Medical Biotechnology Overview

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.