Historical significance and advantages of the method
Before the active introduction of molecular biology methods into clinical practice, the primary source of drugs for the treatment of diabetes mellitus was extracts obtained from the pancreas of pigs or cows. The use of animal-derived insulin saved patients' lives, but had a significant drawback: such preparations caused a considerable number of adverse reactions in the body.
The introduction of recombinant DNA technology marked a true revolution. The first human protein successfully produced by genetic engineering was precisely human insulin. The use of a ready-made recombinant preparation for treating diabetic patients has a major clinical advantage: it causes significantly fewer side effects compared to animal-derived preparations.
Other products of genetic engineering
The success in producing human insulin proved the high efficacy of the technology and paved the way for the synthesis of many other biologically active compounds. Today, a whole range of critically important products are obtained using a similar genetic engineering method, among them:
- Growth hormone (somatotropin).
- Interferon.
- Various vaccines.
- Silk protein and other compounds.
Preparatory stage: vector and target gene
The process of obtaining recombinant insulin begins with the careful preparation of two basic components: the vector, which will deliver the genetic material, and the genetic insert itself (the gene).
- Vector preparation. A plasmid is used as the carrier, which is carefully isolated from a bacterial cell. To enable the plasmid to accept new genetic material, its circular structure is purposefully cleaved by specialized restriction enzymes.
- Insert preparation. Concurrently, work is carried out with human material. Messenger RNA (mRNA) carrying information about the structure of insulin is extracted from human pancreatic cells. Since it is impossible to insert an RNA molecule directly into a DNA plasmid, the process of reverse transcription is employed. Using the enzyme reverse transcriptase (or revertase), a complementary DNA (cDNA) molecule is synthesized using the mRNA template. It is this cDNA that acts as our required gene.
Creation of recombinant DNA and protein expression
Once the vector and insert are ready, the stage of direct assembly and launching of the biological "factory" begins.
- Creation of recombinant DNA. The cDNA obtained in the previous stage is firmly integrated into the cleaved plasmid vector.
- Transformation. The engineered recombinant plasmid is introduced into a host bacterial cell. A mandatory condition is that this bacterium initially contains no other plasmids to prevent competition of genetic material.
- Cloning and expression. Genetically modified bacteria are placed in a nutrient medium where they are actively cultured. As the bacteria divide, the plasmids replicate along with them. As a result, the cellular machinery of the bacteria begins to synthesize the target product — insulin.
- Product recovery. At the final stage of production, complex isolation and deep purification of the synthesized human insulin are carried out. Only after this is the preparation considered ready for use in the treatment of diabetes mellitus.
Briefly: answers to topical questions
What specific types of restriction enzymes (restriction endonucleases) are used for vector construction?
Restriction endonucleases capable of cleaving DNA to form single-stranded complementary overhangs ("sticky ends") are used for vector construction.
Characteristics of the enzymes used:
- Specifically recognize strictly defined DNA regions (restriction sites).
- Recognize sequences with central symmetry (palindromes).
- Cleave phosphodiester bonds in the chain.
For the successful construction of a recombinant molecule, the cleavage of the vector (plasmid) and the target DNA must be performed using the exact same restriction enzyme.
The rule of two "R"s: Restriction enzymes cleave the plasmid, while Revertase triggers reverse transcription (from mRNA to DNA).
Class and exam questions
Why did modern medicine abandon animal insulin?
Pancreatic extracts from pigs or cows caused adverse reactions. Recombinant human insulin produces significantly fewer side effects.
How is the human insulin gene obtained for insertion into a vector?
mRNA is isolated from pancreatic cells, after which complementary DNA (cDNA) is synthesized using the enzyme revertase (reverse transcriptase).
What role do bacteria play in the process of genetic engineering?
The bacterium acts as the host cell. The recombinant plasmid is introduced into it, after which the bacteria are cultured, and their cellular machinery begins to synthesize human insulin.
What else to study on this topic
- Structural features and functions of bacterial plasmids
- Mechanism of action of restriction enzymes
- The process of reverse transcription and the work of revertase
- Methods of bacterial cell transformation
- Procedures for the isolation and purification of recombinant proteins
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