ism (i. e. , Saccharomyces carlsbergensis, or brewer's yeast) and one of its corresponding enzymes. The experiments on this organism and enzyme are not limited to the materials suggested and can be easily adapted to the desired technical level and available budget. Similarly, the subse- quent cloning experiments suggest that use of particular vectors and strains, but, as indicated, alternative materials can be used to success- fully perform the laboratory exercises. We would like to thank the corporate sponsors of the Biotechnology Training Institute for providing the materials and expertise for the devel- opment of our programs, and thus for the materials in this manual. These sponsors include: * Barnsteadffhermolyne, Dubuque, IA * Beckman Instruments, Somerset, NJ * Bio-Rad Laboratories, Hercules, CA * Boehringer Mannheim Corporation, Indianapolis, IN * Coming Costar Corporation, Cambridge, MA * FMC BioProducts, Rockland, ME * Kodak Laboratory Products, New Haven, CT * Labconco, Kansas City, MO * MJ Research, Cambridge, MA * Olympus Instruments, Lake Success, NY * Pharmacia Biotech, Piscataway, NJ * Savant, Inc. , Farmingdale, NY * VWR Scientific, Philadelphia, P A We would also like to thank the following individuals for their input, comments, and suggestions: Tom Slyker, Bernie Janoson, Steven Piccoli, John Ford,JeffGarelik, Yanan Tian, and Douglas Beecher. Special thanks to Alan Williams for his critique of the chromatography experiments and Shannon Gentile for her work in the laboratory. We would especial- ly like to thank Maryann Burden for her comments and encouragement.

Peptide therapy has become a key strategy in innovative drug development, however, one of the potential barriers for the development of novel peptide drugs in the clinic is their deficiencies in clearly defined chemistry, manufacturing and controls (CMC) strategy from clinical development to commercialization. CMC can often become a rate-limiting step due to lack of knowledge and lack of a formal policy or guidelines on CMC for peptide-based drugs. Regulators use a risk-based approach, reviewing applications on a case-by-case basis. Peptide Therapeutics: Strategy and Tactics for Chemistry, Manufacturing, and Controls covers efficient manufacturing of peptide drug substances, a review of the process for submitting applications to the regulatory authority for drug approval, a holistic approach for quality attributes and quality control from a regulatory perspective, emerging analytical tools for the characterisation of impurities, and the assessment of stability. This book is an essential reference work for students and researchers, in both academia and industry, with an interest in learning about CMC, and facilitating development and manufacture of peptide-based drugs.

Protein engineering endeavors to design new peptides and proteins or to change the structural and/or functional characteristics of existing ones for specific purposes, opening the way for the development of new drugs. This work develops in a comprehensive way the theoretical formulation for the methods used in computer-assisted modeling and predictions, starting from the basic concepts and proceeding to the more sophisticated methods, such as Monte Carlo and molecular dynamics. An evaluation of the approximations inherent to the simulations will allow the reader to obtain a perspective of the possible deficiencies and difficulties and approach the task with realistic expectations. Examples from the authors laboratories, as well as from the literature provide useful information.

The intent of this work is to bring together in a single volume the techniques that are most widely used in the study of protein stability and protein folding. Over the last decade our understanding of how p- teins fold and what makes the folded conformation stable has advanced rapidly. The development of recombinant DNA techniques has made possible the production of large quantities of virtually any protein, as well as the production of proteins with altered amino acid sequence. Improvements in instrumentation, and the development and refinement of new techniques for studying these recombinant proteins, has been central to the progress made in this field. To give the reader adequate background information about the s- ject, the first two chapters of this book review two different, yet related, aspects of protein stability. The first chapter presents a review of our current understanding of the forces involved in determining the conf- mational stability of proteins as well as their three-dimensional folds. The second chapter deals with the chemical stability of proteins and the pathways by which their covalent structure can degrade. The remainder of the book is devoted to techniques used in the study of these two major areas of protein stability, as well as several areas of active research. Although some techniques, such as X-ray crystallography and mass spectroscopy, are used in the study of protein stability, they are beyond the scope of this book and will not be covered extensively.

