Production of recombinant proteins through fermentation technology involves several key steps: 1. Selection of a host cell, such as E. coli or yeast, for gene expression. Prokaryotic and eukaryotic cells each have advantages and disadvantages as hosts. 2. Cloning the gene of interest into an expression vector and transforming it into the host cell. Coordinated expression of multiple genes may be required to produce complex proteins. 3. Export and secretion of the recombinant protein from the host cell, which can be facilitated by adding a signal sequence. 4. Purification of the recombinant protein from other secreted proteins, often using affinity tags fused to the protein that enable single-step purification methods.

1. Production of
Recombinant Protein
Through Fermentation
Technology
Presented by :- Miss. Priyal Sethiya
M. Sc (Biotech.)
2nd Sem.
Guided by :- Dr. Ashish Warghane.
Assistant Professor,
Faculty of Life Sciences,
Mandsaur University, M.P., India

2. Content
What is Recombinant protein ?
01
Why we need Recombinant protein ?
02
Steps for Producing Recombinant Protein.
03
04
Necessity of Producing Recombinant Protein.

3. Content
Selection of Host Cell for gene Expression.
05
Gene Expression to Produce Recombinant Prote
in.
06
Export and Secretion of Recombinant Protein.
07
08
Overview of Production Recombinant Protein.

4. Content
09 Purification of Recombinant Protein.

5. Necessity of producing recombinant protein

6. -Problems -
 Not enough can be pr
oduce -2.6million peop
le suffer from disease.
 Some people think it i
s ethically wrong.
 May not work as well.

7. Benefits:-
 Baby cow stoma
ch no longer ne
eded to Make re
nnet for cheese.
 Horseshoe crab
blood no longer
needed for bact
eria toxin detecti
on.
 Pig pancreases
no longer neede
d for insulin pro
duction.

8. What is recombinant protein ?
Recombinant protein is a
manipulated form of protein.01
Modified gene sequence and ma
nufacturing useful commercial p
roducts.
02
Prepared by recombinant DNA ,
which is cloned in expression s
ystem and translated into mRNA
.
03

9. Why we need recombinant protein ?
Why we need
Recombinant
Protein ?

10. Steps of producing recombinant protein
0
Obtaining the cDNA
and creating the
expression clone.
Cloning Expressing the
protein in a suitable
system.
Protein purification
and characterization.
Small scale- test
expression.
01 02 03
0405

11. Overview of producing recombinant protein

12. Selection of host cell for gene
expression.
 The nature of a host cell
or an organisms as
improtant as nature of a
vector.
 Improtant of good host
includes its suitable
cultivation in the
laboratory, beside
incroporating the
vectors genetic material.
 Several prokaryotic and
eukaryotic organisms
are employed as host to
express foregin gene.

14. Prokaryotic hosts:-
• The bacterium extensive
use of Ecoli is mainly due
to it's high rate of reprodu
ction.
• Better knowledge of its bi
ochemistry, physiology a
nd molecular biology.
• Disadvantage is little exp
ression, Absence of post
translation modifications,
No sulphide bond formati
on in cytosol.
Eukaryotic hosts:-
• Production upto grams
and translation occurs t
o mammalian cells.
• Don't undergo hyperglyc
osylation & occurrence
of post-translation modif
ication.
• Include yeast cells, Bac
ulovirus...
• Disadvantages are not s
uitable for higher densit
y culture, low secretion
efficiency.

15. Gene expression to produce protein
Coordinated
Expression
By Coordinating expression
of acloned gene and a
selection markers, the
production of recombinant
protein can be increased.
Some proteins are
composed of two or more
subuntis. Each subunits are
separately synthesizy be
correspond of colned gene,
then they are mixed to form
multimeric protein in-vitro.
Expression of
two cloned
genes

16. Two - vector
expression
system
Two expression vector, each
carrying a cloned gene for
each subunit, are co-
tranfected into host cells.
They then assemble to form
a functional protein.
The two gene can be placed
under the independent
control of promoter and
polyadenylation sequences
to produce the assembled
protein with two subunit.
Two gene
expression vector

17. Export and Secretion
of recombinant protein
.• The yield of production of recombinant p
rotein is efficient if they are quickly expo
rted & secreted into the environment.
• Further the recovery and purification of f
oreign protein is easier from the exporte
d proteins.
• Some species of bacterium, Bacillus su
btilis normally seceret large quantities of
Extracellular proteins.

18. Export and Secretion
of recombinant protein
.
• A short DNA sequence called signal sequ
ences from such species is introduce into
other B.subtilis.
• These bacteria produce Recombinant D
NA tagged with signal peptide, which pro
mote export and secretion.
• The signal peptide can be removed after
purification of foreign protein.

20. Purification of recombinant protein
There are several techniques
use for the purification of
recombinant protein from a
mixture of secreted proteins.
Protein fusion tags are
dispensable tools used to improve
purification yields,enable protein
purification and accelerate the
characterization of protein
structure and function.
Fusion Protein & Purification:-

21. Purification of recombinant protein
When a protein is fused to a
peptide or protein tag, such as
polyhistidine (His), glutathione-S-
transferase (GST) ,maltose binding
protein (MBP), or Strep-tag II, the
properties of the tag can be
exploited for purification purposes.
Affinity chromatography methods
have been developed for each of
the commonly used tags, and there
is a good chance of a successful
purification of a tagged protein in a
single step.
Affinity tagging:-

22. Purifying Recombina
nt protein

23. Thank you