Genetically Engineered Insulin

The advances in recombinant DNA technology have occurred in parallel with the development of biological processes and genetic medicines. New technologies have produced large amounts of biochemically defined proteins of medical significance, creating enormous potential for pharmaceutical industries. Insulin is one of them. 

1.0Introduction

• Insulin is a source of beta cells and comprises 55 amino acid residues. It is divided into two chains, A (21 AA) and B (30AA), held together by peptide bonds. It is water soluble, has a half-life of 5-10 minutes, and is significant in regulating glucose in our body. It can be produced artificially as well as naturally inside the body. 
• Under artificial conditions, the genes capable of producing Insulin are isolated from human cells and transferred into plasmids of E. coli. Then, the strains are grown in a specific culture media, which produces proinsulin. This may convert into Insulin in the lab via several changes and can be used as natural Insulin to treat various diseases, such as diabetes, and other biochemical reactions.

2.0Genetically Engineered Insulin Definition

Insulin is a hormone that regulates the amount of sugar or glucose in our body and participates in other biochemical activities. Most commonly, it is produced naturally inside the body. Still, due to some genetic and other factors, insulin production is affected, which causes the regulation of sugar levels in the blood to avoid the condition of GEI. They were prepared in the lab under in vitro conditions with the help of recombinant DNA technology (RDT).

3.0Structure of Insulin

4.0Types of Insulin

• There are many types of Insulin. Some work slowly, and some quickly. Slower or long-acting Insulin is also called Basal insulin. Basal insulins deliver a steady supply of Insulin that helps control blood sugar levels over time. 
• Fast-acting Insulin is also called Bolus insulin. Bolus is a fancy word for "extra". 

5.0Insulin Production

Cows and pigs are immunogenic. Yeast cells are costly, and plant cells are not fully developed. Hence, E. coli is preferred. They are most commonly used in insulin production because they contain simple, well-understood genetics, are very easy to manipulate, the culturing cost is minimal, have a high level of expression, are easy to ferment and scale up, and inclusion bodies are easy to purify.

Basic Protocol

1. Isolation:

• To produce Genetically Engineered Insulin, the genes capable of producing insulin are first isolated from human cells and subjected to Recombinant DNA Technology. For this purpose, they are inserted into the plasmids of two different E. coli through transformation. 

2. Insertion:

Insertion of the gene into the plasmid is carried out in two different steps. 

• The first is fragmentation or restriction, in which restriction endonuclease enzymes restrict the plasmid. 
• The second step is ligation, which is done by ligate enzymes. 

3. Cultering:

• Then, the E.coli-containing insulin-producing genes are grown and cultured into fermenters with specific culture media for the growth of E.coli. At that condition, proinsulin is formed inside the bacterial strains as the product of insulin-producing genes. 

4. Collection of Proinsulin:

• After the appropriate growth of E.coli, they break down, and proinsulin is collected containing chains A, B, and C. 

5. Cleavage:

• Site-specific cleavage: Two enzymes, trypsin and carboxypeptidase B, cleave the proinsulin at a specific site and convert it to native insulin and C-peptide. 

6. Purification:

• For purification of C-peptide & human insulin, Reverse Phase Liquid Chromatography is very effectively used due to high pressure, which increases the speed & purity of C Peptide & human insulin

7. Upstream Processing:

Step 1 :-

Obtaining the human insulin gene Two general strategies are commonly used to obtain the human insulin gene. They are Complementary DNA (cDNA) obtained from messenger RNA (mRNA) of the two chains using enzyme reverse transcriptase cloning of cDNA of both chains using polymerase chain reactions (PCR). This involves amplification of the cDNA sequences as not every gene yields measurable amounts of mRNA.

Step 2 :-

Insertion of cDNA of both chains into plasmids. Bacterial plasmids are cut using specific restriction enzymes to insert the two DNA molecules into separate plasmids. Each cDNA is extended at its 5' terminus with an ATG (methionine) initiation codon for the start of translation and a translation termination signal at its 3' with the sticky ends EcoRI and BamHI (later as restriction sites). Two vector plasmids are made for both the cDNA. 

They are inserted in the plasmids at the EcoRI and BamHI sites next to the lacZ gene, which encodes for the enzyme β-galactosidase. In E. coli, β-galactosidase is the enzyme that controls the transcription of the genes. The insulin gene must be tied to this enzyme to make the bacteria produce insulin. The cut plasmids are re-ligated by specific DNA ligases. 

Step 3 :-

Transfection Recombinant plasmids enter the bacteria in a process known as transfection. Methods such as CaCl2 treatment and electroporation can be used. These cells are later known as transformed cells. 

Step 4 :-

Media and equipment preparation The LB broth is prepared using the LB powder. It is autoclaved, and ampicillin and lactose are added (after the sterilization to prevent denaturation or destruction). 

