Productn of Insulin, HGH - 2014

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Transcript of Productn of Insulin, HGH - 2014

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    Production of Human Insulinusing

    rDNA Technology

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    Expression vectorsTo produce the product of a cloned gene for further studies

    1) Expression vectors with a strong promoter

    More mRNA More protein

    2) Expression vectors with an inducible promoter

    Foreign proteins when overexpressed could be toxicKeep the gene expression off till it is time to turn it on

    a. Drug-inducible (e.g. IPTG or arabinose)b. Heat-inducible

    3) Expression vectors with a fusion tag for affinity purificationFacilitate the purification of the expressed protein

    1) 6 Histidine tag2) Glutathione transferase tag (GST)3) Maltose-binding protein tag

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    The pharmaceutical industry produces a wide range ofproducts using recombinant DNA techniques

    Human proteins can becloned

    Gene recovered fromhuman genome (as

    genomic DNA or cDNA) Placed in an expression

    vector using ligationtechniques andrestrictionendonucleases

    Transform bacteria (e.g.Escherichia coli orBacillus spp .)

    Grow in batch culture inlarge industrial scalevats

    Purify protein product

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    Fusion Tags

    Tag Purification Commercial vectors

    6-10 his tag Ni column (IMAC)immobilized metal affinitychromatography

    pPRO, pBAD, PET

    GST(Glutathione STransferase)

    glutathione

    MBP (maltose binidngprotein

    Maltose column

    Tags can be removed by including a protease cleavage site: Factor Xa, thrombin,enterokinase

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    Therapeutics

    Human proteins produce fewer side effectsthan proteins from other animals (e.g. porkinsulin vs. human insulin)

    Hormones or hormone-like compounds areused for human therapy

    Enzymes Antisense RNA

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    A. Insulin

    Made by pancreatic beta cells Enables cells to take up glucose from the bloodstream to

    use in production of ATP Insufficient insulin causes diabetes (insulin dependent

    diabetes mellitus IDDM) Must inject insulin to avoid physiological complications Cells cannot take up glucose Insufficient ATP is made Glucose spills into urine (excreted by kidneys; kidney

    tries to dilute glucose by excreting large amounts ofwater)

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    Complications of diabetes (60 x 106

    worldwide cases) Retinopathy Kidney failure

    Nerve disorders Circulatory diseases (including gangrene and stroke) Diabetic coma (pH imbalance caused by fat metabolism

    producing ketones)

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    Frederick Sanger

    In 1958 british molecular biologistFrederick Sanger determined theprimary structure of insulin. Itwas the first protein to have itssequence be determined. Hewas awarded the Nobel Prize inChemistry for his work.

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    Structure of Insulin

    Insulin is made up of 2 chains = 51 amino acids total A chain = 21 amino acids B chain = 30 amino acids

    S - S __l____________l____ A

    S S ___S___________S____________________ B

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    Two disulfide bonds hold A and B together (interchain disulfide bond) One disulfide bond within the A chian (intrachai disulfide bond) Insulin processing

    Preproinsulin Contains a signal sequence + 3 sections of amino acids

    Signal sequence is removed after targeting to RER Translation continues on RER > forming proinsulin

    Removal of 33 amino acids at Golgi Apparatus, and joining of A and B chains to form insulin

    Preproinsulin too difficult forE. coli to produce Simplification had to be made!

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    Recombinant expression of Insulin

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    Before recombinant insulin was available, insulin wasobtained from cows or pigs pancreases (7-10 lb pancreatictissue per patient per year)

    Cow (Bovine) = 3 amino acid differences Pig (Porcine) = 1 amino acid difference Amino acid differences can stimulate allergic responses

    Therefore human insulin is preferred

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    B. Human Growth Hormone (HGH)

    HGH promotes overall body growth by increasing: amino acid uptakeby cells, protein synthesis and fat utilization for energy Dwarfism caused by insufficient production of HGH by the pituitary

    gland Growth retardation

    Chubby face Unusual body properties as an adult ~ 4 feet tall only IQ = Normal

    HGH can treat dwarfism to help undersized children reach theirnormal height and size

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    Recombinant Expression of Human growth hormone

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    Old method

    Purification of HGH from cadaver pituitary glands 8 cadavers/year for 8 10 years per patient

    Risky to use brain tissue Prion disease transmission: Creutzfeldt-Jacob Disease (CJD)

    Muscle wasting Convulsions Tremors

    Dementia 24 cases reported by 1993 in France from cadaver HGH

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    New method

    Gene production for HGH synthesis Protropin Genentech Humatrope Eli Lilly

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    Recombinant Expression of Hepatitis B

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