Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Microbial Production of Therapeutic Agents
Conclusion

Through the cloning of specific genes and their subsequent expression in Bacteria, a variety of Proteins have been produced that can be used as therapeutic agents. Most of these proteins are of eukaryotic origin; thus, to isolate the target Gene, an mRNA preparation enriched with the fractions of interest is first obtained, followed by the Construction of a cDNA library and the insertion of the corresponding DNA into a suitable expression vector. By swapping regions of related genes encoding similar Protein domains, or by directly replacing segments of the cloned gene that encode functional PARTS OF THE protein, novel variants of these Proteins can be engineered. Certain Enzymes can also be utilized as therapeutics. For instance, recombinant DNase I and alginate lyase are administered as an aerosol to reduce the viscosity of mucus accumulating in the Lungs of cystic fibrosis patients.

With the advancement of Recombinant DNA technology and The Development of Methods FOR PRODUCING Monoclonal Antibodies, along with the elucidation of immunoglobulin Structure and function, there has been growing interest in using specific antibodies to treat various diseases. Working with antibody genes is facilitated by the fact that individual domains of the antibody molecule perform distinct Functions.

Drugs or Enzymes can be conjugated to monoclonal antibodies or their Fv fragments specific to surface proteins of target Cells, such as tumor cells. In this case, the drug can be in an inactive form. If repeated administrations of such complexes are intended, their immunoglobulin component must be a human antibody or antibody fragment; this prevents the development of cross-reactive immune responses and patient sensitization. If rodent monoclonal antibodies are to be used for this purpose, their structure should be humanized as much as possible. To achieve this, it is sufficient to replace the CDR regions of human antibodies with the corresponding fragments of rodent antibodies. Recently, the Selection and synthesis of human monoclonal antibodies has been successfully achieved using E. coli.

Introduction/32.html">Genetic Engineering METHODS enable The production of unique therapeutics consisting of a complex of a toxin and a protein that binds to specific cells, such as HIV-infected ones. Although this approach is still under development, its Prospects are highly promising.

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Review Questions

1. You need to clone and express a DNA fragment encoding human interferon. You do not have the required DNA probe for Hybridization, but you have managed to isolate a human cell line in which interferon synthesis can be induced to a level approximately 100 times higher than the Background rate. What cloning and expression strategy would you choose for this DNA?

2. What is the Fc fragment of an antibody molecule? The Fab fragment? The Fv fragment? The CDR region?

3. How is the coordinated synthesis of antibody light and heavy chains achieved in E. coli?

4. What is The Role of DNase I and alginate lyase in the treatment of cystic fibrosis?

5. How can the synthesis of alginate lyase encoded by a cloned gene be detected in transformed E. coli cells?

6. WHAT IS A combinatorial cDNA library?

7. How can bacteriophage M13 be used to select Fv fragments that bind to specific target Antigens?

8. What are disulfide-stabilized and single-chain Fv molecules?

9. How can therapeutic agents be produced by conjugating enzymes to monoclonal antibodies or their Fv fragments?

10. How can murine monoclonal antibodies that are structurally as close as possible to human antibodies be produced? Why are they necessary?

11. Describe a method for producing a therapeutic agent that "labels" and destroys specific cells.