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Why Humulin or recombinant insulin is considered as a significant milestone in recombinant technology. As we all know insulin is a hormone that regulates blood glucose level that is essential for normal functioning of life. it is a drug that is used to treat diabetes for that the previous sources were the insulin from cow and pig for that we isolated the pancreas, inside the pancreas there's a region called islands of Langerhans, within that there are beta cells that secretes insulin. We were using that insulin to treat diabetes. Now with recombinant DNA technology, we have this insulin gene inserted inside a bacterium and bacterium act as mega factories synthesizing large quantities of insulin within bioreactors. This recombinant insulin is called Humulin, recombinant insulin or Humulin is the first approved marketable drug developed through recombinant DNA technology. Large quantities can be produced within a short time as the demand for insulin is on a rise for treating diabetes there is no risk of transferring infections no allergic reactions associated with cow and big insulin no ethical issues especially slaughtering issues concerning the use of animals so all these problems are avoided with the development of Humulin or recombinant insulin. Now let us discuss the most important discovery that lead to the development of this recombinant insulin. It was Frederick sanger who discovered the structure of insulin. Insulin is a protein consists of two chains, A chain and B chain, A chain is made up of 21 amino acids whereas B gene is made up of 30 amino acids that is joined by disulfide bond, for this discovery he was awarded with Nobel prize in 1958. so from this discovery we came to know that insulin is a very simple protein which is made up of 51 amino acids now moving into the steps in recombinant DNA technology. The first step is identification and isolation of gene of interest or DNA fragment to be cloned. Here our gene of interest is insulin gene. second step is insertion of this isolated gene into a suitable vector then we need to introduce this vector into a suitable host the process called as transformation then we need to select the transformed host cell then expression of the introduced gene inside the host for producing our gene product or insulin in large amount and finally we need to purify the protein or insulin. Now let us move into the detail of recombinant insulin production. step one identification and isolation of gene of interest. From where we get this gene, so we have cDNA library, chemical synthesis is possible, PCR amplification is another possibility. cDNA library is widely used as it is devoid of introns so that gene will be directly expressed inside a bacterial cell which doesn't have intron removal mechanism. The most common method adopted for insulin production is chemical synthesis of A and B gene. As we know that insulin is made up of two chains A chain and B chain so we will be chemically synthesizing A and b gene. step 2 insertion of this isolated gene into a suitable expression vector so in this case our intention is to express that gene inside bacterium so we have a plasmid vector we have human insulin gene the gene for a and gene for b chain so we'll be using two separate cultures for A chain and B gene, then using a restriction enzyme we'll be making a cleave or cut in inside the vector and this gene is inserted into the vector we'll be getting a recombinant DNA molecule and finally the cut ends are sealed with ligase. Now we have a recombinant vector we will be making a recombinant vector using ‘A’ gene and will be making a recombinant vector with B gene. Now we have a recombinant DNA molecule, human DNA that is joined to bacterial plasmid DNA forming a recombinant DNA that is why this technology is called as recombinant DNA technology or recombined, two DNA from different sources are recombined to form a new DNA as our intention is expression of insulin gene there should be control elements like promoter in the case of insulin production normally the insulin gene is inserted close to a beta galactosidase gene forming a fusion protein later then there will be antibiotic resistance gene that is present in all markers for selection of transformed colonies so there is a promoter sequence of E coli then a beta galactosidase gene by the side of this beta galactosidase gene insulin gene is inserted we'll in two cultures in the first culture we'll be having this insulin gene A subunit in the second culture to the side of beta galactosidase gene will be having insulin gene B sub unit like this. so we will be getting a fusion protein later now step three step three is introduction of this vector into a suitable host the most common host is E coli. In the case of insulin production then now we have yeast, mammalian cell lines etc so we have our insulin gene that is inserted into a vector this is our recombinant DNA molecule with either A gene or B gene of insulin so we have introduced that into a suitable host that is E coli so this is a recombinant vector inside the host with our gene of interest that is insulin gene so there are different gene transformation methods like electroporation, micro injection, calcium chloride mediated transformation etc you can refer our previous video on recombinant DNA technology for more now we have a genetically modified e coli with our insulin gene this process is called as transformation. Transforming the E coli with a foreign gene so this is the case with insulin so we have the recombinant vector with a gene in one culture a gene in one culture and we have the recombinant vector with b gene in the second culture. Step four is selection of transformed host cell so after the transformation experiment we will be having three types of colonies, first one majority will be non-transformed bacterial cell, then transformed with unchanged vector and the third one transformed with the vector that is recombined or recombinant vector so this is the colony that we need to select. so there are different ways by which we can select this the first selection is by simply growing these in antibiotic containing medium as this vector is having an antibiotic resistance marker, all the colonies that will be growing will be having this vector the second step is to find out the colony that is transformed with unaltered vector and to find out the colony with recombinant vector so we have different methods like antibiotic resistance in selective medium visible characters, assay for biological activity, colony hybridization, blotting test etc we have given a five-minute video on the selection of transformed colonies you can refer that for more. Step five is expression of insulin gene in the host now this protein is expressed inside the host it is a fusion protein as you can see this is beta galactosidase gene that is fused to the A insulin gene h in gene it's in protein so here you can see this is the beta galactosidase gene with b chain of insulin this is called the fusion protein now we have this recombinant vector inside the host and it will express it undergoes transcription followed by translation finally forming the protein product as here is beta galactosidase gene we can induce this transcription and translation process by adding the substrate of beta galactosidase that is lactose ensuring efficient translation to form the protein. so this is one of the advantage of this recombinant fusion protein. Now step six is purification of the protein so now we have in two different cultures, we have this protein beta galactosidase insulin A fusion protein here we have in the second culture beta galactosidase insulin B fusion protein. so this is what is happening this gene coding the target protein and this is the fusion protein this is also called as tag protein as a way of tagging the gene of interest, so we have a fusion protein lysing the cell purifying the protein and protein is passed through affinity chromatography column, it will be having an antibody to this beta galactosidase protein, so beta galactosidase will bind to this so later we can elute this so we have only these protein that is beta galactosidase insulin fusion protein, this beta galactosidase gene often this site is having residues like methionine so we can use cyanogen bromide which will cleave in the methionine. so that we'll be getting the purified protein that is A and B chain separately here there will be a methionine residue so that cyanogen bromide will cleave here so that we'll be getting this purified A chain and B chain separately in different cultures and the final step is this purified A chain and B chain is mixed in vitro in appropriate conditions to form this di-sulfide bond finally forming the active insulin. so now we have in large fermentation tanks these protein chains are produced then further purified and produced in large amounts and is marketed as Humulin. This recombinant insulin was first developed by Genentech, a company located in USA.