Recombinant protein

1. RECOMBINEN
T PROTEINS

2. CONTENTS
 Introduction
 History
 Definition
 PCR
 Application

3. INTRODUCTION
 Proteins: Most abundant organic molecules of
the living system.
 Proteins are result of expression of
recombinant DNA within living cells.
 Recombinant DNA: Such that it takes genetic
material from one source and recombining it
in vitro with another source followed by
introducing recombined material into host cell

4. CONTI…
 Once a recombinant DNA is inserted into
bacteria, these bacteria will make protein
based on this rDNA.

5. HISTORY
 The method of recombinant DNA was initially
planned by a graduate student, Peter Lobban,
along with a biochemist, A. Dale Kaiser at the
Stanford University. During then years,1972–
74, the method was then acknowledged by
Stanley Norman Cohen, an American
geneticist Chang, Herbert Boyer, a addressee
of the 1990 National Medal of Science.

6. CONTI…
 In 1973, they published their predictions in journal
"Enzymatic end-to-end joining of DNA molecules"
which explained the methods to separate and
intensify genes or DNA segments and introduce
them into an additional cell with accuracy. In 1977,
an advance in the field of recombinant DNA
technology took place when Herbert Boyer
created the biosynthetic "human" insulin, a group
of biosynthetic human insulin products.

7. DEFINITION
 Proteins that can result from the expression of
recombinant DNA within living cells are
termed recombinant proteins.

8. FIRST RECOMBINENT
EXPRESSION
 Rise of the recombinant protein expression
systems. ... Following the
first recombinant expression in Escherichia
coli, which took place in 1976 when scientists
at Genentech – the first genetic engineering
company – produced the human protein
Somatostatin, protein production systems that
used bacteria continued to improve ...

9. PROTEIN EXPRESSION AND
PURIFICATION
 Isolation of genes.
 Insertion of isolated gene to expression
vector.
 Transfer of recombinant vector into host cell
through transformation.
 Identification and isolation of cells containing
recombinant vector.
 Growth of cells through fermentation.
 Isolation and purification of proteins.

10. PRODUCTION OF
RECOMBINENT PROTEINS
There are basically two methods for producing
recombinent proteins
One is the molecular cloning a laboratory method
used to make recombinent DNA.
The other method is the Polymerase Chain Reaction
used to proceed the replication of any specific DNA
sequence selected.
The basic difference between the Two methods is
that molecular cloning incorporates the replication
of the DNA within a living cell, whereas PCR
replicates DNA in the test tube, without living cells.

11. OVER EXPRESSION OF
RECOMBINANT PROTEIN
 The genes that code for the proteins are put into
special vectors, or units of DNA. Vectors are selected
that will create huge quantities of the preferred protein.
This is known as “over expression”. Over expression is
performed in unique host cells. Occasionally, the hosts
are bacteria or yeast. The hosts are frequently insect or
mammalian cell lines, in cases where the proteins are
from mammals. Various kits are commercially present
to assist both the cloning of the gene, and the
following recombinant protein fabrication.


12. CLONING PROCESS
 The typical cloning etiquette any DNA fragment
fundamentally involves seven steps:
 Preference of host organism and cloning vector,
 Preparation of vector DNA,
 Preparation of DNA to be cloned,
 Formation of recombinant DNA,
 Foreword of recombinant DNA into the host organism,
 Selection of organisms having recombinant DNA,
 Screening for clones with preferred DNA inserts
and biological characteristics.


13. VECTORS
 Self-replicating DNA molecules used to
transfer foreign DNA segments between host
cells.
 An ideal vector should be small in size, with
single restriction endonuclease site.
 Three types of vectors plasmids,
bacteriophages , cosmids.

14.  Plasmids
 Bacteria contain extra-chromosomal molecules
of DNA called plasmids which are circular.
 pBR322 of E.coli is most popular and widely
used plasmid vector.
 Bacteriophages
 Bacteriophages or simply phages are the
viruses that replicate within the bacteria.
 Phages can accept foreign DNA fragments of
10-20 kb length.

15.  Cosmids:
 These are specialized plasmids containing
DNA sequence namely cos-sites.
 Cosmids can carry larger fragments of foreign
DNA compared to plasmids. i.e 20-50kb.

16. POLYMERASE CHAIN
REACTION
 A method for amplifying DNA segments using
cycles of denaturation, annealing to primers,
and DNA ploymerase-directed DNA synthesis.
 PCR copies a DNA molecule without
restriction enzymes, vectors or host cells.
 Faster and easier than conventional cloning.

17. FIRST STEP IN PCR
 Denaturation:
 DNA is heated to break the hydrogen bonds
between the two polynucleotide strands.
 Two single-stranded DNA molecules serve as
templates.

18. SECOND STEP IN PCR
 Annealing:
 Short nucleotide sequence (primers for DNA
replication) are mixed with the DNA and bind
to complementary regions on single-stranded
DNA.
 Takes place at lower temperature.
 Primers are 20-30 nucleotides long,
synthesized in the laboratory.

19. THIRD STEP IN PCR
 DNA Synthesis:
 The enzymes Taq polymerase is added to
synthesize a complementary DNA strand.
 Taq is a DNA polymerase from a bacterium
found in hot springs.
 These three steps make up one PCR cycle.

20. APPLICATIONS OF
RECOMBINANT PROTEINS
 Several proteins are created from recombinant DNA
(recombinant proteins) and are used in medical applications,
particularly as medications and vaccines.
 On the other hand, various proteins can also be got
from biological sources made from animal extracts or man-made,
for instance, the human growth hormone, a protein-
based peptide hormone which stimulates growth, cell
reproduction, etc.
 Recombinant DNA is used to recognize, map and sequence
genes, when scientists are learning separate genes or the entire
genomes.
 Recombinant proteins are extensively used as reagents in
laboratory experiments and to make antibody probes for
investigative protein synthesis within cells and organisms.

21. CONTI…
 Recombinant DNA is extensively used in
biotechnology, medicine and research.
Currently, recombinant proteins are found
fundamentally in every western pharmacy, doctor's or
veterinarian's office, medical testing laboratory, and
biological research laboratory.
 On top of that, organisms that have been maneuvered
with the help of recombinant DNA technology, and
products which are got from those organisms are found
in many farms, supermarkets, home medicine cabinets
and even pet stores.

22.  Recombinant proteins are obtained from the
recombinant DNA technology. This technology
involves the transfer of specific genes from an
organism into another organism using vectors
and restriction enzymes as molecular tools.

23. RECOMBINANT PROTEINS
AND THERE THERAPEUTIC
USE
 Ten recombinant proteins used in medical
practice are
 Insulin: Treatment for type-I diabetes mellitus.
 Interferon-𝜶: Used for chronic hepatitis C
 Interferon-𝜷: Used for herpes and viral
enteritis.
 Coagulation factor VII: Treatment of
haemophilia A

24.  Coagulation factor IX: Treatment of
haemophilia B
 DNAase I: Treatment of cystic fibrosis.
 Anti-thrombin III: Prevention of blood clot.
 Interferon B : For treatment of multiple
sclerosis.

25.  Human recombinant growth hormone: For
promorting growth in an individual.
 Tissue plasminogen activator: Treatment of
acute myocardial infection.