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What is the primary function of insulin in the body?
What is the primary function of insulin in the body?
Which of the following biopharmaceuticals was the first therapeutic product produced using recombinant DNA technology?
Which of the following biopharmaceuticals was the first therapeutic product produced using recombinant DNA technology?
What percentage of the general population is affected by diabetes mellitus?
What percentage of the general population is affected by diabetes mellitus?
Why did early administration of animal insulin pose challenges for diabetic patients?
Why did early administration of animal insulin pose challenges for diabetic patients?
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What are the components of human insulin in terms of amino acid chains?
What are the components of human insulin in terms of amino acid chains?
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What complications are associated with uncontrolled diabetes?
What complications are associated with uncontrolled diabetes?
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What is one reason for using recombinant DNA technology in the production of insulin?
What is one reason for using recombinant DNA technology in the production of insulin?
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What is considered the root cause of diabetes mellitus?
What is considered the root cause of diabetes mellitus?
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What is the primary role of human growth hormone (hGH) in the body?
What is the primary role of human growth hormone (hGH) in the body?
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What condition is associated with insufficient hGH in young children?
What condition is associated with insufficient hGH in young children?
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Which method was traditionally used to treat children with pituitary dwarfism?
Which method was traditionally used to treat children with pituitary dwarfism?
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What molecular challenge was faced with recombinant technology in hGH production?
What molecular challenge was faced with recombinant technology in hGH production?
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What is the purpose of cutting the base sequence encoding the signal peptide in hGH production?
What is the purpose of cutting the base sequence encoding the signal peptide in hGH production?
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How many amino acids does the human growth hormone consist of?
How many amino acids does the human growth hormone consist of?
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Which bacterial species is typically used for the culture and production of biologically functional hGH?
Which bacterial species is typically used for the culture and production of biologically functional hGH?
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What component of the hGH synthesis process is removed during secretion to release active hGH?
What component of the hGH synthesis process is removed during secretion to release active hGH?
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What is the primary role of reverse transcriptase in the production of synthetic insulin?
What is the primary role of reverse transcriptase in the production of synthetic insulin?
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Why is plasmid chosen as a preferred vector for insulin production?
Why is plasmid chosen as a preferred vector for insulin production?
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Which host organism is commonly used for synthetic insulin production due to its inability to perform post-translational modification?
Which host organism is commonly used for synthetic insulin production due to its inability to perform post-translational modification?
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What is the function of specific ligases in the recombinant DNA process?
What is the function of specific ligases in the recombinant DNA process?
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What is the significance of including a bacterial promoter in the expression vector for insulin production?
What is the significance of including a bacterial promoter in the expression vector for insulin production?
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Which media is primarily used for the cultivation of Escherichia coli in synthetic insulin production?
Which media is primarily used for the cultivation of Escherichia coli in synthetic insulin production?
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Which raw material is directly responsible for producing cDNA from mRNA in the insulin production process?
Which raw material is directly responsible for producing cDNA from mRNA in the insulin production process?
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What is a key disadvantage of using bacterial cells for insulin production compared to yeast?
What is a key disadvantage of using bacterial cells for insulin production compared to yeast?
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What is the primary purpose of lysing the cells in downstream processing?
What is the primary purpose of lysing the cells in downstream processing?
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Which of the following methods is NOT used for lysis of cells?
Which of the following methods is NOT used for lysis of cells?
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What substance is used to cleave the protein chains at methionine residues?
What substance is used to cleave the protein chains at methionine residues?
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What type of chromatography is used at the end of the downstream processing to obtain highly purified insulin?
What type of chromatography is used at the end of the downstream processing to obtain highly purified insulin?
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Which two agents are used for the formation of disulfide bonds between insulin chains?
Which two agents are used for the formation of disulfide bonds between insulin chains?
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What is the key difference between the Chain A and Chain B process and the Proinsulin process?
What is the key difference between the Chain A and Chain B process and the Proinsulin process?
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What is added towards the end of the insulin manufacturing process to ensure its longevity?
What is added towards the end of the insulin manufacturing process to ensure its longevity?
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What type of bonds connect the A and B chains in the final insulin structure?
What type of bonds connect the A and B chains in the final insulin structure?
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Which components are present in the LB media used for insulin production?
Which components are present in the LB media used for insulin production?
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What is the role of lactose in the fermentation broth?
What is the role of lactose in the fermentation broth?
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Which method involves creating a single-chain precursor before cleaving it to produce insulin?
Which method involves creating a single-chain precursor before cleaving it to produce insulin?
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What happens to bacterial cells that do not successfully transform?
What happens to bacterial cells that do not successfully transform?
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What is β-galactosidase's role in the insulin production process?
What is β-galactosidase's role in the insulin production process?
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During the large scale fermentation, which factors need to be monitored for optimal bacterial growth?
During the large scale fermentation, which factors need to be monitored for optimal bacterial growth?
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What is the purpose of adding ampicillin to the fermentation broth?
What is the purpose of adding ampicillin to the fermentation broth?
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What is the significance of the triplet codon for methionine in the context of insulin gene transcription?
What is the significance of the triplet codon for methionine in the context of insulin gene transcription?
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Study Notes
Medical Biotechnology
- Biopharmaceuticals are protein-based products derived from genetically altered bacteria or fungi (also called biotech drugs).
