10.4 Formation of Human Insulin Protein in Bacteria

Human Insulin is produced by recombinant DNA technology, which involves a series of steps. Insulin is a hormone produced by the beta cells of the islets of Langerhans in the pancreas. The gene for insulin is called INS and is located on human chromosome 11. The production of human insulin in bacteria involves the isolation of the insulin gene, insertion into a plasmid vector, transformation into bacteria, and extraction and purification of the insulin protein.

Definition

Human Insulin: Human Insulin is produced by recombinant DNA technology, which involves the following steps.

Insulin Gene: The insulin gene, which produces human insulin, is isolated from a human DNA sample. Insulin consists of two peptide chains (α and β) linked by disulfide bonds. Insulin is produced by endocrine β-cells in the islets of Langerhans of pancreas. Gene for insulin is called INS (word insulin derived from the Latin word insula, means "island") and is located on the human chromosome 11 at position 11p15.5 (on short arm (p), region 11, band 15 and sub band 5). The gene is 1.5 Kbp long having about 1,425 bp with three exons and two introns.

Main Idea

• Identification and isolation of the insulin gene
• Selection of suitable plasmid for insertion of insulin gene
• Creation of recombinant DNA
• Insertion of recombinant plasmids into host cells
• Production of insulin chains
• Extraction and purification
• Folding and disulphide bridging of both insulin chains
• Purification and testing
• Packaging for storage, shipment, and usage

Core Concept

• Insulin = hormone produced by beta cells of islets of Langerhans
• Insulin gene = INS; located on chromosome 11 at 11p15.5
• Insulin gene = 1.5 Kbp long; 1,425 bp; three exons; two introns
• Insulin = two peptide chains (α and β) linked by disulfide bonds
• α chain = 21 amino acids
• β chain = 30 amino acids
• **Plasmids used** = pBR322 and pUC18/pUC19
• Recombinant DNA = insulin gene inserted into plasmid
• Host cells = E. coli
• Transformation = insertion of recombinant plasmids into host cells
• Production = insulin chains produced as fusion proteins
• Extraction = insulin chains separated from fusion proteins
• Folding = disulphide bridges formed
• Purification = insulin purified and tested
• Packaging = insulin packaged into vials or pens

Detailed Explanation

1. Identification and Isolation of the Insulin Gene

• The insulin gene, which produces human insulin, is isolated from a human DNA sample.
• Insulin consists of two peptide chains (α and β) linked by disulfide bonds.
• Insulin is produced by endocrine β-cells in the islets of Langerhans of pancreas.
• Gene for insulin is called INS (word insulin derived from the Latin word insula, means "island") and is located on the human chromosome 11 at position 11p15.5 (on short arm (p), region 11, band 15 and sub band 5).
• The gene is 1.5 Kbp long having about 1,425 bp with three exons and two introns.

2. Selection of Suitable Plasmid for Insertion of Insulin Gene

• The production of human insulin by recombinant DNA technology involves plasmids that are specifically engineered to efficiently transfer insulin gene in host cells e.g. Escherichia coli.
• Two commonly used plasmids in the production of insulin are:
• Both above mentioned plasmids have smaller size, origin of replication, higher copy number and multiple unique restriction sites, which simplifies the insertion of the insulin gene.
• Human genes for chain α and β of insulin may be inserted into these plasmid, for expression in bacterial cells to synthesize insulin chains.

3. Creation of Recombinant DNA

• Bacteria e.g. E. coli are not so advanced cells so cannot directly produce eukaryotic proteins like insulin, so two separate synthetic genes are created for the α and β chains of insulin.
• These genes for both chains (α and β) of insulin and separately inserted into two separate bacterial plasmid vectors i.e. pBR322 plasmid.
• These genes are inserted into the plasmid by the side of β-galactosidase gene.
• The plasmids are engineered with a promoter sequence that helps to initiate the auto-production of insulin chains when inside the bacterial cells.

4. Insertion of Recombinant Plasmids into Host Cells

• The recombinant plasmids are then separately inserted into E. coli (or another bacterial strain) through a process called transformation.
• The bacteria take up the recombinant plasmids and become recombinant bacteria.

5. Production of Insulin Chains

• The bacteria are allowed to grow in large fermentation tanks containing suitable culture medium and optimum conditions.
• When bacteria grow, recombinant plasmid DNA is replicated resulting in multiplication (cloning) of insulin gene.
• Bacteria also express the insulin genes for production of insulin.
• The recombinant bacteria produce pro-insulin chains i.e. fused β-galactosidase-A chain and β-galactosidase-B-chain separately.
• Both chains are produced as part of a fusion proteins, which help in protecting the insulin chains from bacterial protease enzymes during bacterial synthesis.

6. Extraction and Purification

• For extraction of insulin, the bacterial cells are harvested and the pro-insulin chains combined with fusion proteins (β-galactosidase) are isolated.
• These pro-insulin chains A and B are separated from Fusion proteins, β-galactosidase by chemical treatment with cyanogen bromide.
• The removal of pro-insulin chains from fusion protein β-galactosidase is possible because, an extra codon is added before methionine codon at N-terminal of each gene for A and B-chain.