The first volume in this Methods Molecular Biology series, Proteins (1984), concentrated on basic techniques for the analysis and purification of peptides and proteins. As the series developed, more specialized volumes on proteins were introduced, such as those on Immunochemical Protocols (vol. 10), Practical Protein Chro- tography (vol. 11), Analysis Glycoprotein Biomedicine (vol. 14), Protein-DNA Interactions (vol. 30), Biomembrane Protocols (vols. 19 and 27), Analyses and Methods (vol. 17), and Optical Spectroscopy, Microscopy, and Macroscopic Techniques (vol. 22). Further specialist volumes on peptides, monoclonal antibodies, immunoassays, ELISA, protein engineering, protein stability, mass spectrometry of proteins, automated sequence analysis, and protein NMR are currently in preparation. Since it is now a decade since the initial volume was published, it seems an especially appropriate moment to extensively reorganize, update, and revise the earlier volume. In an attempt to be more c- prehensive in our coverage, this current volume, Basic Protein and Peptide Protocols, is totally committed to basic analytical methods; a planned companion volume will later concentrate on preparative techniques. Those analytical techniques requiring expensive speci- ized instrumentation, such as NMR, mass spectrometry, X-ray cr- tallography, spectroscopy, and automated sequence analysis, are not described here, but in the appropriate specialized volumes listed above.

Why a Second Edition?
The Second Edition provides practical answers to the general question, "How can I obtain useful sequence information from my protein or peptide?" rather than the more specific question asked in the first edition, "How can I obtain the N-terminal sequence?" Important new methods include ways of dealing with blocked N termini, computer analysis of protein sequences, and the recent revolution in mass spectrometry.
Key Features
Highlights of the Second Edition:
* Mass spectrophotometric characterization of proteins and peptides
* N-terminal sequencing of proteins with blocked N termini
* Internal amino acid sequence analysis after protease digestion in-gel and on-blot
* Improved microscale peptide purification methods
* Computer analysis of protein sequences
* New protocols tested and refined through everyday use in authors' laboratories
* Updated reference chapter covering all aspects of protein microsequencing

An intimate relation between hormones and lipoprotein metabolism has been known for a long time especially from hormone-deficiency or -overproduction syndromes. The mechanisms through which hormones influence lipid metabolism have become a field of major interest in sci ence. The more we learn about cellular hormonal actions the better we understand regulatory processes and phenomena occuring in patients. The present book summarizes data discussed at an International Meet ing at Marburg, Germany, on "Hormones in Lipoprotein Metabolism." It was an attempt to pull together knowledge in basic science from the mode of action of hormones all the way to clinical appearance of hor monal disorders involving lipoprotein metabolism. Thus data on molec ular biology, on hormonal regulation of apolipoprotein synthesis, on lipoprotein receptors and enzyme induction are discussed together with the large field of oral contraceptive use, postmenopausal estrogen sub stitution, lipid disorders in diabetes mellitus, in thyroid dysfunction, in adrenal insufficiency and in glucocorticoid application. We are aware of the fact that such an overview cannot be complete but should serve as a collection of data and ideas for those interested in hormonal regulation of lipid metabolism. We thank all authors for taking an extra effort in writing up their presentations and thus making this edition possible. We also thank Bristol-Myers-Squibb for generous support of the meeting and of the final publication. Marburg, Germany ARMIN STEINMETZ JURGEN SCHNEIDER HANS KAFFARNIK Contents Molecular Biology Chairmen: D. J. Rader, A."

One of the phylogenetically oldest hormones, somatostatin is a regulatory peptide with remarkable characteristics. It is a nonclassical neurotransmitter discovered less than 20 years ago both in the central nervous system and in the gastroenteropancreatic system. It regulates the secretion of both pituitary and digestive hormones, it ensures nutrient homeostasis and it has therapeutic uses. This volume deals withall these aspects.

A Safety Considerations Many techniques described here involve a number of hazards, such as high electrical current and voltage, radioactivity and highly toxic chemicals. It is absolutely essential that the instructions of equipment manufacturers be followed, and that particular attention be paid to the local and federal safety regulations. B Introduction The expression of prokaryotic and eukaryotic genes has been shown most often to be regulated at the level of mRNA synthesis. Thanks to the rapid development of methods for dissecting DNA sequences, cis-acting regulatory elements such as promoters and enhancers have been recognised. More recently, the widely expressed intuition that discrete sequences within these elements constitute binding sites for sequence-specific binding proteins has been confirmed, especially through the use of "footprinting" assays (for examples, Galas and Schmitz, 1978). This and similar assays have already resulted in the recognition, isolation and analysis of DNA-bind ing proteins for several genes. Excellent reviews exist of the structural studies on these transcription regulatory proteins and related DNA elements (for example, Glover, 1989 and Johnson and McKnight, 1989), to which the reader is referred for detailed information. To set the scene for applications of the techniques described in this volume, only the barest outline of previous studies is presented here. Protein-DNA interactions are dependent on very specific tertiary configurations of the binding protein which allow the closest contact with the DNA helix.

