Genetic Engineering and Human Hormones

Page 1: Basic Terminology

• Genetic engineering: A process used to alter the genetic makeup of an organism.

• Production of insulin: The process of manufacturing insulin through recombinant DNA technology.

• Human hormones: Chemical messengers produced by various glands that regulate physiological processes in the body.

Page 2: Learning Objectives

At the end of this module, participants will be able to:

• Understand the definition, function, importance, and history of insulin.

• Describe insulin production using recombinant DNA (rDNA) technology.

• Understand the definition, function, importance, and history of human growth hormone (HGH).

• Describe HGH production using rDNA technology.

• Describe the genetic engineering methods for the production of other human hormones.

Page 3: Outline of the Learning Module

1. Introduction

   • Structure and function of insulin.

   • Genetic engineering of insulin.

2. Structure and function of HGH

3. Genetic engineering of HGH

4. Genetic engineering of other human hormones.

Page 4: Introduction to Insulin

• The earliest use of biotechnology in pharmaceutical manufacturing was the application of recombinant DNA technology to modify Escherichia coli bacteria to produce human insulin, initiated at Genentech in 1978.

• Insulin is a hormone produced by beta (β)-cells in the islets of Langerhans of the pancreas. It was discovered by Sir Edward Sharpey Schafer in 1916 while studying these islets.

• Individuals who do not produce adequate amounts of insulin suffer from diabetes.

Page 5: Diabetes Mellitus (DM)

• Diabetes Mellitus: A condition characterized by a lack of insulin, leading to an inability to control blood sugar levels.

• Insulin Therapy: Essential for individuals with diabetes to maintain health and proper glucose levels in the bloodstream.

Page 6: Structure of Insulin

• Chemical characteristics: Insulin is a small and simple protein comprising 51 amino acids, structured as follows:

  • Chain A: 21 amino acids

  • Chain B: 30 amino acids

• The A and B chains are linked by disulfide bonds.

• Chemical Structure View:

  • Human Insulin Sequence:
    A Chain: ] Ser, Leu, Pro, Thr, Ser, Tyr, Leu
B Chain: ] Val, Val, Phe, Val, Gly, Arg, Gly, Glu

  • Amino Acid Sequence Representation (simplified):

    • A: 21 amino acids (Ser, Leu, ...)

    • B: 30 amino acids (Val, Val, ...)

Page 7: Genetic Engineering for Insulin Production

• Microorganisms Used:

  • Fungi

  • Yeast

  • Main Microbes:

  • E. coli (most commonly utilized bacteria)

Page 8: Main Process of Insulin Production

• The main process involves utilizing microorganisms, particularly E. coli, to produce insulin through genetic modifications.

• Types of Bacteria: Various strains of bacteria may be used for insulin production.

Page 9: Steps in Producing Insulin Using rDNA Technology

Step 1: Isolate the Human Insulin Gene

1. A plasmid, a small piece of DNA in bacteria, is cut by a specific enzyme.

2. The human insulin gene is then inserted into this plasmid.

3. The engineered plasmid is inserted into a new bacterium.

4. This new bacterium then divides and begins to produce insulin.

Page 10: Step 1 Details (Preparing)

• The human insulin gene is obtained from the mRNA sourced from islet of Langerhans cells.

• Messenger RNA: A type of RNA that carries the genetic code for protein synthesis from DNA to ribosomes.

• The isolated gene contains the code necessary for insulin production.

• Plasmid DNA is extracted from the bacterial cells.

• Commonly, E. coli is utilized in insulin production, but yeast may also be employed.

Page 11: Step 2 (Cutting)

• The plasmid DNA is cleaved by a Restriction Enzyme, which cuts DNA at designated recognition sequences known as restriction sites, creating an open segment referred to as a plasmid ring.

• This step utilizes the same restriction enzyme to ensure matching sticky ends for DNA recombination.

Page 12: Step 3 (Combining)

• The opened plasmid ring receives the human insulin gene, and the ring is subsequently closed.

• The incorporation of the human insulin gene into the bacterial plasmid constructs a new recombinant plasmid.

• This recombinant plasmid is then mixed with bacteria.

Page 13: Step 4 (Inserting)

• Transformation: The resulting recombinant plasmid is reintroduced into the bacterial host cells.

• The modified E. coli cells now contain the gene for human insulin production.

Page 14: Production Phase

• Bacterial cells require nutrients to grow and thrive.

• While these cells proliferate, they activate the gene for human insulin, subsequently producing insulin within the cells.

• Every division of the bacterial cells also replicates the human insulin gene in the newly formed cells.

Page 15: Visual Representation of Insulin Production

• Illustrates the relationships:

  • Human cell DNA -> DNA Plasmid -> Restriction enzyme -> Insulin gene -> Transformation into bacterial cell -> Bacteria producing human insulin.

Page 16: Human Growth Hormone (HGH)

• Growth Hormone: A crucial hormone secreted from somatotropic cells in the anterior pituitary gland, responsible for various bodily functions including normal growth and muscle development.

• Roles of HGH:

  • Development of muscles and bones.

  • Regulation of growth in height, among other physiological functions.

Page 17: Structure of HGH

• The major isoform of human growth hormone comprises 191 amino acids, with a molecular weight of 22,124 daltons.

• Its three-dimensional structure is upheld by two disulfide bridges and features four helical structures.

Page 18: Genetic Engineering of HGH Production

• Transformation processes typically involve:

  1. Isolation of the mRNA for HGH from human cells (specifically pituitary gland cells).

  2. Generation of cDNA from the HGH gene using reverse transcriptase.

  3. Insertion of the HGH cDNA gene into a bacterial vector.

  4. Transformation into E. coli cells, which express the HGH gene.

Page 19: Final Steps in HGH Production

• The human growth hormone is extracted from the transformed bacteria post-expression.

• This hormone is then utilized for patient treatments through injection.

• Conceptual analogy: Simulating the isolation process through selected actions (like removing paper clips in a lab scenario).

Page 20: Other Human Hormones Produced via Genetic Engineering

1. Gonadotropin-Releasing Hormone:

   • Host: Escherichia coli

   • Engineering Approach: Heterologous expression using a T7 RNA polymerase-based system with emphasis on plasmid stability and yield.

2. Human Parathyroid Hormone:

   • Host: Escherichia coli

   • Utilized recombinant strains (e.g., BL21 (DE3)) with a plasmid encoding fusion genes for production.

Page 21: References

1. T.A. Brown; Gene Cloning and DNA Analysis, Sixth Edition, Blackwell Publishing Ltd, 2010.

2. Online Biology Notes: http://www.onlinebiologynotes.com/human-insulin-production-by-genetic-engineering/

3. Glick, B.R., et al.; Molecular Biotechnology: Principles and Applications of Recombinant DNA, 4th ed.

4. Information on insulin from Novo Insulin, Grolier Electronic Publishing Inc., 1992.

5. Course resource: https://nptel.ac.in/courses/102103013/35

Page 22: Conclusion

• Session closure with a prompt for questions related to the covered material on genetic engineering of insulin and human hormones.