives generated from haemopoietic stem cells. The differentiation of a stem cell into any single mature blood cell type is a complex multistage process, promoted by a cocktail of various growth and differentiation factors.

Chapter 09

Figure 9.1 Signal transduction mechanisms induced by type I (α and β) and type II (γ) interferons. Binding of the interferon to the appropriate receptor results in receptor dimerization, with subsequent activation of JAK-STAT pathways. Refer to text for specific details.

Figure 9.2 Outline of the manufacture of the IFN-α product Intron A. The interferon protein is expressed intracellularly in soluble form.

Figure 9.3 Overview of the manufacture of Betaferon, a recombinant human IFN-β produced in E. coli. Fermentation is achieved using minimal salts/glucose medium and product accumulates intracellularly in inclusion body (IB) form. During downstream processing the IBs are solubilized in butanol, with subsequent removal of this denaturant to facilitate product refolding. After two consecutive gel filtration steps, excipients are added, the product is filled into glass vials and freeze-dried. It exhibits a shelf-life of 18 months when stored at 2–8°C.

Figure 9.4 Schematic representation of the IL-2 receptor. Refer to text for details.

Figure 9.5 Activation of T cells by interaction with macrophage-displayed antigen. Activation results in IL-2 production, which acts in an autocrine manner to stimulate further T-cell growth and division. IL-2 thus represents the major regulatory molecule responsible for stimulation of cell-mediated immunity.

Figure 9.6 The production of platelets from bone marrow stem cells. Stem cells undergo differentiation under the influence of various combinations of cytokines, thereby producing red blood cells (erythrocytes), white blood cells (leukocytes) or platelets. This process is known as haemopoiesis. Thrombopoiesis (the production of platelets) is described here. Megakaryocytes are the immediate progenitor cells from which platelets are derived. Platelet production entails the budding off of small membrane-bound vesicles from the megakaryocyte surface. These vesicles (platelets) then enter circulation. A combination of cytokines serve to promote platelet production. The interleukins and granulocyte-macrophage colony stimulating factor (GM-CSF) appear to promote differentiation of earlier cells in the sequence, whereas thrombopoietin (TPO) mainly stimulates the final stages of platelet production. Characteristic cytokine redundancy likely results in several of these regulatory factors displaying overlapping activity in the context of platelet production. The scheme, as presented is likely an oversimplification of the actual process. IL, interleukin; CFU, colony-forming unit; BFU, burst-forming unit.

Figure 9.7 Three-dimensional structure of TNF-α. Image from the RCSB PDB (www.pdb.org) of PDB ID: 1TNF (M.J. Eck, S.R. Sprang (1989) The structure of tumor necrosis factor-alpha at 2.6 A resolution. Implications for receptor binding. J.Biol.Chem.264: 17595-17605).

Figure 9.8 Schematic overview of the production of Beromun, a recombinant human TNF-α product produced intracellularly in engineered E. coli cells.

Chapter 10

Figure 10.1 Some of the areas of speciality that constitute modern medical laboratories. Such laboratories may be located within a hospital or may be stand-alone, private testing companies. The exact organization and profile of testing undertaken will vary from laboratory to laboratory.

Figure 10.2 IVD sectors. Refer to text for specific details.

Figure 10.3 Major global IVD companies who, between them, account for almost two-thirds of global IVD sales. In terms of global market, the USA accounts for in the region of 45% of all sales, with Europe accounting for a further 30% of sales. Additional major IVD companies include Bayer Healthcare, LifeScan Inc. and Becton Dickinson.

Figure 10.4 Overview of optical and electrochemical approaches to analyte detection. Refer to text for details.

Figure 10.5 Detection and quantification of a specific analyte, A, by an enzyme-based end-point method. The enzyme, when incubated under appropriate assay conditions, completely or virtually completely converts A into B in a stoichiometric fashion. In this case, one molecule of A is converted into one molecule of B (a). The amount of substrate consumed or product formed is quantified (b). In this case, A absorbs (at the wavelength chosen) whereas B does not. The change in absorbance recorded can be directly related to analyte concentration by Beer’s law.

Figure 10.6 Generalized reaction in which an enzyme interconverts A + B and C + D. The equilibrium constant (K) value for the reaction is calculated according to the formula presented in (b). A reaction which, on reaching equilibrium, has almost completely converted A + B into C + D will display a high K value. If the reaction does not go to near completion, it can essentially be pulled to completion by addition of a chemical which reacts with one of the products (C or D). This is represented as F in diagram (c). In this example, D is removed almost as soon as it is formed, so its concentration continually remains low.

Figure 10.7 Generalized example of a calibration curve used to correlate reaction rate with analyte (i.e. substrate) concentration. In this case the analyte absorbs at 500 nm, whereas the reaction product does not. Assaying a sample containing an unknown concentration of analyte will yield a ΔA/10 s value, which can then be correlated with the analyte concentration by reference to the standard curve.

Figure 10.8 Generalized structure of a plasma lipoprotein.

Figure 10.9 Generalized schematic diagram of a biosensor. Refer to text for specific details.

Figure 10.10 Diagrammatic representation of an enzyme electrode.

Figure 10.11 Simplified overview of surface plasmon resonance (SPR)-mediated detection of antibody–antigen binding. Light of a fixed wavelength is directed at (and reflected from) a thin metal film as shown. Binding of the target analyte to the metal surface (via the immobilized antibody) slightly changes the refractive index of the surface layer. This in turn causes a reduction in intensity of the reflected light, which can be measured quantitatively by an appropriate detector.

Figure 10.12 The main immunoassay formats currently in use. Classical immunoassays, and to a limited extent agglutination-based assays, are typically undertaken in clinical chemistry laboratories. Membrane-based systems and biosensors tend to be used mainly for point-of-care testing or home use. Examples of all such systems/formats are considered in this chapter.