Case Study - Insulin

Question 1

Give a summary of the Biotechnology Dogma

Answer 1

-DNA is replicated
- DNA is transcribed to produce RNA
- RNA is translated to produce Proteins
- Therapeutic proteins equal cash

Question 2

Give a summary of the Recombinant DNA Dogma

Answer 2

• Recombinant DNA is replicated
• Recombinant DNA is transcribed to produce RNA
• Recombinant RNA is translated to produce Recombinant Proteins
• Recombinant therapeutic proteins equal more cash

Question 3

What year was Human insulin approved?

Answer 3

1982

Question 4

What is Insulin?

Answer 4

Two amino acid chains held together by disulphide bonds

• A chain: 21 amino acids
• B chain: 30 amino acids

The sequence is highly conserved among mammals – biologically active in-between species.

Hydrogen bonding at the c-terminus of the B chain can lead to the formation of dimers and hexamers (in presence of zinc ions)

Involved in the regulation of carbohydrate and fat metabolism
Diabetes mellitus is a metabolic disease in which the body cannot control its insulin synthesis
Patients can be treated with an injection of external insulin

Question 5

What is the molecular weight of insulin

Answer 5

5808 daltons

Question 6

Where is insulin synthesised?

Answer 6

Synthesised by the beta cells in the pancreas

• Synthesised in the beta cells of the Islets of Langerhans (the endocrine / hormone-producing region)
• Initially synthesised as ONE 86 amino acid polypeptide precursor called preproinsulin
• Processed in the cell to form proinsulin
• Undergoes subsequent modification to form insulin
• Excreted from secretory granules in the cell

Question 7

Draw the structure of insulin with its amino acid names.

Answer 7

Week 8 session 8 slide 11

Question 8

Name the 20 amino acids and their properties

Answer 8

WATER LIKING (HYDROPHILIC):
Polar, charged: (HALGA)
-Histidine (His, H)
-Arginine (Arg, R)
-Lysine (Lys, K)
-Glutamate (Glu, E)
-Aspartate (Asp, D)

Polar, uncharged: (STAG)
-Serine (Ser, S)
-Threonine (Thr, T)
-Asparagine (Asn, N)
-Glutamine (Gln, Q)

WATER HATING (HYDROPHOBIC):
Hydrophobic, aliphatic:
-Glycine (Gly, G)
-Alanine, (Ala, A)
-Valine (Val, V)
-Leucine (Leu, L)
-Isoleucine (Ile, I)
-Methionine (Met, M)
-Proline (Pro, P)
-Cysteine (Cys, C)

Hydrophobic, aromatic:
-Phenylalanine, (Phe, F)
-Tyrosine (Tyr, Y)
-Tryptophan (Trp, W)

Question 9

Draw and explain the insulin biosynthesis

Answer 9

https://www.youtube.com/watch?v=EEliypf07JU (quick video)

https://www.youtube.com/watch?v=WJLVFtvxTr8 (more detailed video)

1. Pre-proinsulin mRNA is translated and transcribed in beta cells (in the rough ER)
2. Pre-proinsulin (110 amino acids) consists of 4 parts:
   - Signal peptide a.k.a leader sequence (N-terminal) (36 aa)
   - A polypeptide chain (21 aa)
   - B polypeptide chain (30 aa)
   - C polypeptide chain (23)
3. When entering its secretary vesicle the signal peptide is cleaved by a signal peptidase this then produces proinsulin
4. After the removal of the N-terminal, A and B chains connected with 3 disulfide bridges (DBS) are made 2 interchain DBS and 1 intrachain DBS.
5. These vesicles contain various endopeptidases which cleave off the C-chain polypeptide to produce mature insulin.
6. The histidine molecules in the B-chain then interact with zinc molecules with 6 insulin molecules per zinc ion forming hexamer crystals. This then makes the insulin ready to be secreted

Question 10

What is the role of the signal peptide in pre-proinsulin?

Answer 10

The signal peptide also known as leader sequence is a bit of DNA which leads the mRNA of pre-proinsulin into the lumen of the rough endoplasmic reticulum.

Question 11

What are the therapeutic uses of insulin?

