Download - Insulin & human growth hormone by E.coli
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Insulin & Insulin & human growth human growth hormonehormone by E.coliby E.coli
Edited by Prof. Dr. Sudjadi
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DAFTAR PUSTAKADAFTAR PUSTAKA
BR GLICK & JJ PASTERNAK MOLECULAR BIOTECHNOLOGY
DJA CROMMELIN & RD SINDELAR PHARMACEUTICAL BIOTECHNOLOGY
Sudjadi, Bioteknologi Kesehatan
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OverviewOverview Part A rDNA Objectives Steps in rDNA Processing Genetic Engineering Advances Part B Genomic library
– Plasmid– Restriction enzim, ligase
Part C The Use of rDNA to Produce Human
Insulin and Human Growth Hormon
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Genetic engineering plays a very important role, not only in scientific research, but also in the diagnosis and treatment of disease.
PART A
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rDNA is NOT Whole Animal rDNA is NOT Whole Animal CloningCloning
Recombinant DNA is a tool in Recombinant DNA is a tool in understanding the structure, understanding the structure, function, and regulation of function, and regulation of genes and their productsgenes and their products
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The objectives of Recombinant DNA technology include: – Identifying genes– Isolating genes– Modifying genes– Re-expressing genes in other hosts or
organisms
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These steps permit scientists and clinicians to:– Identify new genes and the proteins
they encode– To correct endogenous genetic
defects– To manufacture large quantities of
specific gene products such as hormones, vaccines, and other biological agents of medical interest
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Process ExampleProcess Example
THINK ABOUT THIS ?What is DNA Ligase Used for?What is a Restriction Enzyme?
Why Are Plasmid’ Used as Cloning Vectors?
Production Human Insulin in E.coli
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Part BPart B
Genomic library
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Prinsip kloning
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PEMOTONGAN HpaI
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PEMOTONGAN EcoRI
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Cloning VectorsCloning Vectors A vector is used to amplify a single molecule
of DNA into many copies. A DNA fragment must be inserted into a cloning vector. A cloning vector is a DNA molecule that has an origin of replication and is capable of replicating in a bacterial cell.
Most vectors are genetically engineered plasmids or phages. There are also cosmid vectors, bacterial artificial chromosomes, and yeast artificial chromosomes.
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PENAPISANPENAPISAN
KOMPLEMENTASIPELACAK DNAANTIBODI
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Genetic engineering has Genetic engineering has permitted:permitted:
Isolation of large quantities of pure protein:Insulin, growth hormone, follicle-stimulating hormone, as well as other proteins, are now available as recombinant products. Physicians will no longer have to rely on biological products of low purity and specific activity from inconsistent batch preparations to treat their patients.
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PART C PART C Case Study: The Use of Case Study: The Use of
Recombinant DNA to Recombinant DNA to Produce Human InsulinProduce Human Insulin
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Recombinant human proteins as Recombinant human proteins as therapeutic agentstherapeutic agents
Human proteins produce fewer side effects than proteins from other animals
(e.g. pork insulin vs. human insulin)
-- Hormones or hormone-like compounds
-- Enzymes
-- Antibodies
-- Proteins with vaccine action
Replacement therapy of genetic diseases
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Why synthesize human Why synthesize human insulin?insulin?
Patients’ immune systems do not produce antibodies against human insulin as they do with bovine or porcine insulin
Need for a more reliable and sustainable method of obtaining the product
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Structure of InsulinStructure of Insulin
Two polypeptide chains; one with 21 amino acids and the second with 30 amino acids
Chains are linked via a disulfide bond
Gene encoding the insulin protein is found on chromosome 11
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In reality both chains are present in In reality both chains are present in the same protein the same protein
www.blc.arizona.edu/.../rick/ biomolecules/protein.html
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First recombinant human insulin First recombinant human insulin Human gene library was screened
and INS gene subcloned into a plasmid expression vector using lac operon to promote transcription
Expression in E. coli resulted in inclusion bodies packed full of insulin
Isolation of insulin from inclusion bodies is timely and expensive
http://www.apsu.edu/reedr/Reed%20Web%20Pages/Chem%204310/Lectures/recomb1.jpg
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HumilinHumilin (Eli Lilly, 1986) – (Eli Lilly, 1986) – first industrially produced human insulinfirst industrially produced human insulin24 aa Signal peptide leader was added to 5’ end. 24 aa Signal peptide leader was added to 5’ end.
secretion of insulin secretion of insulin into culture mediuminto culture medium instead instead of its retention inside inclusion bodiesof its retention inside inclusion bodies
(Signal leader detaches as insulin is transported across (Signal leader detaches as insulin is transported across cell membrane..) cell membrane..)
A chain synthesized in one E. coli strainA chain synthesized in one E. coli strainB chain synthesized in a different E. coli strainB chain synthesized in a different E. coli strain
Chains are purified separately then joined Chains are purified separately then joined togethertogether
Continuous culture techniques used Continuous culture techniques used
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Human Growth Hormone (HGH) Human Growth Hormone (HGH) ((somatotropin)somatotropin)
HGH promotes overall body growthHGH promotes overall body growth by increasing amino acid uptake by cells, protein synthesis and fat by increasing amino acid uptake by cells, protein synthesis and fat
utilization for energyutilization for energyInsufficient production of HGH by the pituitary gland = dwarfismInsufficient production of HGH by the pituitary gland = dwarfism
Chubby faceChubby face ““Baby fat” around waistBaby fat” around waist Unusual body properties as an adultUnusual body properties as an adult ~ 4 feet tall only~ 4 feet tall only IQ = NormalIQ = Normal
HGH helps undersized children reach their normal height and sizeHGH helps undersized children reach their normal height and size
http://www.hopkinsmedicine.org/hmn/F01/photos/36.jpg
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Pressure to use HGH to stimulate growth Pressure to use HGH to stimulate growth in youngsters who have no deficiency in youngsters who have no deficiency
but whose parents want them to grow up tall.but whose parents want them to grow up tall.
In the summer of 2003, the U.S. FDA approved In the summer of 2003, the U.S. FDA approved the use of human growth hormone (HGH) forthe use of human growth hormone (HGH) for
boys predicted to grow no taller than 5′3″ and boys predicted to grow no taller than 5′3″ and for girls, 4′11″ for girls, 4′11″
even though otherwise perfectly healthy.even though otherwise perfectly healthy. If adult suffers with hypopituitarism,
HGH therapy is also available as normally payable under an insurance policy.
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Pre-recombinant era of HGH Pre-recombinant era of HGH productionproduction
HGH could be purified from cadaver pituitary glands
8 cadavers/year for 8 – 10 years per patientCreutzfeldt-Jacob Disease (CJD) is one of the risk of using brain tissue
24 cases reported by 1993 in France from cadaver HGH
umed.med.utah.edu/.../lectures/ inf/baringerimg/brain.htm
news.bbc.co.uk/1/hi/ health/2136673.stm
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10_25_cDNA.jpg10_25_cDNA.jpg
Making cDNA from mRNA using reverse transcriptase and DNA polymerase
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Protropin (Genentech) and Protropin (Genentech) and Humatrope (Eli Lilly) Humatrope (Eli Lilly)
Native human GH contains signal peptide needed to direct nascent polypeptide to RER in
eukaryotesSignal peptide is bad for E.coli
(it can not synthetize this protein)
Signal sequence removed be EcoRI cleavageCoding sequence for first 24 aa also lost in the
process of removal24 amino acids added back by synthetic DNA linker
Expressed in E. coli successfully
hGH therapy is very expensive, costing anywhere from $800 to $2,500 a month
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