productn of insulin, hgh - 2014
TRANSCRIPT
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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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