1. Recombinant Hormones and Drugs

2.  Many human disorders traced to absence or malfunction of a protein normally synthesized in the body  eg. Sickle cell anemia (abnormal hemoglobin) or Diabetes mellitis (insufficient or incorrectly folded insulin). “Could the gene for any given protein be cloned and expressed in E. coli?"

3. Recombinant Insulin Ideal candidate! - human protein is NOT modified after translation - relatively small protein comprising two polypeptides (the A chain and the B chain) held together by disulphide bonds. In humans these are synthesized as a precursor called preproinsulin.

4. Insulin  Insulin is a rather small protein, with a molecular weight of about 6000 Daltons.  A chain is 21 amino acids and B chain is 30 amino acids in length.  The figure shows a molecular model of bovine insulin, with the A chain colored blue and the larger B chain green.

5. In vivo synthesis of human insulin  In humans these are synthesized as a precursor called preproinsulin. A chain (blue), B chain (green) and the C chain (white). Disulphide bonds are shown in red.

6. rec. Insulin production  Synthesize trinucleotides representing all possible codons   ligate together in order dictated by amino acid sequence   Create two plasmids encoding gene for each chain under control of strong lac promoter  protein expressed as fusion protein.   Disulphide bond formation

7. Other recombinant proteins 1. Somatostatin – anti growth hormone - important in the treatment of a variety of growth disorders (eg. Acromegaly, a condition characterized by uncontrolled growth) 2. Somatotrophin – human growth hormone (HGH) - used in the treatment of growth disorders notably dwarfism. 3. Interferons - Purified interferons may be useful as antiviral agents in the treatment of diseases as diverse as cancer and the common cold.

8. Advantages and Disadvantages Proteins are synthesized in continuous culture(s) of E. coli BUT, Problems with side effects, contaminants, ease of purification and production in quantity.

9. Recombinant Vaccines  Vaccination programs make use of an antigenic preparation, called a vaccine, that after injection into the bloodstream stimulates the immune system to synthesize antibodies that will protect the body against subsequent infection.  Antigenic material : usually an inactivated form of the infectious agent eg. Vaccines for viral diseases are “dead” virus particles.

10. Problems!  1. Inactivation process must be 100% efficient (only one live virus particle could result in infection).  2. Need large amounts of virus particles for vaccine production, usually obtained from cell culture ( some viruses do not grow in cell culture though!). BUT, the whole viral particle is not always necessary!

11. Isolated virus coat proteins as vaccines.

12. Methods for production of recombinant vaccines 1. Cloning in expression vectors  Genes coding for the antigenic proteins of a particular virus could be identified and inserted into an expression vector.  Strategy successful with viruses for foot-and-mouth disease and hepatitis B. 2. Recombinant vaccinia viruses  Recombinant vaccinia viruses could be used as live vaccines against other diseases.  eg. gene encoding for a virus coat protein (eg influenza hemagglutinin gene) can be ligated into the vaccinia genome  immunity against both small pox AND influenza.

14. 3. Antigenic peptides  The antigenic component of a virus particle can be reduced beyond even the purified coat.  Short isolated segments of these coat proteins are also capable of stimulating synthesis of antibodies that provide protection against the live virus.

15. Peptides can be used generate vaccines.

16. Future prospects for gene cloning  1. Production of non-protein products by recombinant methods eg. Pharmaceuticals.  2. Animal and plant cell culture Alternatives to E. coli: yeast, plant cells, animal cells (eg. CHO cells).  3. Transformed plants and animals Introduce desirable characteristics such as nitrogen fixation, disease and pest resistance into plants.

17. Other breakthroughs and uses for gene therapy.  Some recombinant DNA products being used in human therapy. Some recombinant DNA products being used in human therapy.