E.Coli in Recombinant Human Insulin Production

Introduction:

The demand for insulin is on the rise worldwide as the increasing rate of diabetes shows no signs of slow down. According to the CDC, more than 100 million U.S. adults have been diagnosed positive for diabetes. The insulin hormone is produced by the pancreas to regulate the sugar levels in the blood. 1930 to 1970 - diabetics used insulin that was harvested from animals such as pigs and cows, which could cause many allergic reactions in patients. It wasn’t until 1982 when the first recombinant human insulin derived from Escherichia coli was available to diabetic patients. This was done by inserting the human insulin gene into the plasmid of the E. coli bacteria which could be translated into insulin. This breakthrough allows insulin to become readily available to patients at a more affordable price. The purpose of this review is to explore how Escherichia coli is used to produce recombinant human insulin and why it is a preferred organism for insulin production.


Why E.coli is considered the best option for insulin production?

Escherichia coli is the preferred organism for insulin production for many different reasons. E. coli has the fastest reproduction rate as under the right conditions it can double its numbers every 20-30 minutes. It is also resistant to antibiotics such as ampicillin and tetracycline which allows insulin manufactures to easily inhibit the growth of unwanted microbes when it is fermented on a large scale. E. coli is easy to handle which makes it cost-efficient to maintain. E. coli also produces the highest yield of insulin compared to other organisms used for its production. All of this makes the production of insulin using E. coli the most profitable for manufacturers.


Structure of Insulin:


Insulin is composed of two peptide chains namely - Alpha and Beta.

  • Alpha and Beta chains are linked together by two disulfide bonds and another disulfide bond is formed within the Alpha chain.

  • Alpha chain contains 21 amino acids and Beta chain contains 30 amino acids, in total there are 51 amino acids present.

Steps for Human Insulin Production:

1) Isolation of genetic material :

  • We already know that the genetic material of all organisms is Nucleic Acid whereas in some it is DNA and in others it is RNA.

  • In normal cells, DNA exists both within the cell membrane and also along with other macromolecules such as RNA, polysaccharides, proteins, and lipids.

  • Other macromolecules that are present with DNA, can be removed with enzymes or chemical treatments. The addition of ethanol purifies the DNA.

2) Restriction enzyme :

  • Restriction enzymes act as molecular scissors that cut the DNA at specific locations.

  • This process is necessary for rDNA technology.

  • DNA has 2 strands, hence it allows to cut both the strands. The cut could be blunt or sometimes it is uneven too (i.e, producing sticky ends).

3) Amplification using PCR :

  • PCR is a technique used for the production of multiple copies of DNA.

  • The fragments of DNA can be amplified using PCR and then ligated with a vector that was excised using the same restriction enzyme.

4) Ligation of DNA molecule :

  • The purified DNA and the vector of interest are then cut with a Restriction enzyme. This provides fragments of DNA and the plasmid.

  • The process of joining these two pieces is done using the enzyme DNA Ligase.

5) Insertion of rDNA into host :

  • Here, rDNA is inserted into the host cells i.e. E-coli.

  • Bacterial cells do not accept foreign DNA easily so they are first treated to make them competent so that they can accept new DNA. Then, multiple copies are produced.

Industries producing insulin:

The American biotechnology company Genentech had developed Humulin which was licensed by Eli Lilly and it was the first product to be marketed plus created through recombinant DNA technology.


Blog Author:- Vibhawari Rajendra Lavane




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