7. Folding and Disulphide Bridging of Both Insulin Chains

• After detachment from fusion proteins, A and B chains of insulin are chemically joined in-vitro to form the functional insulin.
• Disulphide bridges at cysteine residues of both insulin peptide chains are formed by treating with sodium disulphonate and sodium sulphite, resulting in biologically active insulin.

8. Purification and Testing

• The finally produced insulin is further purified to remove any impurities or by-products of bacterial gene expression.
• After purification, the insulin is tested for quality, effectiveness, safety and possible side effects.

9. Packaging for Storage, Shipment and Usage

• After confirmation of best quality, insulin is packaged into vials or insulin pens containing instruction of storage and usage for injection.
• Thus recombinant human insulin is pure, safer and much effective than insulin previously derived from animals.

Science Titbits

• Insulin was discovered by Edward S. Schafer in 1916.
• It is the major regulator of blood sugar in the body.
• Fredrick Sanger and his team in 1954 first time described the complete structure of insulin.
• Initially, β-cells synthesize pre-pro-insulin polypeptide, having 109 amino acids.
• Out of 109 amino acids, 23 amino acids act as signal molecules needed for entry of the pre-pro-insulin through cell membrane.
• When inside the cell, it become inactive pro-insulin with 86 amino acids, that finally converted to active insulin with 51 amino acids by some proteolytic enzymes.
• Active insulin consists of two polypeptide chains, chain α has 21 amino acids and chain β has 30 amino acids, both linked together by disulphide bonds.

Summary Table of Steps

Intermediate Level

Steps of Insulin Production

Plasmids Used

Insulin Chains

Key Enzymes and Chemicals

Formula / Mathematical Form

Not applicable to this topic.

Solved Example

Not applicable to this topic (descriptive topic).

Important Points

• 1. Human insulin is produced by recombinant DNA technology.
• 2. Insulin is produced by beta cells of islets of Langerhans.
• 3. Insulin gene is called INS.
• 4. Insulin gene is located on chromosome 11 at 11p15.5.
• 5. Insulin gene is 1.5 Kbp long.
• 6. Insulin gene has 1,425 bp.
• 7. Insulin gene has three exons and two introns.
• 8. Insulin consists of two peptide chains: α and β.
• 9. α chain has 21 amino acids.
• 10. β chain has 30 amino acids.
• 11. Chains are linked by disulfide bonds.
• 12. Plasmids used: pBR322 and pUC18/pUC19.
• 13. pBR322 has ampicillin and tetracycline resistance.
• 14. pUC18/pUC19 have ampicillin resistance.
• 15. Two separate synthetic genes are created for α and β chains.
• 16. Genes are inserted into two separate plasmids.
• 17. Genes are inserted by the side of β-galactosidase gene.
• 18. Recombinant plasmids are inserted into E. coli.
• 19. Bacteria produce insulin chains as fusion proteins.
• 20. Cyanogen bromide separates insulin chains from fusion proteins.
• 21. Sodium disulphonate and sodium sulphite form disulphide bridges.
• 22. Insulin is purified and tested.
• 23. Insulin is packaged into vials or pens.
• 24. Recombinant insulin is pure, safer, and more effective.
• 25. Insulin was discovered by Edward S. Schafer in 1916.

Remember

• - Insulin = hormone produced by beta cells of islets of Langerhans
• - Insulin gene = INS; located on chromosome 11 at 11p15.5
• - Insulin gene = 1.5 Kbp long; 1,425 bp; three exons; two introns
• - Insulin = two peptide chains (α and β) linked by disulfide bonds
• - α chain = 21 amino acids
• - β chain = 30 amino acids
• - **Plasmids used** = pBR322 and pUC18/pUC19
• - Recombinant DNA = insulin gene inserted into plasmid
• - Host cells = E. coli
• - Transformation = insertion of recombinant plasmids into host cells
• - Production = insulin chains produced as fusion proteins
• - Extraction = insulin chains separated from fusion proteins
• - Folding = disulphide bridges formed
• - Purification = insulin purified and tested
• - Packaging = insulin packaged into vials or pens

Common Mistakes

• 1. Confusing insulin and pro-insulin — Pro-insulin is inactive; insulin is active.
• 2. Mixing up the chains — α chain = 21 amino acids; β chain = 30 amino acids.
• 3. Forgetting the location of the insulin gene — Chromosome 11 at 11p15.5.
• 4. Mixing up the plasmids — pBR322 (ampicillin and tetracycline); pUC18/pUC19 (ampicillin).
• 5. Forgetting the role of β-galactosidase — Fusion protein.
• 6. Mixing up the chemicals — Cyanogen bromide for separation; sodium disulphonate and sodium sulphite for disulphide bridges.
• 7. Believing bacteria directly produce insulin — They produce insulin chains as fusion proteins.
• 8. Mixing up the steps — Isolation, plasmid selection, recombinant DNA, transformation, production, extraction, folding, purification, packaging.
• 9. Thinking animal insulin is better — Recombinant insulin is pure, safer, and more effective.
• 10. Forgetting the discovery of insulin — Edward S. Schafer in 1916.