There is increasing evidence for the clinicial value of the apo lipoprotein measurements. Besides cholesterol in plasma and li poprotein fractions, which is currently used as an indicator of cardiovascular risk, the measurement of the AI and B apolipopro teins can provide additional information about the patients' clinical status. Several studies show that apo B is higher and apo AI is lower in patients with angiographically documented coronary heart dis ease than in symptomatic patients without coronary heart disease. Moreover, discriminant analysis indicated that the concentration of Apo AI and B in plasma are better discriminators than lipo protein cholesterol for identifying patients with coronary heart disease. In some studies the apo Bjapo AI ratio appears to be a more powerful predictor than individual lipoproteins. In a recent study carried out in men, apolipoproteins AI and B were better correlated with the severity of cardiovascular disease than HDL and LDL cholesterol. The predictive power of apolipoproteins could however not be demonstrated in all studies and the value of apolipoprotein measurements in the field of clinical chemistry is still controversial. This is probably due to discrepancies between the results of various studies, arising from differences in the type of immunoassays, the lack of universal reference materials, differ ences between study protocols, variations in the selection of patients and in the grading and interpretation of coronary lesions."

In recent years there has been a tremendous increase in our understanding of the functioning of the cell at the molecular level. This has been achieved in the main by the invention and development of new methodology, parti- larly in that area generally referred to as "'genetic en- neering." While this revolution has been taking place in the field of nucleic acids research, the protein chemist has at the same time developed fresh methodology to keep pace with the requirements of present day molecular bi- ogy. Today's molecular biologist can no longer be content with being an expert in one particular area alone. He/she needs to be equally competent in the laboratory at h- dling DNA, RNA, and proteins, moving from one area to another as required by the problem he/she is trying to solve. Although many of the new techniques in molecular biology are relatively easy to master, it is often difficult for a researcher to obtain all the relevant information nec- sary for setting up and successfully applying a new te- nique. Information is of course available in the research l- erature, but this often lacks the depth of description that the new user requires. This requirement for in-depth pr- tical details has become apparent by the considerable - mand for places on our Molecular Biology Workshops held at Hatfield each summer.

Since the publication of the bestselling second edition of John Walkera (TM)s widely acclaimed Protein Protocols Handbook, there have been continual methodological developments in the field of protein chemistry. This greatly enhanced third edition introduces 57 critically important new chapters, as well as significantly updating the previous edition's tried-and-true methods. Although the timely new chapters are spread throughout all of the book, the vital section on post-translational modifications has been expanded most to reflect the increasing importance of these modifications in the understanding of protein function.

Each readily reproducible method follows the highly praised format of the Methods in Molecular Biologya"[ series, offering a concise summary of its basic theory, a complete materials list, a step-by-step protocol for its successful execution, and extensive notes on avoiding pitfalls, or on modifying the method to function within your own experimental circumstances. The expert authors of each chapter have demonstrated a hands-on mastery of the methods described, fine-tuned here for optimal productivity.

Comprehensive, cutting-edge, and highly practical, The Protein Protocols Handbook, Third Edition is today's indispensable benchtop manual and guide, not only for all those new to the protein chemistry laboratory, but also for those established workers seeking to broaden their armamentarium of techniques in the urgent search for rapid and robust results

This book reviews the principles of design and examples of successful implementation of proteinkinase inhibitors (PKI), and offers a comprehensive and authoritative overview of the history and latest developments in the field. Chapters written by experts from industry and academia cover the function, structure and topology of Proteinkinases, molecular modelling, disclose how to achieve high level of selectivity for kinase inhibitors, and exploit kinase inhibitors for cancer treatment. Particular attention is given to Inhibitors of c-Jun N-terminal kinase 3, and to covalent Janus Kinase 3 Inhibitors. A case study on Receptor Tyrosine Kinases EGFR, VEGFR, PDGFR is also presented in this book. Given its breath, this book will appeal to medicinal chemists, students, researchers and professionals alike.