- Examples include recombinant human insulin, human growth hormone, and other similar products.
- Recombinant DNA technology modifies Escherichia coli bacteria to produce human insulin.
- Human insulin, marketed as Humulin, was the first therapeutic product produced using recombinant DNA technology.
- Human insulin consists of 51 amino acids arranged in two polypeptide chains (A and B).
- Chain A has 21 amino acids, and chain B has 30 amino acids.
- Both chains are held together by disulfide bonds.
- Diabetes mellitus affects about 2-3% of the general population, characterized by increased blood glucose concentration (hyperglycemia).
- Insufficient or inefficient insulin production causes diabetes.
- Insulin facilitates the uptake and utilization of glucose for energy release.
- High blood glucose concentrations (usually exceeding 180 mg/dl) lead to glucose excretion through urine.
- Serious diabetic complications include kidney damage (nephropathy), eye damage (retinopathy), nerve diseases (neuropathy), and circulatory diseases (atherosclerosis).
Problems with Animal Insulin
- Early insulin treatments used animal insulin (isolated and purified from pigs and cows).
- Slight amino acid differences between animal and human insulin caused allergic reactions in some diabetics.
- Large numbers of animals needed for extracting insulin (e.g., 70 pigs for one year's supply for one diabetic patient).
Raw Materials
- Raw materials are the basic components for producing synthetic insulin.
- Recombinant plasmid preparation is a crucial part of the process.
- Isolate the human insulin gene from human DNA; often, mRNA encoding insulin is used (pancreatic B cells produce lots of mRNA).
- mRNA is used to make c-DNA (complementary DNA) of the insulin gene.
Vectors
- Plasmids are the most common vectors for producing insulin in bacterial host cells.
- Plasmids replicate independently, leading to higher yields.
- Plasmids carry foreign DNA using the bacterial replicating system.
- Plasmids usually include selective markers for eliminating undesired cells.
- Crucial to include a bacterial promoter sequence in front of the insulin gene (expression vector) for bacteria to produce the protein of interest.
Specific Enzymes
- Reverse transcriptase synthesizes c-DNA from mRNA templates, essential for insertion into the vector.
- Specific restriction enzymes cut DNA at specific sites within the vector for the desired insulin gene insertion.
- Specific ligases join the DNA fragments once the gene is inserted.
Fermentation Process
- Escherichia coli bacteria are commonly used as the host organism for producing synthetic insulin because they lack post-translational modification.
- Post-translational conversion of the translated protein into insulin needs to be done chemically.
- Post-translational modification in eukaryotes (e.g., yeast) simplifies the process.
- LB medium containing Bacto-tryptone, yeast extract, sodium chloride, dH2O, and pH 7.5 supports bacterial growth.
- Ampicillin and lactose in the LB medium select for transformed bacteria (contain ampicillin resistance gene and lac Z gene).
Methods of Producing Insulin
- Two methods exist for producing insulin through genetic recombination:
- Method A: Individual production of 'A' and 'B' insulin chains, followed by chemical combination.
- Method B: Creating a single-chain precursor (proinsulin) and then cleaving a 35-amino acid peptide to separate the 'A' and 'B' chains.
Downstream Processing
- Isolating crude products from tanks involves enzyme digestion, freezing/thawing, and sonication.
- Lysosome enzymes digest bacterial membranes.
- Detergents further aid in separating the cell wall membrane.
- Purifying recombinant insulin chains from impurities using chromatographic methods like gel filtration and ion exchange, and exploiting differences in hydrophobicity to remove impurities.
- Fusion with β-galactosidase: Protein chains are fused because there's no termination/disruption in the synthesis.
- Cyanogen bromide cleaves the protein at methionine residues allowing insulin chains to be isolated.
Synthesis of Active Insulin
- Two chains (A and B) form disulfide bonds using sodium dithionate and sodium sulfite.
- Reduction-reoxidation reactions (using beta-mercaptoethanol and air oxidation) create the disulfide bonds, yielding Humulin (synthetic human insulin).
Purified Insulin Production
- PR-HPLC (reverse-phase high-performance liquid chromatography) further purifies insulin by removing remaining impurities.
Human Growth Hormone (hGH)
- Produced by the pituitary gland, hGH regulates growth and development by increasing cellular uptake of amino acids and protein synthesis.
- Insufficient hGH in children causes pituitary dwarfism.
- Traditionally, hGH was extracted from deceased human brains.
Limitations in hGH Production
- hGH has 191 amino acids, tagged with a 26-amino acid signal peptide.
- Original hGH process needs signal peptide removal after synthesis in the body.
- Recombinant DNA technology struggles to remove the signal peptide in E. coli.
- Bio-tech resolved the signal peptide problem by cutting cDNA encoding the signal peptide (and its neighbours) using the restriction enzyme EcoRI.
- The modified cDNA now produces hGH without the signal peptide.
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Description
Explore the fascinating world of medical biotechnology, focusing on biopharmaceuticals like recombinant human insulin and human growth hormone. Learn about the role of recombinant DNA technology in producing these vital drugs and understand the impact of diabetes mellitus on glucose metabolism. This quiz covers key concepts and applications of biotechnology in medicine.
