Production Of Recombinant Proteins

While the choices of microbial and eukaryotic expression systems for production of recombinant proteins are many, most researchers in academic and industrial settings do not have ready access to pertinent biological and technical information since it is normally scattered throughout the scientific literature. This book closes the gap by providing information on the general biology of the host organism, a description of the expression platform, a methodological section -- with strains, genetic elements, vectors and special methods, where applicable -- as well as examples of proteins produced with the respective platform. The systems thus described are well balanced by the inclusion of three prokaryotes (two Gram-negatives and one Gram-positive), four yeasts, two filamentous fungi and two higher eukaryotic cell systems -- mammalian and plant cells. Throughout, the book provides valuable practical and theoretical information on the criteria and schemes for selecting the appropriate expression platform, the possibility and practicality of a universal expression vector, and on comparative industrial-scale fermentation, with the production of a recombinant Hepatitis B vaccine chosen as an industrial example. With a foreword by Herbert P. Schweizer, Colorado State University, USA: "As a whole, this book is a valuable and overdue resource for a varied audience. It is a practical guide for academic and industrial researchers who are confronted with the design of the most suitable expression platform for their favorite protein for technical or pharmaceutical purposes. In addition, the book is also a valuable study resource for professors and students in the fields of applied biology and biotechnology."

An Increasing Number Of Recombinant Therapeutic Proteins Are Currently Being Developed, Tested In Clinical Trials And Marketed For Used. Most Of The Recombinant Therapeutic Proteins Are Being Successfully Produced Into Escherichia Coli And Pichia Pastoris Expression System. These Two Expression Systems Are Very Much Efficient And Cost Effective. This Book Takes A Close Look Of These Two Expression Systems And Fermentation Conditions, Purification Strategies Of Different Recombinant Proteins. This Book Also Discusses The Market Size And Cost Analysis For The Production Of Different Therapeutic Proteins And Some General Experimental Protocols For Production. Contents Part I: Recombinant Protein Expression Into Escherichia Coli And Fermentation Conditions; Chapter 1: Introduction; Chapter 2: Construction Of Efficient Expression Vector (Plasmid); Chapter 3: Factors Affecting Transcription, Promoters, Upstream Elements, Transcriptional Terminators, Transcriptional Antitermin, Tightly Regulated Expression Systems; Chapter 4: Mrna Stability; Chapter 5: Factors Affecting Translation, Mrna Translational Initiator, Translational Enhancers, Translational Termination; Chapter 6: Expression Of Target Protein And The Compartments Of Expression, Cytoplasmic Expression, Periplasmic Expression, Extracellular Secretion; Chapter 7: Fusion Proteins; Chapter 8: Post-Translational Protein Folding; Chapter 8: Codon Usage; Chapter 10: Protein Degradation; Chapter 11: Fermentation Conditions For High-Density Cell Cultivation (Hdcc), Growth Medium, Efficient Production Of Recombinant Protein In Hdcc, Nutrient Feeding Strategy In Hdcc; Chapter 12: One Examples Of Protein Production Using E. Coli Expression System; Chapter 13: Conclusion. Part Ii: Recombinant Protein Expression Into Yeast, Pichia Pastoris And Fermentation Conditions; Chapter 1: Introduction; Chapter 2: Why P. Pastoris? Chapter 3: Construction Of Expression Strains, Expression Vectors, Alternative Promoters, Host Strains, Methanol Utilisation Phenotype, Protease-Reduced Host Strains, Integration Of Expression Vectors Into The P. Pastoris Genome, Generating Multicopy Strains; Chapter 4: Post-Translational Modifications Of Secreted Proteins, Secretion Signal Selection, N-Linked Glycosylation; Chapter 5: Production Of Recombinant Proteins In Fermenter Cultures Of The Yeast, Pichia Pastoris, Conceptual Basis For The P. Pastoris Expression System, High-Level Expression In Fermenter Cultures, Protein-Specific Adjustments To Improve Yield, Glycosylation Of Recombinant Proteins, Secretion Signals; Chapter 6: One Examples Of Protein Producing Using P. Pastoris Expression System, Chapter 7: Conclusion. Part Iii: Purification Strategies For Recombinant Proteins; Chapter 1: Purification Of Proteins; Chapter 2: Conventional Chromatography, Ion Exchange Chromatography, Reversed Phase Chromatography, Gel Permeation Chromatography, Affinity Chromatography, Affinity Tags, Cleavage, Conclusion. Part Iv: Market Size And Cost Analysis For The Production Of Therapeutic Proteins; Chapter 1: Market Size Of Therapeutic Proteins; Chapter 2: Outline Structure Of A Productin Unit And Cost Analysis For The Production Of Three Therapeutic Proteins. Part V: General Experimental Protocols; Chapter 1: Different Experimental Protocols, Preparation Of Genome Dna For E. Coli, A Differnt Method For Preparation Of Genomic Dna From Bacteria, Preparation Of Proteins From Periplasm (Osmotic Shock Method), Preparation Of Proteins From Outer Membrane, Transformation Of Plasmid Dna Into E. Coli (Calcium Chloride/Heat Shock Method), Transformation Of Plasmid Dna Into E. Coli (Electroporation), Sds-Page For Large Proteins, Sds-Page For Small Peptide, Pcr Amplification Of Dna, Protein Quantification: Brandford Method, Trans-Bloting For Protein, Restriction Enzyme Digestion Of Dna, Phenol/Chloroform Extraction Of Dna, Ethanol Precipitation Of Dna, Agarose Gel Electrophoresis, Transformation Of E. Coli By Electroporation (Alternative Method), Wizard Tm Pcr Preps Dna Purification System For Rapid, Purification Of Dna Fragments, Alternate Method For Purifying Dna From Agarose Gels, Southern Blotting, Rt Pcr Protocol, Using Superscript Reverse Transcriptase, Preparation Of Sequencing Gels, Isolation Of Rna From Mammalian Cells Using Rnazoltm (Teltest), Preparation For Yeast Transformation, Yeast Transformation, Digesting Prsq-Ura3 With Bamhi, Genomic Dna Preparation Of Yeast, Ligation (Circularisation) Of Genomic Dna Fragments, E. Coli Transformation (Alternate Method), Dna Miniprep From E. Coli (Alternate Method), Basic Plasmid Dna Isolation Protocol, Identification And Determination Of Amount Rec-Hum Proteins Via An Immunoenzymatic Test (Elisa), Determination Of Host Dna Contaminant Into R Hu Protein Through Dot Blot Method, Protocols For Down-Stream Processing.

