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The chloroplast of eukaryotic green algae, Chlamydomonas reinhardtii, is a relatively new and unexplored platform for the production of human therapeutics, vaccines, and human monoclonal antibodies. To accurately determine the viability of algae as a production platform, not only does the titer of correctly folded proteins have to be competitive to existing platforms (insect, yeast, plants, and bacteria) but the ease of downstream processing must also be taken into consideration. Two proteins expressed in the chloroplast of C. reinhardtii were studied, a malaria vaccine candidate (Plasmodium falciparum surface protein 25 (Pfs25)) and a single-chained antibody fragment ([lowercase Alpha symbol]CD22scFv). The first part of this study (Chapter II) had two objectives: (i) increase the accumulation of Pfs25 and (ii) characterize the Pfs25-aggregates. By suspending algal cells in fresh media after 5 days of growth, the accumulation of Pfs25 increased 1.7-fold. The Pfs25-aggregates were separated by size exclusion chromatography (SEC) and found to range in size from 25 kDa to >600 kDa; even in the presence of reducing agents and detergent the higher molecular (MW) aggregates remained intact. Due to the wide variances of Pfs25, evaluating the ease of downstream processing of algae with Pfs25 would prove to be difficult. Previously, our lab used [lowercase Alpha symbol]CD22scFv to compare three pretreatment methods (ammonium sulfate precipitation at pH 8.0, isoelectric precipitation at pH 4.5, and chitosan precipitation at pH 5.0) on residual chlorophyll, DNA, host cell protein, and [lowercase Alpha symbol]CD22scFv yield. However, the pretreatment methods were compared using anti-FLAG-affinity resin (a very specific yet highly expensive resin). Therefore, the second part of this study (Chapter III) used [lowercase Alpha symbol]CD22scFv to (i) test the effect of pretreatment methods on [lowercase Alpha symbol]CD22scFv adsorption to Capto Q and Phenyl Sepharose resins and (ii) develop a purification process resulting in >98% purity. Isoelectric precipitation at pH 4.5 and anion exchange chromatography (AXC) combination was determined to be the best combination for initial recovery and concentration of [lowercase Alpha symbol]CD22scFv. After isoelectric precipitation and AXC, anti-FLAG-affinity resin was the only resin scouted that showed a >98% [lowercase Alpha symbol]CD22scFv purity and 77% yield. The electronic version of this dissertation is accessible from http://hdl.handle.net/1969.1/155514
Fundamentals of Recombinant Protein Production, Purification and Characterization is organized into nine chapters in a logical fashion that cover an introduction to recombinant proteins and expression in different host expression systems, extraction, purification and analysis of proteins. This important reference features protocols, along with the advantages and disadvantage of each expression hosts and characterization technique (presented in tabular format) and offers detailed coverage of all aspects of protein production and processing (upstream and downstream processing) in one place. Finally, the book ends with different characterization techniques. Production of recombinant proteins for biotechnological and therapeutic applications at a large scale is an essential need of mankind. With the huge application potential of therapeutic and industrial proteins, there has been increasing demand for effective and efficient bioprocessing strategies. Recent progress around recombinant DNA technologies and bioprocessing strategies has paved the way for efficient production of recombinant proteins. Important factors such as insolubility and cost of production need to be considered for large scale production of these recombinant proteins. Includes step-by-step reproducible protocols while also providing updated information on the rationale and latest developments in expression systems Can also be used as a handbook for protein expression and purification as expression systems and chromatographic methods are explained in detail Consists of notes on troubleshooting from the eminent researchers in the field Provides comprehensive information on protein production, purification and characterization in a single volume Describes different purification methods for comparatively difficult to obtain proteins Brings the topics of recombinant protein expression, purification and characterization together, thereby making it the first resource on how to solve problems with respect to upstream and downstream processing of heterologous proteins
Advances in genetic engineering have enabled scientists to manipulate all three domains of life, and as a community we have utilized this knowledge to improve important platforms of biotechnology like agricultural crops. Now, photosynthetic microalgae present an opportunity to address several global problems including those concerning renewable energy, climate change, food and medicine. Presented here are several contributions to our understanding of the molecular genetics and biology of C. reinhardtii, and applications of these toward developing the species for recombinant therapeutic protein production. A review of the current methods of producing high-value recombinant proteins in microalgae is presented, along with a rapid screening method for revealing strains of C. reinhardtii capable of producing these proteins. The unique biochemical environment of the chloroplast is challenged to produce novel targeted anti-cancer immunotoxins that cannot be produced in existing expression systems. Additionally, the potential for edible microalgae to serve as vehicles for oral delivery of therapeutic proteins in enteric diseases is addressed. Finally, efforts toward improving C. reinhardtii as a platform for producing therapeutic proteins are presented in the form of several genetic toolsets. Fluorescent proteins were used to explore protein targeting within the cell, quantitate changes in gene regulation, and as a biomarker for generating improved strains with production platform qualities.
