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What is the general process of making a therapeutic protein?
Results from an improved understanding of the disease or the discovery of the potential of a protein, forming a hypothesis on a protein and its therapeutic potential against a disease, design, preparation, and experimentation to test the hypothesis and seek supporting evidence
Describe the steps in preparing a therapeutic protein
Obtain gene sequence information from the National Center for Biotechnology Information website, gene sequence preparation through polymerase chain reaction based on gene amplification, design primers based on reverse transcription-based DNA amplification, clone the gene into gene expression plasmid, produce the therapeutic protein
What methods are available to produce recombinant proteins?
Production using microbial reactors, mammalian cell-derived bioreactors, animal bioreactors "pharming", plants
How is the E. coli expression system used to produce proteins?
Most common microbial species to produce heterologous proteins of therapeutic interest, heterologous protein: protein that does not occur in host cells
What are the advantages and disadvantages of the E. coli expression system?
Advantages: serves as a model system for prokaryotic genetics because its molecular biology is well characterized, high-level expression of heterologous proteins, easy/simple process: rapid growth, simple inexpensive media, fermentation technology established for large scale; Disadvantages: E. coli cells do not have signal peptide for protein secretion so there is intracellular accumulation of proteins in the cytoplasm, accumulation of partially folded proteins into inclusion bodies, cannot undertake post-translational modification, complicated purification procedure needed to remove lipopolysaccharide from expressed protein
Define inclusion body
Bacteria can have a variety of small bodies called inclusion bodies, contain aggregated heterologous proteins that are not properly folded, heterologous proteins in the inclusion bodies can be refolded into proper structure with proper procedure like treatment of high concentration urea with rapid dialysis, prevention of inclusion body formation is possible by growing bacteria at lower temperatures or including a fusion partner or a high-level co-expression of molecular chaperones
How is the yeast expression system used to produce proteins?
Molecular biology is well characterized with a long history of industrial applications, fast growth in inexpensive media, outer cell wall protects them from physical damage, suitable industrial-scale fermentation equipment/technology is already available, post-translational modifications of proteins are present, especially glycosylation
What are the advantages and disadvantages of the yeast expression system?
Advantages: well-characterized molecular biology, long history of industrial applications, fast growth in inexpensive media, outer cell wall protects from physical damage, suitable industrial-scale fermentation technology available, post-translational modifications including glycosylation; Disadvantages: glycosylation pattern differs from the pattern observed in the native glycoprotein, low expression level of proteins (<5%)
What are examples of yeast systems in therapies?
Major therapeutic proteins produced in yeast for general medical use include insulin, colony-stimulating factor, and hirudin
How is the mammalian cell expression system used to produce proteins?
Suitable for production of glycoproteins, especially glycosylation
What are the advantages and disadvantages of using the mammalian cell expression system to produce proteins?
Advantages: suitable for production of glycoproteins, especially glycosylation; Disadvantages: very complex nutritional requirements, slow growth rate and long cultivation time, far more susceptible to physical damage
What are the advantages and disadvantages of using the insect cell-based system to produce proteins?
Advantages: use of a strong promoter derived from the viral polyhedron results in high-level intracellular protein expression (~30-50% of total intracellular protein), cultivation at high growth rate and less expensive media than mammalian cell lines, no infection of human pathogens (e.g., HIV); Disadvantages: low level of extracellular secreted target protein, incomplete and different glycosylation patterns, most commonly used for lab-scale protein production, no therapeutic protein approved so far for human use
How is the transgenic animal system used to produce proteins?
Direct microinjection of exogenous DNA into an egg cell, stable integration of the target DNA into the genetic complement of the cell, after fertilization, the ova are implanted into a surrogate mother, transgenic animal harbors a copy of the transferred DNA; Other approaches: use of replication-defective retroviral vectors for consistent gene delivery and chromosomal integration, use of nuclear transfer technology (e.g., transgenic sheep Polly and Molly producing human blood factor IX in the 1990s)
What are the advantages and disadvantages of using the transgenic animal system to produce proteins?
Advantages: goats and sheep are attractive host systems, high milk production capacities (700-800 L/year for goats), ease of handling and breeding, ease of harvesting crude product via milking, pre-availability of commercial milking systems with maximum process hygiene, low capital investment and running costs, high expression levels of proteins (>1 g protein/L milk), ease of downstream processing due to well-characterized properties of major native milk proteins; Disadvantages: variability of expression levels (1 mg/L ~ 1 g/L), different post-translational modifications (especially glycosylation) from human patterns, significant time lag between the generation of a transgenic embryo and routine product recovery, gestation period of 1 to 9 months, requirement of successful breeding before lactation, overall time lag of 3 years for cows and 7 months for rabbits, no therapeutic proteins produced in transgenic animal milk approved for general medical use
What other transgenic animals can be used for developing protein therapies?
Rabbits: cheaper housing costs and shorter generation time; Chickens: recombinant drugs produced in their eggs by the Roslin Institute
What are the advantages and disadvantages of using the transgenic plant system?
Advantages: low cost of plant cultivation, inexpensive and well-established harvest equipment/methodologies, ease of scale-up, proteins expressed in seeds are generally stable, plant-based systems are free of human pathogens (e.g., HIV); Disadvantages: variable/low expression levels of proteins, potential occurrence of post-translational gene silencing (a sequence-specific mRNA degradation mechanism), different glycosylation pattern from humans, seasonal/geographical nature of plant growth
How are therapeutic proteins purified?
Size and density: centrifugation, size chromatography; Charges: ion exchange column; Hydrophobicity: hydrophobic column chromatography; Isoelectric points: precipitation; Affinity to a substance: affinity chromatography; Sensitivity to salt concentration: salt out
What is the general process of preparing a candidate protein?
Obtain gene coding sequence, amplify gene of interest, clone gene into a plasmid for protein expression, transfer the gene expression vector into E. coli, yeast, mammalian cells, or transgenic animals/plants, large-scale production of therapeutic protein, purification and characterization
What methods are commonly used to evaluate the activity of a candidate therapeutic protein?
Cell culture system: effects on cell growth, effects on activation of a biochemical signal pathway; Diseased animal model: effects on disease development; Clinical evaluation: assessment of improvement of pathological indicators