The elucidation of the ultrastructure, cytochemistry and reactivity of cutaneous recep- tors is of particular importance in the unsolved biological problem of sensory transduc- tion. Topographically and with regard to evolution, cutaneous receptors are the most widespread primary sense organs. Because of their simple structure they are particular- ly useful in the study of the functional morphology of receptor end-organs. Research into receptor structures in classic neuromorphology revealed magnificent patterns and pictures of receptors, showing their great diversity and their presence in all human and animal systems. Since the end of the sixties, ultrastructural studies have led to the discovery of a large amount of new information. The findings concerning the cellular organization of the inner core (pease and Quilliam, 1957), axon terminals (Cauna and Ross, 1960) and capsule cells (Polacek and Mazanek, 1966) of encapsulated receptors have opened up new possibilities for a revaluation of receptor cell units and their relationships.

This text offers in-depth perspectives on every aspect of protein structure identification, assessment, characterization, and utilization, for a clear understanding of the diversity of protein shapes, variations in protein function, and structure-based drug design. The authors cover numerous high-throughput technologies as well as computational methods to study protein structures and residues. A valuable reference, this book reflects current trends in the effort to solve new structures arising from genome initiatives, details methods to detect and identify errors in the prediction of protein structural models, and outlines challenges in the conversion of routine processes into high-throughput platforms.

While the genomic revolution has quickly led to the deposit of more than 30,000 structures in the protein data bank (PDB), less than one percent of those contributions represent membrane proteins despite the fact that membrane proteins constitute some 20 percent of all proteins. This discrepancy becomes significantly troublesome when it is coupled with the fact that 60 percent of current drugs are based on targeting this group of proteins, a trend that does not seem likely to reverse. Structural Genomics on Membrane Proteins provides an excellent overview on novel research in bioinformatics and modeling on membranes, as well as the latest technological developments being employed in expression, purification, and crystallography to obtain high-resolution structures on membrane proteins. This cutting-edge work also explains the difficulties facing researchers-both technical and ethical-that have slowed the process. Structural Genomics on Membrane Proteins provides researchers with an unprecedented look at the novel technologies that will ultimately allow them to conquer the last frontier in structural biology, leading to accelerated breakthroughs in drug discovery.

The handling and analysis of data generated by proteomics investigations represent a challenge for computer scientists, biostatisticians, and biologists to develop tools for storing, retrieving, visualizing, and analyzing genomic data. Informatics in Proteomics examines the ongoing advances in the application of bioinformatics to proteomics research and analysis. Through computer simulations, scientists can determine more about how diseases affect cells, predict how various drug interventions would work, and ultimately use proteins as therapeutic targets. This book first addresses the infrastructure needed for public protein databases. It discusses information management systems and user interfaces for storage, retrieval, and visualization of the data as well as issues surrounding data standardization and integration of protein sequences recorded in the last two decades. The authors subsequently examine the application of statistical and bioinformatic tools to data analysis, data presentation, and data mining. They discuss the implementation of algorithms, statistical methods, and computer applications that facilitate pattern recognition and biomarker discovery by integrating data from multiple sources. This book offers a well-rounded resource of informatic approaches to data storage, retrieval, and protein analysis as well as application-specific bioinformatic tools that can be used in disease detection, diagnosis, and treatment. Informatics in Proteomics captures the current state-of-the-art and provides a valuable foundation for future directions.

This monograph is neither a historical outline of the development of the concepts of protein biosynthesis and the structure and functions of the ribosomes, nor an exhaustive survey of the literature on these questions. The monograph is based upon an analysis of the modern trends in this field. The purpose of the monograph was to formulate more or less generalized representations of the structure and" function of the ribosome, as we envision it at the present day. It may be that this attempt is premature for a number of reasons, and the concepts outlined here will very soon be revised. Nonetheless, despite this risk, we believe it to be advisable to undertake this attempt for the following reasons: firstly, the undertaken analysis could aid in the comprehension of the substantial mass of extremely scattered experimental data on the ribosomes presently available; secondly, in any event, even if most of the concepts outlined rapidly become obselete, they can still serve as a stimulus for a whole series of experiments; and thirdly, we hope that some of the concepts outlined will still remain essentially correct and relatively stable. In view of the aforementioned, we should make the following reservations. First of all, we made no attempt to cite all the literature on the problems discussed, but considered it sufficient to illustrate the various premises with one or several sample references.