Inoculation is done by adding the transformed bacteria to the media. The bioreactor is also prepared. Parts of the bioreactors are fixed and checked, such as the calibration of the pH electrode, pO2 probe, exhaust condensers, and air inlet. The bioreactor is then sterilized. 

Step 5:-

Fermentation This stage consists of minor scaling (enrichment liquid culture in shake flask) to large scaling (fermentor). The two chains are grown separately. Small scaling (early stage) uses shake flasks to do the enrichment culture method for selecting the desired type of E. coli for fermentation. 

The fermentation broth contains two unique components - an antibiotic known as ampicillin and lactose. Bacterial cells that have successful transformation will contain the plasmid gene, which includes the ampicillin resistance gene, and the lacZ gene, which encodes for β-galactosidase in the presence of lactose. 

Therefore, These cells can grow in the ampicillin environment, and the transcription of the lacZ gene will result in the human insulin chain DNA transcription. Bacterial cells that have failed the transformation do not contain the ampicillin resistance gene and the lacZ gene. As a result, the growth of these cells will be suppressed by ampicillin and will not replicate during the fermentation process. 

They are moving on to the large scale, where transfected bacterial cells are transferred from the small flask and replicated under optimal conditions such as temperature and pH in fermentation tanks. 

This step involves process monitoring and control. The bacterial cell processes turn on the gene for human insulin chains, producing insulin chains in the cell.

8. Downstream Processing 

Step 6 :-

Isolation of crude products Cells are removed from tanks and are lysed using different methods such as enzyme digestion, freezing and thawing and sonication. For enzyme digestion, the lysosome enzyme is used to digest the bacterial cells' outer layer, and a detergent mixture is subsequently added to separate the cell wall membrane. 

Step 7 :-

Purification of crude product Centrifugation is conducted to help separate the cell components from the products. Stringent purification of the recombinant insulin chains must be taken to remove any impurities. 

This uses several chromatographic methods such as gel filtration and ion-exchange, along with additional steps which exploit differences in hydrophobicity. 

Step 8 :-

Obtaining of insulin chains The proteins isolated after lysis consist of the fusion of βgalactosidase and insulin chains because there is no termination or disruption to the synthesis of these two proteins as the genes are linked together; therefore, cyanogen bromide is used to split the protein chains at methionine residues, allowing the insulin chains to be obtained.

Step 9 :-

Synthesis of active insulin Two chains (A and B) form disulfide bonds using sodium dithionite and sodium sulphite, and the chains are joined through a reaction known as reduction-reoxidation under beta-mercaptoethanol and air oxidation, resulting in Humulin-synthetic human insulin. 

Step 10 :-

PR-HPLC to obtain highly purified insulin. Lastly, reverse-phase high-performance liquid chromatography (PR-HPLC) is performed to remove almost all impurities and produce highly purified insulin. The insulin can then be polished and packaged to be sold in other industries. 

9. Storage of Gel:

Saline dilution and Zn+3 complexion are used to keep valuable insulin longer by blocking its immediate use by cells and stabilizing the protein. The insulin is stored at 40 C, so the solution cannot ultimately come out.

6.0Type 1 Diabetes

• Diabetes mellitus, commonly referred to as diabetes, is a metabolic disease characterized by issues with insulin secretion, action, or both that lead to hyperglycemia. There are two primary types of diabetes: Type 1 and Type 2.
• Type 1 diabetes occurs due to impaired insulin secretion caused by damage to the β cells in the pancreas.
• Type 2 diabetes is caused by increased insulin resistance or decreased insulin secretion from the pancreas.
• Diabetes can lead to long-term complications, including damage and dysfunction in the eyes, kidneys, nerves, heart, and blood vessels, potentially resulting in disability and death. Experimental animal models are frequently used in research to understand the disease's pathogenesis better and explore prevention and treatment options.

7.0Insulin Plant

• Costus igneus Nak is commonly referred to as the fiery costus, step ladder, spiral flag, or insulin plant. Native to South and Central America, this plant has recently been introduced to India as an herbal remedy for diabetes
• BASAGLAR is a prescription long-acting insulin used to control high blood sugar levels in adults with type 1 or type 2 diabetes and children with type 1 diabetes.

8.0Function of Insulin

Insulin maintains glucose homeostasis by keeping plasma glucose levels within an optimal range throughout the day. Its main effects include: 

(i) stimulating glucose oxidation and glycogen storage in the liver, as well as converting glucose into triglycerides and promoting protein synthesis; 

(ii) facilitating glucose uptake and glycogen storage in muscle tissue; and

(iii) promoting glucose uptake and conversion into triglycerides for storage in fat tissue.