This book reviews preparation of expression vectors, generation of high-yielding clones, scale-up, disruption of yeast cells to enable isolation of recombinant protein prior to purification and more, in the popular Methods in Molecular Biology format."

More then 20 years have passed now since the first recombinant protein producing microorganisms have been developed. In the meanwhile, numerous proteins have been produced in bacteria, yeasts and filamentous fungi, as weIl as higher eukaryotic cells, and even entire plants and animals. Many recombinant proteins are on the market today, and some of them reached substantial market volumes. On the first sight one would expect the technology - including the physiology of the host strains - to be optimised in detail after a 20 year's period of development. However, several constraints have limited the incentive for optimisation, especially in the pharmaceutical industry like the urge to proceed quickly or the requirement to define the production parameters for registration early in the development phase. The additional expenses for registration of a new production strain often prohibits a change to an optimised strain. A continuous optimisation of the entire production process is not feasible for the same reasons.

Since newly created beings are often perceived as either wholly good or bad, the genetic alteration of living cells impacts directly on a symbolic meaning deeply imbedded in every culture. During the earlier years of gene expression research, te- nological applications were confined mainly to academic and industrial laboratories, and were perceived as highly beneficial since molecules that were previously unable to be separated or synthesized became accessible as therapeutic agents. Such were the success stories of hormones, antibodies, and vaccines produced in the bacterium Escherichia coli. Originally this bacterium gained fame among humans for being an unwanted host in the intestine, or worse yet, for being occasionally dangerous and pathogenic. H- ever, it was easily identified in contaminated waters during the 19th century, thus becoming a clear indicator of water pollution by human feces. Tamed, cultivated, and easily maintained in laboratories, its fast growth rate and metabolic capacity to adjust to changing environments fascinated the minds of scientists who studied and modeled such complex phenomena as growth, evolution, genetic exchange, infection, survival, adaptation, and further on—gene expression. Although at the lower end of the complexity scale, this microbe became a very successful model system and a key player in the fantastic revolution kindled by the birth of recombinant DNA technology.

