Protein Expression and Purification Series - Bio-Rad

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CHAPTER 1 BACKGROUND Protein Expression and Purification Series Choice of Cell Type The biotechnology industry uses several cell types, both prokaryotic (bacteria) and eukaryotic (animal, fungi, plant), to synthesize recombinant proteins. The choice of the host cell depends on the protein expressed. Bacteria can express large amounts of recombinant protein, but the expressed proteins sometimes do not fold properly. In addition, bacterial cells cannot carry out the post-translation modifications that are characteristic of some of the proteins made by eukaryotic cells. The most important post-translation modification is glycosylation, the covalent addition of sugar residues to the amino acid residues making up the protein. Glycosylation can change the structure and thus affect the activity of a protein. Many mammalian blood proteins are glycosylated, and the addition of these sugars often changes the rate of turnover (half-life) of the protein in the blood, because proteins that are misfolded will be quickly degraded. If glycosylation is important for the function of the protein, mammalian cells are the cell type of choice, but these cells produce less protein and are more expensive to grow. In the early days of the biotechnology industry Escherichia coli (E. coli) was the bacterial host of choice. This species had been used as the primary experimental system to study bacterial genetics for decades. More was known about the molecular biology of E. coli than any other species, and many genetic variants were available. In addition E. coli grows quickly, can reach high cell concentrations, and can produce large quantities of a single protein. It is also relatively inexpensive to grow. Today E. coli remains the bacterial system of choice, and many companies produce recombinant proteins using this bacterial species. Insulin, the first protein produced by genetic engineering, was produced in E. coli. Blockbuster products like human growth hormone and granulocyte colony stimulating factor (which increases white cell production in cancer chemotherapy patients) are also produced using this bacterial species. In general, if a protein’s properties allow it to be produced in bacteria, then E. coli is the system of choice. For recombinant protein expression in lower eukaryotic cells, two yeast species are commonly used: Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris). S. cerevisiae is the yeast species used to make bread, wine and beer. Baker’s yeast is used in research laboratories as a model system to study the genetics of eukaryotic cells. P. pastoris is a yeast species initially discovered by the petroleum industry. It divides rapidly, grows to a very high cell density, and can produce large quantities of a single protein. In addition, it can be genetically engineered to secrete the protein into the surrounding medium to allow easier recovery. Both species can glycosylate proteins, although the glycosylation patterns may differ from mammalian patterns. The sugars that are added to the protein and their position on the amino acid chain may differ between yeast and mammalian cells. Despite these advantages, relatively few biotech companies use yeast as a production system. The exceptions are the vaccine that immunizes against hepatitis B virus and the vaccine Guardasil that immunizes against HPV, the human papillomavirus. If a protein has a very large and complex structure, or if that protein requires glycosylation to be active, then the protein must be produced in a mammalian cell line. Chinese hamster ovary cells (CHO) is the cell line that is almost always used. CHO cells bear relatively little resemblance to the cells of the hamster from which they were derived in the 1950s; they have adapted to growth in cell culture medium. Cell lines are established when cells from a multicellular organism are separated from one another by a protein-digesting enzyme and grown as if they are really a unicellular organism. The cells require a rich medium that provides them with all of the amino acids, vitamins, and growth factors that they need to grow. This complexity means that mammalian growth medium is many times more expensive than the media used to grow either bacterial or yeast cells. The CHO cell lines can be adapted for growth in suspension culture. The CHO cell lines are most often engineered to synthesize the protein of interest on the ribosomes attached to the rough endoplasmic reticulum, to package and glycosylate the protein in the Golgi apparatus, and to eventually secrete the protein into the extracellular medium where it is easier to purify. CHO cells can glycosylate proteins with a mammalian glycosylation pattern. If glycosylation is important 34 Chapter 1: Recombinant Protein Expression & Purification
