The document discusses recombinant proteins, including their production through recombinant DNA technology. It describes how genes can be isolated and inserted into expression vectors, which are then transferred into host cells to produce the recombinant protein. Various methods are covered, including molecular cloning and polymerase chain reaction. Examples of recombinant proteins discussed include insulin, interferons, hormones, and monoclonal antibodies that are used for medical applications.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Introduction
Definition
History
Why are the transgenic animals being produced
Transgenic mice
Mice: as model organism
Methods of creation of transgenic mice
knock-out mice
Application of transgenic mice
Conclusion
References
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
Recombinant protein expression in E.coliajithnandanam
Recombinant Protein expression in E.coli, Best suitable strains for protein expression, advantages of using E.coli for choosing the host for protein expression
Introduction
Definition
History
Why are the transgenic animals being produced
Transgenic mice
Mice: as model organism
Methods of creation of transgenic mice
knock-out mice
Application of transgenic mice
Conclusion
References
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
Recombinant protein expression in E.coliajithnandanam
Recombinant Protein expression in E.coli, Best suitable strains for protein expression, advantages of using E.coli for choosing the host for protein expression
Expression and purification of recombinant proteins in Bacterial and yeast sy...Shreya Feliz
This presentation gives the information about bacterial and yeast system as host for expressing recombinant proteins, suitable vectors, strains of host, Pros and cons of this system, different purification techniques and commercially available proteins produced so far by this system.
Recombinant baculoviruses are widely used to
express heterologous genes in cultured insect cells
and insect larvae. For large-scale applications, the
baculovirus expression vector system (BEVS) is particularly
advantageous.
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Historically cell line performance has been enhanced through media, feed and process optimization, primarily through trying to meet the basic nutritional requirements of the cells so that they can sustain high growth and productivity throughout the production runs.
However, the omics (genomics, transciptomics and metabolomics) era, sequencing of the CHO genome and enhancements in genome editing technologies over the past several years have enabled scientists to take a more direct route in cell line optimization through the modification of specific genes that have direct implications on cell culture performance, protein quality attributes and upstream and downstream manufacturing processes. These targets include but are not limited to genes that may be involved in cell cycle regulation, cellular metabolism, cellular transcription and translation, the secretory pathway and protein glycosylation or other post-translational modifications.
In this webinar we will discuss specific genetic modifications that have been made to CHO cell lines and how these modifications can lead to more efficient expression systems.
Genetic engineering principle, tools, techniques, types and applicationTarun Kapoor
Basic principles of genetic engineering.
Study of cloning vectors, restriction endonucleases and DNA ligase.
Recombinant DNA technology. Application of genetic engineering in medicine.
Application of r DNA technology and genetic engineering in the products:
a. Interferon
b. Vaccines- hepatitis- B
c. Hormones- Insulin.
Polymerase chain reaction
Brief introduction to PCR
Basic principles of PCR
Recombinant DNA Technology and Drug DiscoveryAshok Jangra
In these slide I discuss about the various DNA Technology and their biological importance and modern uses of these technologies. Here I also discussed about the oligonucleotide and new pharmaceutical approaches.
Genetic transformation & success of DNA ligation Sabahat Ali
DNA is ligated through DNA Ligase, problems may occur during DNA ligation are
1) vector cyclization
2) vector-vector concatemers
3) target DNA-target DNA ligation
in this topic we will discuss the following contents
Introduction.
Cloning.
Discovery.
Molecules need in rDNA technology.
Enzymes.
Vectors.
Procedure or steps involves in rDNA technology.
Application of rDNA technology.
Advantages and disadvantages.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
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1. SEMINAR ON
1
EVALUATION OF RECOMBINANT PROTEINS
Presented by,
D. Pranitha,
M. Pharmacy 2sem,
Pharmaceutics.
2. CONTENTS
Introduction
Gene expression
Protein Expression and Purification
Production of Recombinant Proteins
Applications
Conclusion
References
2
3. Introduction
• Proteins are the most abundant organic molecules of the living
system. They have significant role in structural and functional
organisation of the cell.
• Proteins that result from the expression of recombinant DNA within
living cells are termed recombinant proteins.
• Recombinant DNA technology involves taking genetic material from
one source and recombining it in vitro with another source followed
by introducing of recombined material into host cell.
• Once a Recombinant DNA is inserted into bacteria, these bacteria
will make protein based on this rDNA.This protein is know as
Recombinant Protein.
3
6. 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 protein.
6
7. Production of Recombinant protein
• There are basically two methods for producing recombinant
proteins.
• One is the molecular Cloning a laboratory method used to make
recombinant 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.
7
8. Cloning process
• Gene of interest is cut out with
restriction enzymes (RE)
• Host plasmid (circular
chromosome) is cut with same
RE
• Gene is inserted into plasmid and
ligated with ligase.
• New (engineered) plasmid
inserted into bacterium
(transform)
8
9. 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
9
Plasmids
Bacteriophages
Cosmids
10. Plasmids
• Bacteria contain extrachromosomal 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.
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.
10
.
.
.
11. Polymerize chain reaction
• A method for amplifying DNA segments using cycles of
denaturation, annealing to primers, and DNA polymerase-directed
DNA synthesis
• PCR copies a DNA molecule without restriction enzymes, vectors,
or host cells .
• Faster and easier than conventional cloning.
