EXPRESSION OF RECOMBINANT PROTEIN

What is
recombinant
protein?
• Recombinant protein is a manipulated
form of protein
• It is generated to produce large
quantities of proteins
• it is also used to modify gene sequence
 What is vector?
The formation of recombinant Protein
is carried out in specialized vehicle
known as vector
What is
recombinant
technology ?
Recombinant technology is the
process involved in the formation of
recombinant protein

It is defined as the joining together

of dna molecules from different
organisms and inserting it into a
host organism to produce new
genetic combinations
What is expression
system?
Expression system are used to express proteins
that are used in a range of methods ranging from
structural biology to in Vivo studies or as
therapeutic agents
 Components of
expression system
An expression system essentially consists of three different
components necessary for the production of recombining
proteins

• firstly , a biological environment, most often a cell, is used

to provide energy and machinery for protein synthesis. It is
also possible to use cellular extracts containing the
necessary components, i.e, cell-free protein expression
system
Secondly, a vector that facilities the introduction
of genetic material into the cell containing
regulatory parts that provide for application of the
genetic material and usually selection markers for
maintenance
• Lastly the expression cassette which is
incorporated into the vector that contains the
open reading frame in coding the Amino acid
sequence for the protein to be expressed the
expression gadget also contains all the
components necessary for transcription and
translation of the protein to be produced.
How does
recombinant protein
expression work?

There are two stages of protein production in cells:

Transcription and Translation, which is known as the central
dogma of molecular biology.
In other words, Transcription and Translation steps belong to the
recombinant protein expression steps.

To make recombinant proteins, the gene is isolated and cloned

into an expression vector. Generating a recombinant protein
requires the protein expression system, protein purification system
and protein identification systems.
Basic steps to get recombinant Protein:

1. Amplification of gene of interest.

2. Insert into cloning vector.
3. Sub cloning into expression vector.
4. Transformation into protein
expressing host (bacteria (E coli),
yeast, mammalian cells or baculovirus-
insect cell system).
5. Test for identification of
recombinant protein.( Western blot or
Fluorescence)
6. Large scale production. (Large scale
fermentation)
7. Isolation and purification.
It is not easy to select a proper
expression host or to use the right
purification method, where the
properties of the interested
recombinant protein should be
considered. Some of the important
factors are listed below:

• Membrane bound
• Solubility
• Single or multidomain
• Size (molecular weight)
• Where it expressed
Summary of Expression of recombinant protein
Expression of recombinant protein refers to the process of
generating large quantities of a protein using genetic
engineering techniques. Recombinant proteins are those that
have been produced through the introduction of DNA encoding
the desired protein into a host cell, which then produces the
protein.
The expression process usually involves the following steps:
Cloning the gene:
The DNA sequence for the protein of interest is cloned into a
vector, such as a plasmid or virus, that can be used to introduce
the DNA into the host cell.
Transfection/transformation:
The vector containing the DNA is introduced into the host cell
by transfection (for animal cells) or transformation (for
bacterial or yeast cells).
Selection of transformed cells:
Host cells that have taken up the vector are selected and
grown in culture.
Expression:
The transformed cells are grown under conditions that allow for
the expression of the protein. The protein can be expressed in
the cytoplasm, the periplasm (for bacterial cells), or the
extracellular space (for animal cells).
Purification:
The protein is purified from the culture using various
chromatography techniques to obtain the desired purity and
quantity.
Characterization:
The protein is characterized using various methods such as SDS-
PAGE, Western blot, and mass spectrometry to confirm its
identity and purity.
 Thank you !
Presented by
Ruqaiya Imran (Roll no B2171050)
Farhana Majeed (Roll no B2171009)