it also include processing of recombinant proteins, extracellular and pericellular production of recombinant proteins

1. Processing of recombinant proteins
Extracellular and pericellular
production of recombinant proteins
Silpa Mohandas
MSc Biotechnology

2. Recombinant proteins
Recombinant protein is a manipulated form
of protein, which is generated in various
ways to produce large quantities of proteins,
modify gene sequences and manufacture
useful commercial products. The formation
of recombinant protein is carried out in
specialized vehicles known as vectors.
Recombinant technology is the process
involved in the formation of recombinant
protein.

4. Recombinant proteins
■ Recombinant Proteins: Proteins are complex biomolecules that
play various fundamental roles in living systems. They are made
up of building blocks called amino acids that are attached to one
another in chains.
■ Proteins occur in different types and serve functions like
structural
support, mediation of cell processes, the regulation of cells and
tissues, and the expression of physical attributes like eye color,
height, and much more.
■ The advent of modern technologies has undeniably brought so
much improvement in the study of protein molecules. For instance
one of the main goals of the biotechnological field is the
production of recombinant proteins.

5. History of recombinant proteins
■ The idea of recombinant DNA and the method of producing it were
made by a graduate student Peter Lobban of Stanford University.
Along with his professor, Dale Kaiser, the two the published their
hypotheses in the journal Enzymatic end-to-end- joining of DNA
molecules (1973) and explained the method of separating and
amplifying genes and inserting them to a new host cell.
■ In 1974, Stanford University applied for patent (with Stanley
Cohen
and Herbert Boyer as inventors) and was awarded in 1980.
■ New advancements in the technology arose such

6. Two methods of producing recombinant proteins
■ The process of producing recombinant proteins is highly
dependent on the process of inserting the DNA segment to the
host’s genome. Two methods are known to produce recombinant
DNA namely :
■ Molecular cloning
■ Polymerase chain reaction

7. 1.Through Molecular Cloning
■ Molecular cloning is a process that is used to insert a recombinant
DNA into a vector (vehicle used to transfer genetic material) that
will replicate the segment inside the host organism. The DNA
segment may be isolated from either a prokaryote or a eukaryote
bearing the gene or protein of interest.
■ Following the isolation of the DNA segment is its cutting (with
restriction enzymes) and inserting into the plasmid vector through
ligation.
■ The resulting product is now called a recombinant plasmid and is
now ready to be inserted into the host (which is in the case of the
illustration below is the bacterium E. coli).

8. production of recombinant proteins

10. Through polymerase chain reaction
■ Also referred to as “Molecular Photocopying “, Polymerase
Chain Reaction (PCR) is a method to quickly amplify
segments of the DNA into million copies. Once amplified,
the copies of the DNA segments produced can now be used
in various laboratory experiments like mapping. Check out
the History of Genetics and the year 1983 section for more
discussion on PCR.
■ Unlike molecular cloning, PCR produces recombinant
DNA
without the need for vectors and host cells.
■ The templates DNA (DNA to be copied) is simply mixed
with the forward and reverse primers, nucleotides, and DNA
polymerase and undergo repeated cycles through PCR.

11. PCR
■ PCR, polymerase chain reaction, is an in-
vitro technique for amplification of a region
of DNA whose sequence is known or which
lies between two regions of known
sequence.
Requirement
■ DNA template
■ Primers
■ Enzyme
■ dNTPs
■ Mg2+
■ buffers

12. ■ Comprises of 3 steps,

17. Insulin
■ Insulin is produced and secreted by beta cells of
Langerhans pancreas, that regulates the use and
storage of carbohydrates.
Structure of Insulin
■ Chemically, insulin is a small and simple protein , consists of
51 amino acids, 30 construct 1 polypeptide chain and 21
■ Amino acids construct the second chains. Both chains are
linked by disulfide bond

18. ■ Synthesis of human insulin using E.coli
Human Insulin is produced by
inserting gene of insulin at
suitable vector of Escherichia coli
(production of Humulin, Eli Lily) by
recombinant DNA Technology
■ Selectively obtaining insulin producing
beta epithelioid cells from a pancreas, a
small circular DNA in a bacteria, called
plasmid, modified by inserting human
DNA gene for a precursor proinsulin

20. ■ Dispersing cells in a
nutrient amino acid rich
medium composition in an
open aeration incubation
vessel maintained at
about32° -39°C

22. Production of recombinant interferon
■ Inside are a group of signalling proteins made and
released by host cell in response to the presence of
pathogens, such as viruses, bacteria, and tumor cells.
■ The genes of all 3 types Humans have been cloned
on micro organisms and expression obtained.
■The problem of both purity and quality resolved by
this technology.