This highly readable textbook serves as a concise and engaging primer to the emerging field of antibody engineering and its various applications. It introduces readers to the basic science and molecular structure of antibodies, and explores how to characterize and engineer them. Readers will find an overview of the latest methods in antibody identification, improvement and biochemical engineering. Furthermore, alternative antibody formats and bispecific antibodies are discussed. The book's content is based on lectures for the specializations "Protein Engineering" and "Medical Biotechnology" within the Master's curriculum in "Biotechnology." The lectures have been held at the University of Natural Resources and Life Sciences, Vienna, in cooperation with the Medical University of Vienna, since 2012 and are continuously adapted to reflect the latest developments in the field. The book addresses Master- and PhD students in biotechnology, molecular biology and immunology, and all those who are interested in antibody engineering.

This textbook introduces readers in an accessible and engaging way to the nuts and bolts of protein expression and engineering. Various case studies illustrate each step from the early sequence searches in online databases over plasmid design and molecular cloning techniques to protein purification and characterization. Furthermore, readers are provided with practical tips to successfully pursue a career as a protein engineer. With protein engineering being a fundamental technique in almost all molecular biology labs, the book targets advanced undergraduates and graduate students working in molecular biology, biotechnology and related scientific fields.

This is the first serious attempt to synthesize all that became known of glutathione over the last three decades. The book contains an update of glutathione biosynthesis with special emphasis on its regulation in adaptive stress responses. Other chapters review glutathione transport systems and glutathione peroxidases and their differences in substrate specificities and localization. Further contributions center on the diversified roles of different glutathione-S-transferases and the roles of nitrosoglutathione and glutaredoxins - a subfamily of redoxins. The book closes with discussions of the analogous or homologous thiol metabolism in pathogens and the potential suitability of involved enzymes as drug targets. Key selling features: Summarizing the way glutathione is involved in stress responses Compiling the multiple ways glutathione affects inflammatory responses Disclosing how glutathione dampens programmed cell death such as ferroptosis Exploring the enigma of how enzymes accelerate glutathione-dependent processes Discussing how detoxification and redox regulation is mediated by glutathionylation Reviewing the ways glutaredoxins catalyze protein disulfide reduction Highlighting the medical impact of glutathione-related metabolic pathways Illustrating the role thiol metabolism of pathogens might play in drug discovery

"Introducing Proteomics" gives a concise and coherent overview of every aspect of current proteomics technology, which is a rapidly developing field that is having a major impact within the life and medical sciences.

This student-friendly book, based on a successful course developed by the author, provides its readers with sufficient theoretical background to be able to plan, prepare, and analyze a proteomics study.

The text covers the following: Separation TechnologiesAnalysis of Peptides/Proteins by Mass SpectrometryStrategies in Proteomics

This contemporary text also includes numerous examples and explanations for why particular strategies are better than others for certain applications. In addition, "Introducing Proteomics" includes extensive references and a list of relevant proteomics information sources; essential for any student.

This no-nonsense approach to the subject tells students exactly what they need to know, leaving out unnecessary information. The student companion site enhances learning and provides answers to the end of chapter problems.

"I think this book will be a popular and valuable resource for students and newcomers to the field who would like to have an overview and initial understanding of what proteomics is about. The contents are well organized and address the major issues."
--Professor Walter Kolch, Director, Systems Biology Ireland & Conway Institute, University College Dublin

Companion Website
www.wiley.com/go/lovric

This book reviews the principles of design and examples of successful implementation of proteinkinase inhibitors (PKI), and offers a comprehensive and authoritative overview of the history and latest developments in the field. Chapters written by experts from industry and academia cover the function, structure and topology of Proteinkinases, molecular modelling, disclose how to achieve high level of selectivity for kinase inhibitors, and exploit kinase inhibitors for cancer treatment. Particular attention is given to Inhibitors of c-Jun N-terminal kinase 3, and to covalent Janus Kinase 3 Inhibitors. A case study on Receptor Tyrosine Kinases EGFR, VEGFR, PDGFR is also presented in this book. Given its breath, this book will appeal to medicinal chemists, students, researchers and professionals alike.