Human need for food and fuel has disturbed the balance of the biosphere thus triggering a catastrophe known as the Anthropocene Extinction. The use of microalgae in the biotechnology field offers multiple solutions that could alleviate the demand human activity imposes on the ecosystem. The most well studied microalga is the model organism Chlamydomonas reinhardtii, resulting in multiple genetic tools available in the alga for recombinant protein expression. However, the yields of recombinant protein expression are not high enough to be commercially viable, therefore this organism is usually not considered as a biotechnological host. To boost the recombinant protein productivity in Chlamydomonas reinhardtii two milestones need to be achieved: increased recombinant protein expression at single cell level, and increased number of cells per unit of culture volume. To accomplish higher transgene expression in C. reinhardtii the GAL4/UAS system was adapted into algal protein expression vectors. This system showed a 10-fold improvement in recombinant mRNA and protein accumulation of a reporter gene under the control of a chimeric promoter 5XUAS-AR1. To accomplish higher number of cells, or biomass, per unit of culture volume an optimized algal fed batch bioreactor was designed. Through media optimization we achieved a 1.67-fold improvement in biomass accumulation which in turn yielded a 3-fold improvement over the highest recombinant protein concentration reported in the literature using C. reinhardtii. Finally, an extremophile green alga from the Chlamydomonas genus was isolated from the wild and used to express recombinant GFP. Said extremophile showed robust growth in open ponds thriving in media at pH 11 while continuing to express recombinant protein for the duration of the experiment. These findings highlight the potential of Chlamydomonas reinhardtii to become a robust biotechnological host at commercial scale.
The subject of aluminium and Alzheimer's disease has been plagued with controversy. This controversy has served to obscure much of the scientific research in this field, and subsequently has obscured the possibility that aluminium is a contributory factor in the aetiology of Alzheimer's disease. This book brings together many of the world's leading scientists researching aluminium and life and contains their critical summaries on the known facts about aluminium toxicity in man and to offer an opinion on the implications of this knowledge on a link between aluminium and Alzheimer's disease. The subject areas of the chapters were chosen to reflect the myriad of ways that aluminium is known to impact upon mammalian physiology and function and range from clinical studies, through animal models of disease to the detailed biochemistry of aluminium toxicity. Chapters are also included on epidemiology and other factors involved in the aetiology of Alzheimer's.This is the first time that this subject has been treated in such a comprehensive manner. The research detailed in each chapter, includes the latest research in the field, it has been critically appraised and this appraisal has been used by each author to present an informed opinion of its relevance to aluminium and Alzheimer's disease. The chapters are much more than reviews; they are a statement of the state of the art and of what the future may hold for research in this field. As a whole they show the high quality of research that has been carried out in our efforts to understand the toxicity of aluminium in man and that we are far away from discounting the possibility that aluminium is a contributory factor in the aetiology of Alzheimer's disease.
Microalgae have been largely commercialized as food and feed additives, and their potential as a source of high-added value compounds is well known. Yet, only a few species of microalgae have been genetically transformed with efficiency. A better understanding of the mechanisms that control the regulation of gene expression in eukaryotes is therefore needed. In this book a group of outstanding researchers working on different areas of microalgae biotechnology offer a global vision of the genetic manipulation of microalgae and their applications.
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.
Many Microorganisms and some macro-organisms can live under extreme conditions. For example, high and low temperature, acidic and alkaline conditions, high salt areas, high pressure, toxic compounds, high level of ionizing radiation, anoxia and absence of light, etc. Many organisms inhabit environments characterized by more than one form of stress (Polyextremophiles). Among them are those who live in hypersaline and alkaline, hot and acidic, cold/hot and high hydrostatic pressure, etc. Polyextremophiles found in desert regions have to copy with intense UV irradiation and desiccation, high as well as low temperatures, and low availability of water and nutrients. This book provides novel results of application to polyextremophiles research ranging from nanotechnology to synthetic biology to the origin of life and beyond.
This Microbiology Monographs volume covers the current and most recent advances in genomics and genetics, biochemistry, physiology, and molecular biology of C. reinhardtii. Expert international scientists contribute with reviews on the genome, post-genomic techniques, the genetic toolbox development as well as new insights in regulation of photosynthesis and acclimation strategies towards environmental stresses and other structural and genetic aspects, including applicable aspects in biotechnology and biomedicine. Advancement in Chlamydomonas biology allowed new understandings in biotechnological and biomedical related aspects.
Protein Byproducts: Transformation from Environmental Burden into Value-Added Products deals with the added value of proteinaceous waste byproducts, discussing in detail the different sources of protein-rich byproducts, their extraction, recovery, and characterization. The book provides thorough insights into different protein modification techniques to extend the product portfolio using these waste byproducts. Divided between three main sections, the book covers various feedstock resources, such as animal-derived/plant-derived proteins, marine waste-derived proteins, protein extraction and recovery methods, and related technical issues including modification and conversion technologies for the production of high value bioproducts. It contains contributions from experts in the fields of applied industrial microbiology, engineering, bioprocess technology, protein chemistry, food chemistry, agriculture, plant sciences, environmental science, and waste management, serving as a comprehensive reference for students and research scientists in the food and agriculture industries. Covers various feedstock resources, protein extraction, recovery methods, and related technical issues Presents modification and conversion technologies for the production of high value bioproducts Exhibits case studies and examples to illustrate both driving forces and constraints in the utilization of these proteinaceous materials Contains contributions from experts in the fields of applied industrial microbiology, engineering, bioprocess technology, protein chemistry, food chemistry, agriculture, plant sciences, environmental science, and waste management Serves as a comprehensive reference for students and research scientists in the food and agriculture industries