How can insulin be genetically engineered

Large quantities can be made quickly. No risk of transferring infections. More effective at treating diabetes as animal insulin is different to human insulin. No ethical issues concerning the use of animals. Vectors take pieces of DNA and insert them into other cells. Viruses and plasmids can act as vectors.

Similarly, ligase enzymes join pieces of DNA together at specific sites. However, there are some disadvantages using E. Among yeast strains, Saccharomyces cerevisiae, Hansenulla polymorpha and Pichia pastoris are very commonly used for production of recombinant proteins [ 21 ],[ 24 ],[ 33 ]-[ 35 ].

Like E. Recombinant proteins produced in yeast are properly folded and glycosylated to a certain extent similar to the one expressed in mammalian cells. Various human therapeutic proteins, including therapeutic monoclonal antibodies are being produced in mammalian cell lines such as Chinese hamster ovary CHO and Baby hamster kidney BHK cells.

Recombinant proteins expressed in mammalian cells are properly folded, glycosylated and generally yield a functionally active protein [ 36 ]. However, the production cost of biopharmaceuticals using mammalian expression system is very high due to expensive culture media. The mean rate of approval is 13 as shown in the Figure 2 [ 5 ]-[ 13 ]. Remarkably, the number is same for both the years and The human insulin is comprised of 51 amino acids and has a molecular weight of Da.

It is produced by beta cells of the pancreas and plays a key role in regulating carbohydrate and fat metabolism in the body. Insulin is synthesized as a single polypeptide known as preproinsulin in pancreatic beta cells. Preproinsulin harbours a residue signal peptide, which directs the nascent polypeptide to the endoplasmic reticulum. The signal peptide is cleaved as the polypeptide is translocated into the human of the endoplasmic reticulum resulting in the formation of proinsulin.

In the Endoplasmic reticulum, the proinsulin is folded in proper confirmation with the formation of 3 disulphide bonds. Folded proinsulin is then transported to the trans-Golgi network, where it is converted into active insulin by cellular endopeptidases called as prohormone convertases PC1 and PC2 and exoprotease carboxypeptidase E. The endopeptidases cleaves at two positions, resulting in the release of a fragment termed as C-peptide. The mature insulin, thus formed consists of an A-chain with 21 aminoacids and a B-chain containing 30 aminoacids and both polypeptides linked together by two disulphide bonds.

Besides, the A-chain has an intrachain disulphide bond [ 37 ],[ 38 ]. However, several disadvantages limit its use for production of recombinant biopharmaceuticals. Various post-translational modifications PTMs such as glycosylation, phosphorylation, proteolytic processing and formations of disulfide bonds which are very crucial for biological activity, do not occur in E.

N-linked glycosylation is the most common posttranslational modification of proteins in eukaryotes. It has been discovered that the bacterium Campylobacter jejuni possess the capability to glycosylate the proteins and it was also shown that a functionally active N-glycosylation pathway could be transferred to E. Although the structure of bacterial N-glycan is different from that observed in eukaryotes, engineering of Campylobacter N-linked glycosylation pathway into E.

Expression of Pglb oligosaccharyltransferase or OTase from C. Recently efforts has been made to produce glycosylated proteins with substrates other than native and non-native to E. The codon usage of the heterologous protein also plays a major role in determining the expression level of recombinant protein. If the codon usage of heterologous protein differs significantly from the average codon usage of the E.

Usually, the frequency of the codon usage reflects the abundance of their corresponding tRNA. Therefore, significant differences in codon usage could result in premature termination of translation, misincorporation of aminoacids and inhibition of protein synthesis [ 49 ].

Expression of heterologous proteins in E. Similarly, co-expression of the genes encoding for a number of the tRNA for rare codon, may enhance the expression of heterologous proteins in E. There are some commercial E. These rare codons have been associated with low expression of proteins in E. The use of protease-deficient E.

Rather than the external parameters, targeted methods such as modifications in protease or secretion pathways can provide the insight into biology of recombinant proteins [ 53 ]. Periplasm has advantages over cytoplasm in less protein concentration and proteolytic activity, improve the production titer [ 55 ], and enhance the solubility of recombinant protein. Altogether, with these advanced modifications and developments ease the process of target protein production thus accelerating the drug development [ 56 ].

Heterologous proteins generally accumulate in E. Use of molecular chaperones may increase the protein solubility and assist in proper folding of recombinant protein. Some of the chaperones prevent aggregation of protein and some assist in refolding and solubilization of misfolded proteins. The most important chaperones in E.

These chaperones may be used singly, or in combination to enhance the protein solubility in E. Recombinant human insulin was first produced in E. After expressing independently, the two chains are purified and co-incubated under optimum reaction conditions that promoted the generation of intact and bioactive insulin by disulphide bond formation. The first commercial recombinant insulin was developed for therapeutic use in human by this two-chain combination procedure [ 60 ].

Another approach involves the expression of a single chemically synthesized cDNA encoding for human proinsulin in E. This approach was more efficient and convenient for large scale production of therapeutic insulin as compared to the two chain combination approach and has been used commercially since [ 60 ]. Eli Lilly followed this technology to produce Humulin, the first recombinant insulin approved in , for the treatment of diabetic patients. These first generation recombinant insulins have an amino acid sequence identical to native human insulin and are preferred over animal derived insulin products [ 14 ].

However, advancement in the field of genetic engineering and development of technology to chemically synthesize genes with altered nucleotide sequence, facilitated the development of insulin analogues with altered amino acid sequence. It had been observed that native insulin in commercial preparations usually exist in oligomeric form, as zinc-containing hexamer due to very high concentration, but in blood, biologically active insulin is in monomeric form [ 61 ].

Hence, this oligomeric complex should dissociate so that insulin can be absorbed from the site of injection into the blood. Due to this, subcutaneously injected recombinant insulin usually have a slow onset with peak plasma concentration after 2 hours of injection and longer duration of action that last for hours [ 62 ]. Hence, in order to develop a fast- acting insulin analogue, it was required to modify the amino acids residues whose side chains are involved in dimer or oligomer formation.

It has been shown that amino acids residues in insulin B-chain particularly B8, 9,12, 13, 16 and play critical role in oligomerization [ 63 ],[ 64 ]. Lispro, developed by Eli Lilly, was the first fast acting insulin analogue to obtain regulatory approval in , for therapeutic use [ 60 ].

Insulin Lispro is engineered in such a way that it has similar amino acid sequence as the native insulin but has an inversion of proline-lysine sequence at position 28 and 29 of the B-chain, which resulted in reduced hydrophobic interactions and thus prevented dimer formations.

Another rapid-acting insulin analogue, produced in E. Insulin Glulisine have been generated by replacing B3 asparagine by a lysine and B29 lysine replaced by glutamic acid [ 14 ]. To avoid multiple injection, long-acting insulin analogues with prolonged duration of actions have also generated. Insulin Glargine is one of such long-acting insulin analogues, which was developed by Aventis Pharmaceuticals and approved by regulatory authorities of USA and EU in Insulin Glargine was generated by replacing the C-terminal asparagine of the A-chain with a glycine residue and the C-terminal of the B- chain was modified by adding two arginine residues.

These modifications resulted in increase of the isoelectric point pI from 5. Glargine was produced as proinsulin and expressed in E. However, after subcutaneous administration, it precipitated due to neutral pH in the subcutaneous tissue. Resolubilization of insulin occur slowly, resulting in longer duration for its release in the blood [ 14 ]. Diabetologia 25 , — Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

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