Answer 11

• Used to treat Type I Diabetes (since 1922)
• Original sources involved extraction from either the bovine or porcine pancreas
• A recombinant human insulin was approved for use in 1982

Question 12

What are the choices of Insulin cell selection?

Answer 12

Human tissue:
- Low supply and ethical issues

Mammalian cells:
- Porcine (pork) pancreas can be used to extract insulin
- Differs by only one amino acid (amino acid number 30 (Thr) on the B chain, it is Ala instead)
- Expensive, low supply of raw material

Microbial Cells:
- Commercially both bacteria and yeast are currently used to synthesise insulin
- Usually, due to the commercially sensitive nature of biopharmaceutical production, exact details of systems are not published and openly available
- However, there is still considerable academic work available to view in this area

Question 13

Draw the system of the Expression System

Answer 13

Week 8 Session 8 slide 34

Question 14

Why do insulin production in E.coli rely on the use of synthetic genes?

Answer 14

Insulin production in E.coli relies on the use of synthetic genes due to gene compatibility from human insulin and E.coli

It was possible to make an insulin gene one by one since it was a relatively small number of base pairs as well as the amino acid sequence was known. This approach avoids issues in relation to gene compatibility

Question 15

What was the gene compatibility between human insulin and E.coli?

Answer 15

Prokaryotic and Eukaryotic cells both use genes made from DNA, however, their structure differs significantly.

The codon for human insulin differs from the E.coli codons, that’s why it’s important to have the codons of the desired genes that E.coli would like therefore

Question 16

What was the gene compatibility between human insulin and E.coli?

Answer 16

Prokaryotic and Eukaryotic cells both use genes made from DNA, however, their structure differs significantly.

The codon for human insulin differs from the E.coli codons, that’s why it’s important to have the codons of the desired genes that E.coli would prefer so it would help the process go smoother.

Another reason for Human genes to be compatible with E.coli is Eukaryotic cells are much more complicated and they contain all the eukaryotic genes that help in the eukaryotic secretion that prokaryotes don’t need. Therefore there is a lot of engineering that takes place on the eukaryotic cells before it is inserted into the prokaryotic cell.

Question 17

What is Oligonucleotide Synthesis?

Answer 17

This would produce short pieces of DNA, such as creating primers.

Question 18

Why did they need oligonucleotide synthesis for insulin gene production?

Answer 18

This was how they created the DNA of the insulin to insert it into the E.coli plasmid.

Question 19

What is the manufacturing process of Insulin?

Answer 19

First synthesised in the 1970s by Genentech & Eli Lilley & Co. They did this by the Two Chain Method.

Two-Chain Method:
- A chain and B chain of insulin are produced separately and then chemically fused.
- Purified A and B chains are then incubated under oxidizing conditions to form disulphide bonds that are present in human insulin

Question 20

What is the strategy for synthesising insulin in E.coli?

Answer 20

Plasmid Insertion:
- A-chain and B-chain are inserted onto a plasmid.
- EcoRI met codon + A/B-chain + BamHI

Then they go through transformation into an E.coli cell

Then they go through a selection
- Ampicillin ensures all cells which grow on the plate carry a plasmid.
- IPTG (an inducer) prevents negative regulation of the lacZ gene activating the production of β galactosidase if functional
- X-Gal generates a blue chromogenic product in the presence of the enzyme β galactosidase

Fermentation:
After Selection, they are then grown in two different bioreactors. The ampicillin resistance and lacZ genes present on the plasmids are used for the identification of successfully transformed cells, as they show resistance against the ampicillin present in the growth medium and encode β-galactosidase. These successfully transformed cells are retained for replication under optimal conditions and then transferred into a bioreactor for commercial production. The A and B chains are synthesized as their respective E. coli strains replicate in separate fermenters

Cell Lysis:
The bacterial cells are removed from the bioreactor tank and must be lysed with one of several methods, such as enzyme digestion, sonication, or freezing and thawing of cells. The use of lysosome enzymes is preferred for large-scale operations, as it digests the bacterial outer layer to release the insulin into the surrounding media, so detergents can later be added to remove the cell wall. Gel filtration and ion-exchange chromatography methods are used to remove impurities