This volume provides an overview of the main yeast production platforms currently used and future yeast cell factories for recombinant protein production. Chapters detail approaches of genetic and metabolic engineering, co-factor containing proteins and virus-like particles, glycoproteins, and post-translational modifications of proteins. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Recombinant Protein Production in Yeast: Methods and Protocols aims to provide state of the art background and methods for protein producing yeast platforms, as well as case studies for special applications.

With the advent of recombinant DNA technology, expressing heterologous proteins in microorganisms rapidly became the method of choice for their production at laboratory and industrial scale. Bacteria, yeasts and other hosts can be grown to high biomass levels efficiently and inexpensively. Obtaining high yields of recombinant proteins from this material was only feasible thanks to constant research on microbial genetics and physiology that led to novel strains, plasmids and cultivation strategies. Despite the spectacular expansion of the field, there is still much room for progress. Improving the levels of expression and the solubility of a recombinant protein can be quite challenging. Accumulation of the product in the cell can lead to stress responses which affect cell growth. Buildup of insoluble and biologically inactive aggregates (inclusion bodies) lowers the yield of production. This is particularly true for obtaining membrane proteins or high-molecular weight and multi-domain proteins. Also, obtaining eukaryotic proteins in a prokaryotic background (for example, plant or animal proteins in bacteria) results in a product that lack post-translational modifications, often required for functionality. Changing to a eukaryotic host (yeasts or filamentous fungi) may not be a proper solution since the pattern of sugar modifications is different than in higher eukaryotes. Still, many advances in the last couple of decades have provided to researchers a wide variety of strategies to maximize the production of their recombinant protein of choice. Everything starts with the careful selection of the host. Be it bacteria or yeast, a broad list of strains is available for overcoming codon use bias, incorrect disulfide bond formation, protein toxicity and lack of post-translational modifications. Also, a huge catalog of plasmids allows choosing for different fusion partners for improving solubility, protein secretion, chaperone co-expression, antibiotic resistance and promoter strength. Next, controlling culture conditions like temperature, inducer and media composition can bolster recombinant protein production. With this Research Topic, we aim to provide an encyclopedic account of the existing approaches to the expression of recombinant proteins in microorganisms, highlight recent discoveries and analyze the future prospects of this exciting and ever-growing field.

A single volume collection that surveys the exciting field of plant-made pharmaceuticals and industrial proteins This comprehensive book communicates the recent advances and exciting potential for the expanding area of plant biotechnology and is divided into six sections. The first three sections look at the current status of the field, and advances in plant platforms and strategies for improving yields, downstream processing, and controlling post-translational modifications of plant-made recombinant proteins. Section four reviews high-value industrial and pharmacological proteins that are successfully being produced in established and emerging plant platforms. The fifth section looks at regulatory challenges facing the expansion of the field. The final section turns its focus toward small molecule therapeutics, drug screening, plant specialized metabolites, and plants as model organisms to study human disease processes. Molecular Pharming: Applications, Challenges and Emerging Areas offers in-depth coverage of molecular biology of plant expression systems and manipulation of glycosylation processes in plants; plant platforms, subcellular targeting, recovery, and downstream processing; plant-derived protein pharmaceuticals and case studies; regulatory issues; and emerging areas. It is a valuable resource for researchers that are in the field of plant molecular pharming, as well as for those conducting basic research in gene expression, protein quality control, and other subjects relevant to molecular and cellular biology. Broad ranging coverage of a key area of plant biotechnology Describes efforts to produce pharmaceutical and industrial proteins in plants Provides reviews of recent advances and technology breakthroughs Assesses realities of regulatory and cost hurdles Forward looking with coverage of small molecule technologies and the use of plants as models of human disease processes Providing wide-ranging and unique coverage, Molecular Pharming: Applications, Challenges and Emerging Areas will be of great interest to the plant science, plant biotechnology, protein science, and pharmacological communities.