How can insulin be produced by genetic engineering

Insulin production Before genetic engineering, insulin was obtained from pigs and cattle. Process The human insulin gene is removed using a restriction enzyme. A bacterial plasmid is cut open using the same restriction enzyme. Using the same restriction enzyme to cut both the human DNA and bacterial plasmid results in complementary sticky ends that join by base pairing. A different enzyme is used to join the insulin gene and the bacterial plasmid. At first suitable vector plasmid is isolated from E.

The gene of interest ie. Plasmid and gene of interest are recombined together by DNA ligase enzyme This recombined plasmid is inserted into suitable host cell ie E. The recombinant plasmid were then separately transformed into E. The recombinant host produced pro-insulin chains ie. The single chain proinsulin was purified and converted to active insulin by a trypsin-mediated transpeptidation reaction in presence of threonine ester [ 19 ].

Besides native recombinant insulin, various insulin analogues are also being produced in S. Insulin Aspart is another fast-acting insulin analogue, which was produced in S. Insulin Aspart was generated by replacing proline residue at position 28 with aspartic acid in the B-chain.

This genetic modification resulted in an increase in inter-chain charge repulsion, decrease in self-association and thus causing rapid entry into the blood from the site of subcutaneous injection [ 63 ],[ 75 ]. Insulin Detemir is another recombinant long-acting insulin analogue that was commercially produced in S. Recombinant Detemir have been generated by removing the threonine residue at the 30 position of the B-chain, and a C14 fatty acid chain covalently attached to the lysine residue at the 29 position of the B-chain.

These genetic alterations resulted in the binding of insulin to albumin in plasma, which ensured the slow and constant release of insulin and thus prolonging its duration of action up to 24 hours [ 76 ]-[ 78 ]. Sacharomyces cerevisiae has been reported for the production of more than 40 different recombinant proteins [ 79 ].

A few of which related to diabetes are illustrated in Table 2 , along with different characteristics. Furthermore, a synthetic leader sequence had been developed by Kjeldsen and associates at Novo Nordisk for more efficient protein secretion in yeast [ 79 ],[ 80 ].

Transgenic plants have been utilized to produce recombinant proteins because of their advantage of cost effectiveness, high quality protein processing, absence of human pathogens, ease of production and presence of eukaryotic machinery for posttranslational modifications.

Initially, the human growth hormone was the recombinant protein product extracted from transgenic tobacco plant [ 81 ]. After that, numerous different products have developed from plants such as Hepatitis-B-Virus surface antigen, antibodies, industrial proteins and milk proteins. Recombinant human insulin has been successfully expressed and produced in oilseeds of plant Arabidopsis thaliana [ 27 ].

This technology involved the targeted expression of insulin in subcellular organelles known as oilbodies that allowed very high level of expression with easy recovery of recombinant insulin. Oilbodies are storage organelles inside the oilseeds, which comprises of hydrophobic triacylglycerol core encapsulated by phospholipid membrane and an outer wall of proteins known as oleosins.

Genetically engineered oil seeds have been generated with recombinant protein specifically targeted to oilbodies as oleosin fusion [ 27 ],[ 82 ],[ 83 ]. Then the oilbodies are easily separated from other seed components by liquid-liquid phase separation, which reduced the number of chromatography steps required to obtain purified insulin.

It has been observed that insulin accumulated to high level in transgenic seed 0. Recombinant insulin was cleaved from the oleosin fusion partner and matured with trypsin digestion following oil body purification to yield a biologically active insulin. This study clearly demonstrated that expression of insulin as oleosin fusion protein in plant allow accumulation of large amount of recombinant insulin within the seed and also provide simple downstream purification by centrifugation i.

Subsequent maturation to obtain biologically active insulin can be accomplished using standard enzymatic methods currently used for commercial production of insulin from E. Oilseeds also act as a natural cellular warehouse, where recombinant insulin can be stockpiled until required [ 27 ]. In another approach, transgenic plants have been generated, in which, tobacco and lettuce chloroplasts were transformed with human proinsulin comprised of A, B and C-chains fused with the cholera toxin B subunit [ 28 ].

Oral delivery of unprocessed proinsulin encapsulated in plant cell or by injection into mice revealed lowering of blood glucose levels similar to commercially available insulins. C-peptide of proinsulin, which is not present in current commercially available insulin and insulin analogues derived from E. Very high level of expression of biologically active proinsulin in tobacco and lettuce leaves and long-term stability in dried leaves offers a reliable low-cost technology for both injectable as well as oral delivery of proinsulin.

Dietary and lifestyle changes are causing dramatic increase in diabetes incidence all over the world. Both Type I and Type II diabetic patients use insulin, however late stage Type II diabetes patients require large doses of insulin as they develop insulin resistance.

The dramatic increase in the number of diabetic patients globally and exploration of alternate insulin delivery methods such as inhalation or oral route is bound to escalate the demand for recombinant insulin in near future. Current manufacturing technologies will not be able to meet the growing demand of insulin due to limitation in production capacity and high production cost.

Recombinant human insulin is produced predominantly using E. However, there is an upmost need to increase the production by several fold of a biologically active insulin and its analogues from E.

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