Recombinant Protein Synthesis Steps … - 123seminarsonly.com

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Dec 14, 2012 (4 years and 8 months ago)

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Recombinant Protein Synthesis
Steps



Proteins

Are Any molecule that is involved in the maintenance and
metabolic processes of living organisms. Biomolecules
include carbohydrate, lipid, protein, water and nucleic
acid.Proteins in living organisms are classified according
to their biological roles i.e
Enzymatic,hormonal,structural,receptors,contractile,defen
sive,strorage&transport.

Humans have traditionally used animal and plant sources
to obtain proteins (such as hormones and clotting factors)
in order to treat different diseases and conditions. Many
of these were extracted from human and animal corpses
.


Protein
-
Based Drugs.


Prior to advances in biotechnology such as rDNA
technology, the few protein drugs available were
taken directly from human and animal corpses. For
instance, the human growth hormone was taken
from human corpses and insulin required to treat
diabetes was collected from slaughtered pigs. These
drugs were available in limited supply and they
were expensive, given their sources. Biotechnology
has boosted the production of protein
-
based drugs
in a significant way i.e Recombinant DNA
Technology


The Chemical makeup of DNA and
RNA
-



1.DNA


is made up of many DNA nucleotides
(nucleotides are the building blocks of nucleic
acids). Each DNA nucleotide is composed of
deoxyribose (a pentose, or five carbon sugar),a
phosphate molecule and one of four nitrogenous
bases: Adenine, Thymine, Cytosine and
Guanine. (A, T, C, G). The picture below shows
the double stranded DNA structure and
complementary bases of the DNA bases.




2
.
RNA



is

made

up

of

many

RNA

nucleotides

(nucleotides

are

the

building

blocks

of

Nucleic

acids)
.

Each

RNA

nucleotide

is

composed

of

ribose

(a

pentose,

or

five

carbon

sugar),

a

phosphate

molecule

and

one

of

four

nitrogenous

bases

Adenine,

Uracil,

Cytosine

and

Guanine
.

(A,

U,

C,

G)
.


3.
DNA and RNA Nucleotide Diagrams

The diagrams below show different diagrams of nucleotides,
both RNA and DNA. Be able to recognize the sugar, the five
sided figure in the first two diagrams and the five sided figure
labeled Y in the third diagram, the phosphate (the circle with
the P or Z inside)

and the base (the six sided figure with the
N in its structure, labeled W or X in the third diagram) The last
diagram shows 4 nucleotides, showing bases, sugars and
phospates




RNA & DNA



Recombinant DNA Technology


Genetic engineering,is a technique in which
the gene that encodes for the required
protein is transferred from one organism
into another, which is capable of producing
large amounts of the drug.Recombinant
DNA
-

contains DNA from 2 different
sources. Organisms that have had a foreign
gene inserted into them are called
transgenic organisms


Importance of Recombinant
DNA technology


Objectives

of

DNA

technology

include,identifying,isolating,modifying

&re

expressing

genes

in

other

hosts

or

organism
.

The introduction of rDNA technology or genetic engineering, has provided
a large and cheaper source of protein
-
based drugs .Genetic engineering
plays very important role not only in scientific research but also in
diagnosis and treatment of disease.Genetic engineering produces proteins
that offer advantages over proteins isolated from other biological sources
like high purity,high specific activity,steady supply&batch to batch
consistency

It

permit

scientists

&clinicians

to

identify

new

gene&proteins

they

encode,to

correct

endogenous

genetic

defect&to

manufacture

large

quantities

of

specific

gene

products

such

as

hormones,vaccines

&other

biological

agents

of

medical

interest
.




Use of Recombinant DNA
research


some

of

the

areas

where

Recombinant

DNA

will

have

an

impact,

Better

Crops

(drought

&

heat

resistance)

Recombinant

Vaccines

(ie
.

Hepatitis

B)


Production

of

clotting

factors


Production

of

insulin


Production

of

recombinant

pharmaceuticals


Plants

that

produce

their

own

insecticides


Germ

line

and

somatic

gene

therapy

Prevention

and

cure

of

sickle

cell

anemia


Prevention and cure of cystic fibrosis

.
Different type of protein based drugs produced by recombinant
DNA technology


Cytokines

-

these drugs regulate the immune system. That is, they
are proteins that activate the immune system cells to carry out
different immune functions.

