Tutorial Revision

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Department
Chemical and Biochemical Engineering
Course
Chemical and Biochemical Engineering 2290A/B
Professor
Mita Ray
Semester
Fall

Description
Revision Notes for CBE 2290 a (as discussed in the Tutorials on December 10th 2010). Insulin: Insulin is the sugar regulating hormone present in our body. It is made up of two chains of amino acids, Chain A and Chain B, hence is also a dipeptide hormone. How insulin helps our body :- Blood Glucose rises in the absence or malfunctioning of insulin, resulting in Diabe- tes. Frederik Banting and Charles best discovered insulin in 1922 Insulin was first extracted from pigs. However this insulin, when administered to humans caused severe side ef- fects. This was primarily because of the difference between the chemical structure of human insulin and porcine insulin. The Alanine amino acid in porcine insulin is replaced by threonine in Humans. The first genetically engineered , synthetic ―human insulin‖ was produced in the laboratory by Herbert boyer using E.Coli in 1977. Human insulin has 51 amino acids ; 21 amino acids in the A chain and 30 amino acids in the B chain. The two chains are connected to each other through disulfide linkages. Comparison of the A & B chains of insulin from different sources:- A Chain B Chain Ends with Ala Sheep —————Ala-Gly-Val——————— Ends with Ala Cow —————Ala-Gly-Val——————— Pig —————Thr-Ser-Ile——————— Ends with Ala Rabbit —————Thr-Ser-Ile——————— Ends with Ser Human —————Thr-Ser-Ile——————— Ends with Thr Production of Insulin in Body : Insulin is produced in pancreatic gland of the hu- man body. Beta islets of pancreas produce insulin in response to sugar levels in our body. Insulin is used to move the glucose obtained from food from the blood stream into cells throughout the body, which then use the glucose for energy. The beta cells produce insulin in three stages. Stage 1. Beta cells produce pro-insulin which has three chains Chain A (21amino acids), Chain C 30 amino acids, Chain B 30 amino acids. Proinsulin unlike insulin is a single polypeptide. Stage 2. Enzymatic removal of C-peptide structure (or C-Chain) from proinsulin Stage 3. Chain A & B bind to from Insulin (51 amino acids). What Happens when you eat? Some of the food in the stomach breaks down into sugars- one of these sugars is glucose, the body’s main fuel. Sugar enters the bloodstream, and the level of sugar in your beings to rise. When your body senses an increase in sugar it sends a signal to your processes. Insulin lowers the level of blood sugar by acting as a key to unlock the body’s cells and allowing sugar to pass from the bloodstream into the cells. The level of sugar in the bloodstream falls as the sugar passes into the cells. The body’s cells use the sugar for fuel. (See video for a better understanding of the concept). Four Major Methods of Insulin Production: - 1. Extracting from human insulin—IDEAL but not feasible 2. Chemical Synthesis - Possible by expensive 3. Porcine Pancreas –Commercially developed process 4. Recombinant DNA Technology / Genetically engineering process—Preferred Production of Insulin from Porcine Pancreas :- Step 1. Defrost frozen pancreas Step 2. Mince/Dice pancreas, submerge in Ethanol : aids extraction of hormone from the pancreatic cells Step 3. Adjust pH to 2 Step 4. Add Calcium Carbonate Step 2 &3 inactivate and precipitate trypsin. Trypsin inactivates insulin, hence removal critical Step 5. Concentrate extract by vacuum Step 6. Add salt to precipitate insulin Step 7. Re-dissolve in water Step 8. Re-precipitate at pH 5.3 (isoelectric point of insulin is 5.3). Step 9. Purify using chromatographic techniques (such as Gel filtration, ion exchange chromatography) Step 10. Convert to Human insulin. . Making Recombinant DNA to produce genetically engineered proteins:- Gene is part of the DNA sequence for coding a protein. 1. Restriction enzyme cut required gene from DNA sequence. Example of Restriction enzymes :- 2. Restriction enzymes recognize specific sites on the DNA EcoR1 (recognition site: GAATTC) 3. They produce sticky ends of DNA. BamH1 (recognition site: GGATCC) Hind III (recognition site: AAGCTT) 4. DNA ligase anneals the sticky ends. Transfer & Cloning of the Insulin Gene : Diagrammatic Representation Step 1. Clone the insulin gene into E.Coli. Step 2. Insert the cloned sequence into an antibiotic resistance gene sequence (This destroys the resistance) Step 3. Grow recombinant cells on media which contains the antibiotic cell is resistant to. This aids in identifying the cells that contain insulin gene. Step 4. Harvest the inclusion bodies that are produced. Note: inclusion bodies are aggregates of target protein. They contain denatured or primary structure of pro- tein. PRODUCTION OF INSULIN IN THE INDUSTRY USING RECOMBINANT DNA TECHNOLOGY RAW MATERIALS : Host Cell : E.Coli or S.Cerevisiae Plasmid : contains Trp-LÈ-Met-Pro-insulin gene and is inserted into E.Coli or suitable host cell. Trp-LÈ : promoter sequence which codes for the enzyme required for synthesis of amino acid trypto- phan Met– amino acid methionine STEP 1: Grow r-DNA cells in a bio- reactor Once the plasmid is generated, its Bioreactor inserted into the host cell and cultivated in a bioreactor. Sterilized nutrients, pH 7.0 Broth contains E.Coli r-DNA E.Coli in- cells, inclusion bodies, oculum, Sterile Air. 37˚C depleted nutrients. Mixing STEP 2: Harvest the E.Coli cells to obtain Inclusion bodies A) Broth from bioreactor is centrifuged to harvest the E.Coli cells. B) Cells are homogenize in a high pressure homogenizer (90kPa) and centrifuged to separate the inclusion bodies from the cell debris. C) Inclusion bodies are aggregates containing 80-90% Trp-LÈ-Met-pro-insulin STEP 3: Solubilise inclusion bodies A) Inclusion bodies from previous step are mixed well with Urea & 2-mercap
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