Transcript Vectors

Insulin & human growth
hormone by E.coli
Edited by Prof. Dr. Sudjadi
DAFTAR PUSTAKA

BR GLICK & JJ PASTERNAK
MOLECULAR BIOTECHNOLOGY
 DJA CROMMELIN & RD SINDELAR
PHARMACEUTICAL BIOTECHNOLOGY
 Sudjadi, Bioteknologi Kesehatan
Overview
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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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PART A

Genetic engineering plays a very
important role, not only in scientific
research, but also in the diagnosis and
treatment of disease.
rDNA is NOT Whole Animal
Cloning
Recombinant DNA is a tool in
understanding the structure,
function, and regulation of genes
and their products

The objectives of Recombinant DNA
technology include:
– Identifying genes
– Isolating genes
– Modifying genes
– Re-expressing genes in other hosts or
organisms

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
Process 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
Part B

Genomic library
Prinsip kloning
PEMOTONGAN HpaI
PEMOTONGAN EcoRI
Cloning 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.
PENAPISAN

KOMPLEMENTASI
 PELACAK DNA
 ANTIBODI
Genetic engineering has
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.
PART C
Case Study: The Use of
Recombinant DNA to
Produce Human Insulin
Recombinant human proteins as
therapeutic 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
Why synthesize human
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
Structure 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
In reality both chains are present in
the same protein
www.blc.arizona.edu/.../rick/ biomolecules/protein.html
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
Humilin (Eli Lilly, 1986) –
first industrially produced human insulin
24 aa Signal peptide leader was added to 5’ end.
secretion of insulin into culture medium instead of its
retention inside inclusion bodies
(Signal leader detaches as insulin is transported across cell
membrane..)
A chain synthesized in one E. coli strain
B chain synthesized in a different E. coli strain
Chains are purified separately then joined together
Continuous culture techniques used
Human Growth Hormone (HGH)
(somatotropin)

HGH promotes overall body growth
by increasing amino acid uptake by cells, protein synthesis and fat
utilization for energy
Insufficient production of HGH by the pituitary gland = dwarfism
 Chubby face
 “Baby fat” around waist
 Unusual body properties as an adult
 ~ 4 feet tall only
 IQ = Normal
HGH helps undersized children reach their normal height and size
http://www.hopkinsmedicine.org/hmn/F01/photos/36.jpg
Pressure to use HGH to stimulate growth
in youngsters who have no deficiency
but whose parents want them to grow up tall.
In the summer of 2003, the U.S. FDA approved
the use of human growth hormone (HGH) for
boys predicted to grow no taller than 5′3″ and
 for girls, 4′11″
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even though otherwise perfectly healthy.
If adult suffers with hypopituitarism,
HGH therapy is also available
as normally payable under an insurance policy.
Pre-recombinant era of HGH
production
HGH could be purified from cadaver pituitary glands
8 cadavers/year for 8 – 10 years per patient
Creutzfeldt-Jacob Disease (CJD) is one of the risk of using brain tissue
24 cases reported by 1993 in France from cadaver HGH
news.bbc.co.uk/1/hi/ health/2136673.stm
umed.med.utah.edu/.../lectures/ inf/baringerimg/brain.htm
10_25_cDNA.jpg
Making cDNA from
mRNA using reverse
transcriptase and
DNA polymerase
Protropin (Genentech) and
Humatrope (Eli Lilly)
hGH therapy is very expensive, costing anywhere from $800 to $2,500 a month
Native human GH contains signal peptide
needed to direct nascent polypeptide to RER in
eukaryotes
Signal peptide is bad for E.coli
(it can not synthetize this protein)
Signal sequence removed be EcoRI cleavage
Coding sequence for first 24 aa also lost in the
process of removal
24 amino acids added back by synthetic DNA linker
Expressed in E. coli successfully