Recombinant Protein

Production
Through biotechnology there is a mulitple step
processes are:
1. Isolation of gene of interest
2. Choose of expression system and vector
3. Introduction of gene to expresion vector
4. Transformation into host cell
5. Protein expression
6. Purification of protein

Choice of expression
system
Expression system are the host cells in which protein is
amplified
Choosing the right expression system for the recombinant
protein production is very essential
Basis for choice:
Protein size

Large protein is >100kD (eukaryotes)

Small proteins is <30 kD (prokaryotes)

Protein nature and functionality:
Post translational modification required eukaryotic cell

Post translational modification not necessary

Protein structure
Abilities of host cell

Choosing a vector
Genes that code for recombinant protein are put into unit
of DNA
Vectors are choosen that will produce large amount of
desired protein
An appropriate vector need a good combination of

Strong promoters

Ribosome binding site

Termination sequences

Affinity tag

Multi enzyme restriction site

Origin of replication and independent replication ability

Compatibility with host cell system

Intro of gene to expression vector

Gene of interest is cut out with
restriction enzyme
Host plasmid /circular chromosome is
cut with same restriction enzyme
Gene is inserted into plasmid and
ligated with DNA ligase

Transformation into host cell

It is the insertion of recombinant DNA
into expression system /host cell
Essential for actively expressing the
gene inside the cell to produce
protein
2 routes of transformation
CaCl2 + cold shock
Elctroporation

Electroporation
Electric shock causes disruption in
cell membrane
Pores created which allow DNA
molecules to enter
Reconstruction of membrane occurs
o Works for bacteria,yeast and animal
cell

Protein expression and cell growth

There are several different ways to induce protein
expression
Either a compound is added for induction
Or the temperature is maintained at which promoter is
active
Cells are cultured/grown for mass production in
i. Fermentation flask
ii. Bioreactor
iii. Culture medium
. Which provide suitable environment with enough nutrients
. This allows one cell to divide into two identical copies
which in turn divides further

Purification of protein
Proteins that are secreted outside the
cell are easily separatedly and purified
but for the purification of intra cellular
proteins several approaches are
adopted such as the use of tags
Tags is the recombinant DNA carrying
desired gene e.g
a. Cellulose binding domian tags (CBD)
b. His6 for metal affinity chromatography

Histidine tag purification

The His tag on the protein is
attracted to the metal ion mostly (Ni)
on the agarose bead to assist in
separation.

Summary

Gene of interest
Gene inserted in plasmid
Protein expression in a host organism
Host organism expansion in a bio
reactor
Protein purification
Soluble protein