Hormones or hormone-like compounds -- Enzymes -- Antibodies

polarbearfellowshipBiotechnology

Dec 12, 2012 (4 years and 10 months ago)

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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


Part A

z
rDNA Objectives


Steps in rDNA Processing


Genetic Engineering Advances

z
Part B

z
Genomic library


Plasmid


Restriction enzim, ligase


Part C


The Use of rDNA to Produce Human Insulin



and Human Growth Hormon



Genetic engineering plays a very
important role, not only in scientific
research, but also in the diagnosis and
treatment of disease.


PART

A

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
Hpa
I


PEMOTONGAN

Eco
RI

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″




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

umed.med.utah.edu/.../lectures/ inf/baringerimg/brain.htm


news.bbc.co.uk/1/hi/ health/2136673.stm


10_25_cDNA.jpg

Making
cDNA

from
mRNA

using reverse
transcriptase and
DNA

polymerase

Protropin (
Genentech) and

Humatrope (Eli Lilly)

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

hGH therapy is very expensive, costing anywhere from $800 to $2,500 a month