Protein Expression and Purification Series to the function of the protein, CHO cells should be used. Because CHO cells divide slowly, the production runs are much longer than with E. coli (on the order of weeks rather than days). All equipment and all growth media must be scrupulously sterilized. A single contaminating bacterial cell will overgrow the culture and will lead to that batch being discarded. Although the growth of CHO cells takes longer, uses expensive media, and presents a greater risk of contamination, the isolation of the proteins that these cells produce is usually easier than in bacterial or yeast cells. Interferon beta provides a good example of how the end product influences the choice of expression system for recombinant proteins. Avonex, the human interferon beta-1a form produced in CHO cells, is glycosylated; while Betaseron, the human interferon beta-1b form produced in E. coli, is not glycosylated. Since glycosylation is important for interferon beta-1a function, it is produced in CHO cells. Table 1.2. Advantages and disadvantages of using bacteria, yeast and mammalian cells to produce recombinant proteins. Parameter Bacteria Yeast Mammalian Contamination risk Low Low High Cost of growth medium Low Low High Product titer (concentration) High High Low Folding Sometimes Probably Yes Glycosylation No Yes, but different pattern Full Relative ease to grow Easy Easy Difficult Relative ease of recovery Difficult Easy Easy Deposition of product Intracellular Intracellular or extracellular Extracellular Product Intracellular Often secreted into media Secreted Table 1.3. Examples of pharmaceutical products and the cell line used to produced them. Product Cell Line Insulin Escherichia coli Human growth hormone Escherichia coli Granulocyte colony stimulating factor Escherichia coli Tissue plaminogen activator CHO cells Pulmozyme (DNase) cystic fibrosis CHO cells Erythropoietin induces red blood cell production CHO cells Hepatitus B virus vaccine Yeast Human papillomavirus vaccine Yeast Rituxan rheumatoid arthritis, non-hodgkins lymphoma, leukemia CHO cells Herceptin breast cancer CHO cells Choice of Plasmid Once the cell type has been chosen, the plasmid or vector to express the protein needs to be selected. Different plasmids are used for expression of proteins in bacteria, yeast and higher eukaryotic cells. Some features that need to be considered in the plasmid include: selection system (such as antibiotic resistance), the promoter, the copy number of the plasmid, presence of signal peptide sequence to excrete the expressed protein out of the cell, presence of sequence coding for a protein purification tag or DNA coding for fusion protein partners. Antibiotic resistance is a common component of both prokaryotic and eukaryotic vector systems. The presence of a gene for antibiotic resistance allows for selective retention of the plasmid and suppression of growth of any cells that do not contain the plasmid. However, to ensure safety for expression systems being used for vaccine and therapeutic protein production, other selection systems can be used such as Chapter 1: Recombinant Protein Expression & Purification 35 CHAPTER 1 BACKGROUND
Page 1: Biotechnology Explorer TM Protein E
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CHAPTER 3B ADVANCE PREP INSTRUMENTA
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CHAPTER 4 11 ml CULTURE PROTOCOL Pr
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CHAPTER 5 100 ml CULTURE PROTOCOL P
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CHAPTER 6 HANDPACKING COLUMNs Prote
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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
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CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
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APPENDIX A TIPS AND HELPFUL HINTS P
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APPENDIX A TIPS AND HELPFUL HINTS P
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APPENDIX B RESULTS ANALYSIS Protein
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APPENDIX B RESULTS ANALYSIS Protein
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APPENDIX C RCF TO RPM CONVERSTION P
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APPENDIX D SETTING UP THE SMARTSPEC
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APPENDIX D SETTING UP THE SMARTSPEC
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APPENDIX E MINI-PROTEAN GEL BOX SET
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APPENDIX F BIOLOGIC LP SYSTEM SETUP
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APPENDIX F BIOLOGIC LP SYSTEM SETUP
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APPENDIX G BIOMANUFACTURING Protein
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APPENDIX G BIOMANUFACTURING Protein
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APPENDIX H FOCUS QUESTIONS INSTRUCT
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APPENDIX I GLOSSARY Protein Express
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APPENDIX I GLOSSARY Protein Express
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APPENDIX J REFERENCES, LEGAL NOTES
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Life Science Group Bulletin 1665067
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Protein Expression and Purification Series - Bio-Rad

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