• First Step in PCR: Denaturation
1. DNA is heated to break the hydrogen bonds between the two
polynucleotide strands.
• Two single-stranded DNA molecules serve as templates.
11
12. Second Step in PCR: Annealing
2. Short nucleotide sequences (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.
Third Step in PCR: DNA Synthesis
3. The enzyme 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 .
12
13. Production of recombinant Insulin
Insulin
• Insulin is produced by β cells of islets of Langerhans of
pancreas.
• Human insulin contains 51 amino acids ,arranged in two
polypeptide chains.
• The chain A has 21 amino acids while chain B has 30
amino acids both are held together by disulfide bonds.
15. APPLICATIONS
• Several proteins are created from recombinant DNA
(recombinant proteins) and are used in medical applications.
• Hematopoietic growth factor.
• Interferon’s
• Hormones
• Recombinant protein vaccines
• Tissue/bone growth factors and clotting factors
• Biological response modifiers
• Monoclonal/Diagnostic/Therapeutic antibodies
• Recombinant proteins is extensively used in biotechnology,
medicine and research.
15
16. Hematopoietic growth factor
• Product of blood cells in bone marrow of central axial skeleton
is referred to as medullary hematopoiesis.
• While the mechanism of early stages of lineage commitment by
bone marrow to particular type of blood cells remains elusive,
the later stages of this process is driven by hematopoietic
growth factor
• List of factors of recombinant origin
16
Product Company Indication
Thrombopoietin Phamacia Thrombocytopenia
Erythropoietin Amgen Anaemia
Ancestim Amgen Blood cell transplantation
17. Antibody Structure
•Antibodies are immune system-related proteins called
immunoglobulins. Each antibody consists of four
polypeptides– two heavy chains and two light chains
joined to form a "Y" shaped molecule.
•The amino acid sequence in the tips of the "Y" varies
greatly among different antibodies. This variable
region, composed of 110-130 amino acids, give the
antibody its specificity for binding antigen. The
variable region includes the ends of the light and
heavy chains. Treating the antibody with a protease
can cleave this region, producing Fab or fragment
antigen binding that include the variable ends of an
antibody.
•The constant region determines the mechanism used
to destroy antigen. Antibodies are divided into five
major classes, IgM, IgG, IgA, IgD, and IgE, based on
their constant region structure and immune function.
17
19. Interferons
• In 1957 it was noted that infected by viruses produces protein called
Interferon ,viral resistance to native cells.
• Inability to produce sufficient quality and inadequate purity, limits the
clinical use of interferons.
• The problem of both purity and quality were resolved using
recombinant DNA technology.
IFN ι IFN α IFN ω IFN К IFN β IFN γ
IFN- α1 IFN- α2 IFN-α1a IFN- 1bβ
IFN- α2a IFN-α2b
19
Types of recombinant IFN
20. Hormones
• Initially peptide and hormones used clinically were extracted
and purified from animal or human source.
• Since these extraction may also contain animal proteins or
peptides their use can lead to development of antibodies.
• There were limitations on use due to scarcity, contamination
with other peptides and in some cases ineffectiveness.
• By introduction of recombinant DNA techniques,
synthesizing of proteins and peptides takes place.
• In 1982 first recombinant protein hormone , insulin was
introduced in USA.
20
21. List of hormones of recombinant origin
21
Hormones Company Indication
Human chronic gonadotropin Sereno Breast cancer
Leptin Amgen Diabetes mellitus
Thyroid stimulating hormone Genzyme Recurrent thyroid
cancer
22. Electrophoresis
• Most often used technique for
protein products is sodium dodecyl
sulphate polyacrylamide gel
electrophoresis .
• Proteins are denatured by boiling in
the SDS solution. All charges of
protein are masked by negative
charge of dodecyl sulphate.
• Thus protein moves on
polyacrylamide gel strictly on basis
of size of protein molecule.
• This technique is useful for
determining molecular weight of
proteins.
• For visualization of proteins on the
gel reagents used are silver nitrate,
coomassie brilliant blue dye.
22
23. List of products of recombinant origin
Product Company Indication
Alpha-glucosidase Genzyme Pompe’s disease
Interleukin-4 receptor Immunex Asthma
Tumor necrosis factor receptor Immunex Rheumatoid arthritis
Vascular endothelial growth
factor
Genvec Cardiovascular
disorders
HIV vaccine Chiron AIDS
Prostvac Therion Prostate cancer
Neurex Xoma Cystic fibrosis
23
24. CONCLUSION
• Recombinant technology is mostly used in production of
insulin, human growth hormone, vaccines ,Interferons etc.
• Recombinant proteins are used in medical applications,
particularly as medications and vaccines.
• Development of improved drug delivery system.
• Recombinant technology is that it allows introduction of
modifications into proteins at desired positions.
24
25. REFERENCES
• Daan J.A.Crommelin, Robert D.Sindelar. Pharmaceutical
Biotechnology: An Introduction for Pharmacists and Pharmaceutical
Scientists. 2nd Edition. London: Taylor & Francis Routledge; pg no.5-
18
• U.Satyanarayana. Biotechnology ,pg no.75-85;189-198
WEBSITES
http://www.innovateus.net/science/what-are-recombinant-proteins .
http://en.wikipedia.org/wiki/Recombinant_DNA .
http://www.accessexcellence.org/RC/VL/GG/plasmid.php
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