How is Recombinant Insulin made? Steps in the production of Recombinant Insulin or Humulin

 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.

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

How is recombinant insulin made? Steps in the production of recombinant insulin or Humulin
How recombinant insulin is produced? We will be discussing the basic steps involved in production of recombinant insulin.

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
This is a summarized video explaining recombinant insulin production 
Step II: joining of this gene into a suitable vector (construction of recombinant DNA)

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.

 Step III: Introduction of this vector into a suitable organism

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:

  •  Antibiotic resistance,
  • 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.

 5 Steps in Recombinant DNA Technology or rDNA Technology

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