Let us start with definition of rDNA technology
Definition: It is technique used in genetic engineering that involves the
identification, isolation and insertion of gene of interest into a vector such
as a plasmid or bacteriophage to form a recombinant DNA molecule and production
of large quantities of that gene fragment or product encoded by that gene.
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History
of recombinant insulin or Humulin
Humulin, is the insulin developed using rDNA
technology which is used to treat diabetes. It was developed by David Goeddel
and his colleagues of Genetech of USA and later marketed by Lilly under the
trade name Humulin. It is the first
approved drug created through r DNA technology.
Here insulin is synthesized inside bacterium
where we introduced human insulin gene. Thus bacterial system just works as
biofactories for the synthesis of insulin.
What are the advantageous of recombinant
insulin?
- Large quantities in short time.
- No risk of transferring infections.
- No allergic reactions (compared to cow and pig insulin)
- No ethical issues concerning slaughtering and the use of animals.
Insulin is a hormone that regulates blood
glucose level. Frederick Sanger discovered the structure of insulin. Insulin
protein is made up of 2 chains; A chain and B chain with 21 and 30 amino acids
respectively. The chains are joined by di-sulphide bond. Insulin is a
comparatively simple human protein, enabling its development by r DNA
technology
Step1: Identification and isolation of gene of
interest
From where we get the desired gene?
In the case of insulin, A chain gene, B chain gene from
- cDNA library (as it has no introns)
- Chemical synthesis of gene. This is the most preferred method.
- Isolate the gene from tissues and gene amplification using PCR
What is a Gene Cloning Vector?
A vector is any DNA molecule which is capable of
multiplying inside the host to which our gene of interest is integrated for
cloning. The selection of vector depends upon the size of the fragments to be
cloned.
Common vectors include plasmids (Eg: pBR 322)
In the process, restriction enzymes functions as scissors for cutting DNA
molecules. Ligase enzyme is the joining enzyme that joins the vector DNA with
gene of inertest. The resulting DNA is called the recombinant DNA, chimera or
recombinant vector.
Most common method
In the case of recombinant Insulin, A chain and
B chin are made separately in two cultures
A gene construct with a promoter,
β-galactosidase, insulin A chain gene with other features of vectors like
selectable markers (antibiotic resistance gene for selection of transformed
colonies), ori etc. Insulin gene is placed next to β-galactosidase to form a
fusion protein.
A second construct with a promoter,
β-galactosidase, insulin B chain gene.
Introduction of recombinant vector into host
cell is achieved by different gene transfer methods
Physical gene transfer methods:
Electroporation
Microinjection
Liposome mediated gene transfer etc
Recombinant Insulin is mainly produced in either in E.coli or Saccharomyces cervevisiae (yeast)
Step VI: Selection of transformed recombinant
cells with gene of interest
The number of cells with recombinant vector will
be very less. So the next step is to select the transformed recombinant cells
with our gene of interest from the sea of non transformed cells. Several
methods are employed for selection of transformed cells:
- Visible characters,
- Assay for biological activity,
- Colony hybridization,
- Blotting test.
- Isolation of DNA from colonies and sequencing to find out presence of gene
- DNA isolation followed by PCR amplification using gene specific primers
The selected cells are cultured in large scale.
Step V: Expression of the gene of interest
For expression of the desired gene, expression vector is used (vector with control elements like promoter). Lactose, the substrate of β-galactosidase in the medium induces gene expression thus ensuring efficient transcription of our protein of interest also.
The product is
synthesized in mass cultures in large quantities in fermentation bioreactors. Now
Fusion protein is formed in separate cultures.
Step VI: Purification of Protein
We get
fusion protein upon translation. That is β-galactosidase-Insulin A fusion protein and β-galactosidase-Insulin
B fusion protein.
In
recombinant insulin production, Fusion protein of Insulin A chain and B chain
is formed with fusion partner β-galactosidase. β-galactosidase enables easy
purification by affinity chromatography.
Finally
separate Insulin A chain from β-galactosidase by using cyanogen bromide (CnBr)
which cleaves within metheonine residues. Thus we get insulin A chain and B
chain in separate cultures. Then the purified A chain and B chain are joined by
disulphide bond under appropriate condition to form functional insulin.