Insulin Chain Isolation:
The isolated purified protein has an insulin chain fused with β-galactosidase, as it has been linked with the gene incorporated into the plasmid and translated along with it. Cyanogen bromide is used to separate the insulin chains from β-galactosidase, as it splits the protein at the methionine residue which begins the β-galactosidase protein

Chain Joining:
The insulin chains (A and B) are treated with sodium dithionite and sodium sulfite to form the disulfide bonds that bridge the chains. This whole process is called reduction-reoxidation, and it is induced by β-mercaptoethanol and air oxidation to synthesize human insulin

Question 21

Draw the plasmid map of two chain method

Answer 21

http://www.bmrat.org/index.php/BMRAT/article/view/692#

Question 22

What is the Proinsulin Method?

Answer 22

Again commercialised by Eli Lilley

http://www.bmrat.org/index.php/BMRAT/article/view/692#:~:text=The%20second%20commercial%20method%20for,different%20fermenters%20and%20then%20purified.

Plasmid insertion:
-This recombinant protein is produced by incorporating proinsulin-producing plasmids into E.coli.

Transformation:
- The transformed cells are then grown on tryptic soy broth containing the antibiotic kanamycin monosulfate. The plasmid contains a kanamycin monosulfate resistance gene along with the proinsulin coding genes, so the transformed E. coli can survive in the broth. However, kanamycin monosulfate kills the E. coli cells that have not been transformed

Fermentation:
- The transformed E.coli then go through fermentation.

Cell Lysis:
- Centrifugation - Cell isolation is the first step in down streaming of the insulin made by transformed E. coli cells. This process is also referred to as cell harvesting because the proinsulin inclusion bodies are harvested using both filtration and centrifugation.
- Homogenization - The proinsulin inclusion bodies present inside the cell contain insulin precursor products in the form of proinsulin fusion proteins. Since they are present in dense aggregates, they are protected from being processed into the soluble form within the cytoplasm. To release these inclusion bodies, various methods of cell membrane disruption are available. In this particular process, high-pressure homogenization with a blade-type homogenizer, and chemical alkali treatment are used

Inclusion Body Separation:
- After the E. coli have been lysed, the inclusion bodies need to be isolated from the cell debris. For this purpose, centrifugation can be used for reverse osmosis.
- After the separation of inclusion bodies, proinsulin is in an insoluble form and therefore must be solubilized. This is accomplished through the addition of denaturing agents such as urea or guanidium hydrochloric acid, which will release the fusion proteins

Insulin Chain Isolation:
The isolated purified protein has an proinsulin chain fused with β-galactosidase, as it has been linked with the gene incorporated into the plasmid and translated along with it. Cyanogen bromide is used to separate the insulin chains from β-galactosidase, as it splits the protein at the methionine residue which begins the β-galactosidase protein

Site-Specific Cleavage:
- The Proinsuline then undergoes protease treatment where the C-chain is cleaved off

Question 23

advantages and disadvantages of E.coli

Answer 23

Pros:
- Well-characterized genetics
* Versatile cloning tools
* Rapid growth & very high titres
* Low-cost media

Cons:
* Lack of PTMs
* Lack of disulfide bond formation
* Proteolytic protein maturation
* Inclusion-body formation
* Surface lipopolysaccharide (LPS)
are pyrogenic

Things to consider
* Copy the number of the plasmid
* pUC (500 to 700 copies)
* pBR322 (15 to 20 copies)
* Strength of promoter
* Induction (independent of the
media component)
* Controlling degradation of mRNA

Question 24

Why can’t disulfide bonds form in the cytoplasm or cytosol?

Answer 24

First of all, the thiol-disulfide redox potential of the cytoplasm is too low to provide a sufficient driving force for the formation of stable disulfide.

Second, under physiological conditions, there are no enzymes that can catalyze protein thiol oxidation.

Question 25

What is the formulation of creating disulfide bonds?

Answer 25

R-SH SH-R –oxidation–> R-S-S-R + 2H+ + 2e-

Safwan’s lecture slide 45