Quick Revision

Human Insulin is produced by recombinant DNA technology, which involves the following steps: 1. Identification and Isolation of the Insulin Gene: The insulin gene, which produces human insulin, is isolated from a human DNA sample. Insulin consists of two peptide chains (α and β) linked by disulfide bonds. Insulin is produced by endocrine β-cells in the islets of Langerhans of pancreas. Gene for insulin is called INS (word insulin derived from the Latin word insula, means "island") and is located on the human chromosome 11 at position 11p15.5 (on short arm (p), region 11, band 15 and sub band 5). The gene is 1.5 Kbp long having about 1,425 bp with three exons and two introns. 2. Selection of suitable plasmid for insertion of insulin gene: The production of human insulin by recombinant DNA technology involves plasmids that are specifically engineered to efficiently transfer insulin gene in host cells e.g. Escherichia coli. Two commonly used plasmids in the production of insulin are: 1. pBR322 is one of the most widely used plasmids in genetic engineering. It contains antibiotic resistant genes for ampicillin and tetracycline. 2. pUC18/pUC19: These plasmids are derivatives of pBR322 that have been modified for efficient transformation. They include a gene for ampicillin resistance. Both above mentioned plasmids have smaller size, origin of replication, higher copy number and multiple unique restriction sites, which simplifies the insertion of the insulin gene. Human genes for chain α and β of insulin may be inserted into these plasmid, for expression in bacterial cells to synthesize insulin chains. 3. Creation of Recombinant DNA: Bacteria e.g. E. coli are not so advanced cells so cannot directly produce eukaryotic proteins like insulin, so two separate synthetic genes are created for the α and β chains of insulin. These genes for both chains (α and β) of insulin and separately inserted into two separate bacterial plasmid vectors i.e. pBR322 plasmid. These genes are inserted into the plasmid by the side of β-galactosidase gene. The plasmids are engineered with a promoter sequence that helps to initiate the auto-production of insulin chains when inside the bacterial cells. 4. Insertion of recombinant plasmids into host Cells: The recombinant plasmids are then separately inserted into E. coli (or another bacterial strain) through a process called transformation. The bacteria take up the recombinant plasmids and become recombinant bacteria. 5. Production of insulin chains: The bacteria are allowed to grow in large fermentation tanks containing suitable culture medium and optimum conditions. When bacteria grow, recombinant plasmid DNA is replicated resulting in multiplication (cloning) of insulin gene. Bacteria also express the insulin genes for production of insulin. The recombinant bacteria produce pro-insulin chains i.e. fused β-galactosidase-A chain and β-galactosidase-B-chain separately. Both chains are produced as part of a fusion proteins, which help in protecting the insulin chains from bacterial protease enzymes during bacterial synthesis. 6. Extraction and Purification: For extraction of insulin, the bacterial cells are harvested and the pro-insulin chains combined with fusion proteins (β-galactosidase) are isolated. These pro-insulin chains A and B are separated from Fusion proteins, β-galactosidase by chemical treatment with cyanogen bromide. The removal of pro-insulin chains from fusion protein β-galactosidase is possible because, an extra codon is added before methionine codon at N-terminal of each gene for A and B-chain. 7. Folding and disulphide bridging of both insulin chains: After detachment from fusion proteins, A and B chains of insulin are chemically joined in-vitro to form the functional insulin. Disulphide bridges at cysteine residues of both insulin peptide chains are formed by treating with sodium disulphonate and sodium sulphite, resulting in biologically active insulin. 8. Purification and testing: The finally produced insulin is further purified to remove any impurities or by-products of bacterial gene expression. After purification, the insulin is tested for quality, effectiveness, safety and possible side effects. 9. Packaging for storage, shipment and usage: After confirmation of best quality, insulin is packaged into vials or insulin pens containing instruction of storage and usage for injection. Thus recombinant human insulin is pure, safer and much effective than insulin previously derived from animals.

60-Second Revision

• Insulin = hormone produced by beta cells of islets of Langerhans
• Insulin gene = INS; located on chromosome 11 at 11p15.5
• Insulin gene = 1.5 Kbp long; 1,425 bp; three exons; two introns
• Insulin = two peptide chains (α and β) linked by disulfide bonds
• α chain = 21 amino acids
• β chain = 30 amino acids
• Plasmids used = pBR322 and pUC18/pUC19
• Recombinant DNA = insulin gene inserted into plasmid
• Host cells = E. coli
• Transformation = insertion of recombinant plasmids into host cells
• Production = insulin chains produced as fusion proteins
• Extraction = insulin chains separated from fusion proteins
• Folding = disulphide bridges formed
• Purification = insulin purified and tested
• Packaging = insulin packaged into vials or pens
• Warning: Bacteria produce insulin chains as fusion proteins; they do not directly produce insulin

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