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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.
Recombinant proteins are used for many industrial purposes including food processing, pharmaceuticals, and nutraceuticals. The use of Chlamydomonas reinhardtii, a type of microalgae, as a molecular farming platform has piqued research interests due to their edibility, photosynthetic capabilities, and eukaryotic cellular machinery, attributes that contribute to their potential to produce bioactive recombinant proteins in an economic and scalable way. However, several challenges remain before algae can be considered an industrial organism for expression of recombinant proteins. One area that will require additional improvement is the low product yield in transgenic lines, brought about by a lack of molecular tolls as well as efficient screening methods. To overcome these challenges, we developed a multi-cistronic nuclear transformation vector that utilizes an intracellular fluorescent protein reporter to facilitate the identification of transgenic strains with high expression levels, in a high-throughput fashion. This was made possible by establishing a linkage between an upstream fluorescent reporter gene and a downstream recombinant protein gene: a self-cleaving viral peptide was transcriptional fused between the two recombinant protein genes, allowing the fluorescent reporter to be produced separately from recombinant protein of interest. In this study we show the utility of this polycistronic vector and high-throughput screening method by first showing that both the upstream reporter and downstream protein of interest and produced in equal amounts over a range of expression levels. We then demonstrate that we can use the cassette and fluorescence-activated cell sorting to identify transgenic lines with increasing quantities of secreted recombinant proteins. We also show that mutagenesis can be used to increase the co-expression of FP reporter and RP from our vector after it is successfully transformed in C. reinhardtii.
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
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 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
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.
Chlamydomonas reinhardtii has emerged as a promising alternative host for recombinant protein expression. Despite its advantageous characteristics and low-cost production, its use is hampered by low expression levels of nuclear transgenes. In this thesis we test several strategies designed to reduce or overcome this limitation. As a result, on the base of a secreted fusion protein comprising a small growth factor and a reporter, the use of regulatory and stabilizing regions resulted in expression levels ranging from 1 to 100 μg /L of culture. We report the expression of a fully-assembled monoclonal antibody in Chlamydomonas nucleus, therefore, validating Chlamydomonas as a host for complex protein production. The cassettes and high throughput screenings developed emerge as innovative tools expanding the molecular toolbox available for Chlamydomonas nucleus. In addition, a scalable purification method to recover the target protein from culture medium has been developed and validated indicating a simple downstream processing for secreted recombinant protein production. Finally, we report that Chlamydomonas secreted components induce proliferation of murine fibroblasts and have a synergistic effect with supplied hEGF, unveiling the potential of extracellular components of Chlamydomonas for a variety of applications.
Over the past decade, the transient gene expression (TGE) technology platform has been actively pursued to produce a wide range of therapeutic proteins, monoclonal antibodies, and vaccines for mainly preclinical assessment, due to its short development times and low overall cost. This book updates the latest advances in the field, with focusing on systematic description of the technology from cell lines, cell culture conditions, vector construction, expression strategy, current protocols, optimisation of the procedure, and potential for clinical application. As a conclusion, the author foresees that therapeutic biopharmaceutics will be manufactured for clinical development using TGE technology in the near future because of its fast development time, good protein expression, acceptable quality of product and due to the progress which has been made in analytical methodology and process quality control. The objectives of this book are to summarise current TGE protocols, to describe optimisation of the technology through the latest advances, and to explore clinical applications of the technology. It gives the reader a good insight into the latest development and future application of the technology platform, including: The current protocols from small to large scale for different cells. Optimisation methods in construction designing, transfection procedures, and cell culture conditions. Overall quality of the product from the transient gene expression. Future clinical application of the technology platform.
Altogether, the biochemical, technical and economic limitations on existing proka- otic and eukaryotic expression systems and the growing clinical demand for complex therapeutic proteins have created substantial interest in developing new expression systems for the production of therapeutic proteins. To that end, plants have emerged in the past decade as a suitable alternative to the current production systems, and today their potential for production of high quality, much safer and biologically active complex recombinant pharmaceutical proteins is largely documented. The chapters in this volume, contributed by leaders in the field, sum up the state-- the-art methods for using a variety of different plants as expression hosts for phar- ceutical proteins. Several production platforms are presented, ranging from seed- and leaf-based production in stable transgenic plant lines, to plant cell bioreactors, to viral or Agrobacterium-mediated transient expr ession systems. Currently, antibodies and their derived fragments represent the largest and most important group of biote- nological products in clinical trials. This explains why the potential of most prod- tion platforms is illustrated here principally for antibodies or antibody fragments with acknowledged potential for immunotherapy in humans. In addition, a comparison of different plant expression systems is presented using aprotinin, a commercial phar- ceutical protein, as a test system. Although multiple books and monographs have been recently published on mol- ular pharming, there is a noticeable dearth of bench step-by-step protocols that can be used quickly and easily by beginners entering this new field.
Recombinant proteins have revolutionized the biomedical industry, providing therapeutics for life-threatening diseases and protein reagents for research applications. BioMarin Pharmaceutical Inc. develops recombinant protein therapeutics to treat rare diseases including lysosomal storage disorders (LSDs), a group of about 50 individually rare disorders together affecting 1 in 8,000 live births. With an increase in the number of novel therapeutics in our drug discovery pipeline, there is a high demand to produce a variety of recombinant proteins for early-stage drug development projects. In order to equip our protein production process with the tools and capability for diverse protein expression, it is valuable to expand our expression toolbox with high-expressing platforms. The goal of this project is to expand to our current expression platforms by developing a murine myeloma based expression system with SP2/0 cells as a host. Since the SP2/0 cell line is amongst the most commonly used cell lines for therapeutic and reagent protein production, developing a SP2/0 expression system may offer additional benefits to our recombinant protein production needs including: expression of difficultto-express proteins, improving titers, and extending recombinant cell line stability. A lysosomal enzyme therapeutic candidate is expressed in the SP2/0 cells as a proof-of-concept for developing this protein expression platform. To this end, we have shown that SP2/0 cells can be grown to a high density in commercially available serum-free media with a doubling time of less than twenty four hours. A clone isolation strategy was used to pick the top clone expressing high levels of recombinant protein. Using the highest expressing clone, we developed a high yielding bioprocess at a two liter scale to demonstrate the utility of this system for generating recombinant proteins at large scale. Furthermore, the therapeutic properties of the recombinant protein expressed in SP2/0 cells are similar to the recombinant protein expressed in Chinese hamster ovary (CHO) cell lines, demonstrating similar uptake into diseased cells (Kuptake values) and binding affinity to the receptor responsible for drug mediated cellular uptake. Thus, the SP2/0 expression system proves to be a valuable addition to our expression toolbox for the production of research-grade protein therapeutics for cell-based assays.