24. Production of Human growth hormones
■ Somatostatin and somatotrophin are two proteins
that act in conjunction to controll growth processes in
human body, their malfunctioning leading to painful
disorders and also Acromegaly and dwarfism.
■ Same process of insulin, involves insertion of an
artificial gene into a lac z vector, synthesis of a fusion
protein and cleavage with cyanogen bromide.

25. Recombinant blood clotting factors
■ Human factor VIII is a protein that plays a central
role in blood clotting.
■ Commenest form of haemophilia in human results
from an inability to synthesize factor VIII
■ Production of recombinant human blood clotting
factor includes alternative method pharming
■ The complete human C DNA has been attached to
the promoter for the acidic protein gene of pig, leading
to synthesis of human factor VIII in pig mammary
leading to subsequent secretion of protein in milk.

26. periplasmic production of recombinant
proteins
■ Strategies for the secretory production of recombinant proteins
in Escherichia coli. The Sec system, the twin-arginine
translocation (TAT) system, and the strategies for enhancing
secretory protein production using periplasmic chaperones and
protease-negative mutants are shown.
A) The co-expression of periplasmic chaperones, such as disulfide
-bond formation (Dsb) family proteins, SurA, FkpA, and Skp,
can improve the efficiencies of secretory production and
protein folding .
B) Protease- negative mutant strains can improve secretory production
of recombinant proteins by reducing proteolysis .
C) A novel TAT system can directly secrete the folded proteins .

28. Extracellular production of recombinant proteins
■ Strategies for the extracellular production of recom- binant proteins
by
E. coli.
A) Recombinant proteins can be excreted into the culture medium by
treating cells with various agents or by using L-form cells.
B) Recombinant protein fused to outer- membrane protein F (OmpF) of
E. coli can be excreted into the culture medium .
C) Proteins secreted into the E. coli periplasm can also be released into
the culture medium by co-expression of kil, out genes, the gene
encoding the third topological domain of the transmembrane protein
TolA (TolAIII), or the bacteriocin-release protein gene .
D) The target protein fused to the C- terminal hemolysin secretion signal
can be directly excreted into the culture medium through the

30. Application
Medicine
■ Therapeutic proteins provide important therapies for a variety of
diseases, such as diabetes, cancer, infectious diseases, hemophilia,
and anemia. Common therapeutic proteins include antibodies, FC
fusion proteins, hormones, interleukins, enzymes, and
anticoagulants. There is a growing demand for recombinant
proteins for therapeutic applications.

31. Therapeutic proteins can be classified into four groups
■ Group I: Therapeutic proteins with enzymatic or regulatory
activity. These proteins replace a protein that is deficient or
abnormal, up-regulate an existing pathway, or provide a new
function or activity.
■ Group II: Therapeutic proteins with special targeting activity.
These proteins interfere with a molecule or organism or deliver
other molecules.
■ Group III: Therapeutic proteins as vaccines. These proteins help
protect against foreign agents, autoimmune diseases, and cancer.
■ Group IV: Therapeutic proteins as diagnostics.

32. Research
■ Recombinant proteins help to elucidate the basic and
fundamental principles of an organism. These molecules can be
used to identify and locate the position of the protein encoded
by a specific gene, and to uncover the function of other genes
in various cellular activities such as cell signaling, metabolism,
growth, replication and death, transcription, translation, and
protein modification.

33. Biotechnology
■ Recombinant proteins are also used in industry, food production,
agriculture, and bioengineering. For example, in breeding industry,
enzymes can be added to animal feed to increase the nutritional
value of feed ingredients, reduce feed and waste management costs,
support animal gut health, enhance animal performance and improve
the environment.

34. Limitations of recombinant proteins
1. In some cases, the production of recombinant proteins is complex,
expensive, and time-consuming.
2. The recombinant proteins produced in cells may not be the same
as the natural forms. This difference may reduce the effectiveness of
therapeutic recombinant proteins and even cause side effects.
Additionally, this difference may affect the results of experiments.
3. A major concern for all recombinant drugs is immunogenicity. All
biotechnologically produced therapeutics may exhibit some form of
immunogenicity. It is difficult to predict the safety of novel
therapeutic proteins.

35. Thank you................