Hormones
-

protein drugs that regulate functions in the body. As
drugs, these proteins can be used to elevate levels of certain
hormones, such as estrogen during menopause or growth
deficiency. They can also be used to treat certain diseases such as
diabetes or conditions such as infertility


Clotting factors.

proteins that regulate the clotting of blood. These
drugs are used to treat blood clotting disorders such as hemophilia.

Vaccines
-

proteins that stimulate the immune system to produce
specific antibodies used to prevent or treat diseases.



Monoclonal antibodies
-

proteins that mark a specific foreign material
(such as cancer cells, disease
-
causing bacteria and viruses), for
removal or destruction by other components of the immune system.
These are also used as
effective diagnostic tools

for many specific
genetic diseases and other conditions such as pregnancy


Genetic engineering has
permitted


Identification of mutations
:People may be tested for the presence of
mutated proteins that may be involved in the progression of breast
cancer, retino
-
blastoma, and neurofibromatosis.

Diagnosis of affected and carrier states for hereditary diseases:

Tests exist to determine if people are carriers of the cystic
fibrosis gene, the Huntington’s disease gene, the Tay
-
Sachs
disease gene, or the Duchenne muscular dystrophy gene.


Mapping of human genes on chromosomes:
Scientists are able to link
mutations and disease states to specific sites on chromosomes.

Performing structure and function analyses on proteins:
Researchers
may now employ rational drug design to synthesize drug compounds
that will be efficacious and selective in treating disease.

Transferring genes from one organism to another:
People suffering from
cystic fibrosis, rheumatoid arthritis, vascular disease, and certain cancers
may now benefit from the progress made in gene therapy.

Isolation and alteration of genes:
Once gene modification becomes
successful, alteration of genes to produce a more functional protein
than the endogenous protein may become possible, opening up the
route of gene therapy.


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.




Steps in Synthesizing a Recombinant Protein:


Recombinant Protein synthesis is a complicated
process to illustrate and includes many steps but simply
it can be expressed as under.

.Protein expression,transcription and translation,
Protein
purification (small
-

through large
-
scale)
,
Protein
characterization.Quality assurance .Specialized
services

custom cell
-
based assays & custom
antibodies, libraries, and cloning

protein expression.


.
Recombinant technology begins with protein expression as
following.


These are charts to describe the whole procedure
illustrating recombinant protein production steps.



.isolation of a gene of interest by using restriction
enzymes.insersion of gene of interest into a vector by
using integration enzymes,ligases.( A vector is a
piece of DNA that is capable of independent growth;
commonly used vectors are bacterial plasmids and
viral phages).

introduction or integration of The gene of interest (foreign
DNA)into the plasmid or phage(and this is referred to as
recombinant DNA){Plasmids are circular, double
-
strandedDNA molecules that exist in bacteria and in the
nuclei of some eukaryotic cells.They can replicate
independently of the host cell.The size of plasmids ranges
from a few kb to near 100 kb,Can hold up to 10 kb
fragments,have an origin of replication,

antibiotic resistance genes as markers, and several unique
restriction sites.After culture growth, the clone fragment can
be recovered easily. The cells are lysed and the DNA is
isolated and purified.A DNA fragment can be kept
indefinitely if mixed with glycerol in a

70 degrees C
freezer)}.cloning of vector is done(Before introducing the
vector containing the foreign DNA into host cells to express
the protein)


)(Cloning is necessary to produce numerous copies of the
DNA since the initial supply is inadequate to insert into host
cells.){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 chromosomes,
and yeast artificial chromosomes}.isolation of the vector
having foreign DNA in large quantities.introduction of
vector into the desired host cells such as mammalian,
yeast, or special amounts.


bacterial cells.
.synthesis of foreign protein by host cells from
the recombinant DNA.
.isolation of foreing or recombinant protein
When the cells are grown in vast quantities.

purification of foreign protein in large amounts.

it is
important for saving the integrity and properties of the
proteins. Basically, the purification is based on affinity
chromatography. However, if a high purity grade or
polishing step is required, we combine/associate additional
techniques: ions exchange, gel filtration, hydrophobic
interaction chromatography.

After this other steps are done.