Methods for Obtaining Genetic Material
The first step in creating recombinant molecules is the isolation or artificial synthesis of the target gene. Several main approaches are used in genetic engineering for this purpose:
- Isolation of natural genes. Genetic structures (chromosomes or plasmids) are isolated intact. To precisely extract the required segment, enzymatic cleavage is used with restriction endonucleases (restriction enzymes)—enzymes that cut DNA at strictly defined sites. If the target is RNA, ribozymes are used (enzymes acting analogously to restriction endonucleases).
- Chemical synthesis. Suitable for creating relatively small genes. The process includes three stages: protein analysis with amino acid sequence determination $\rightarrow$ translation of the amino acid sequence into a nucleotide sequence (based on codons) $\rightarrow$ direct gene synthesis on a specialized synthesizer.
- Reverse transcription. Used when working with RNA viruses. Using the enzyme reverse transcriptase, genetic information is copied from viral RNA into complementary DNA (cDNA).
Vector Construction and Expression Systems
After obtaining the target gene, it must be inserted into a delivery system—a vector. Ligation of the desired gene with vector DNA is performed by DNA ligases (the ligation process). Plasmids, bacteriophages, and human, animal, or plant viruses are typically used as vectors.
Next, the recombinant DNA molecule is introduced into a recipient cell (host cell). Upon integration of the hybrid gene, the cell acquires a new property and begins synthesizing a substance atypical for it.
Recipient organisms:
- Bacteria (e.g., E. coli, B. subtilis, pseudomonads).
- Yeasts.
- Viruses.
When choosing a recipient, factors considered include its ability to integrate foreign genes, expression level (the yield of the synthesized product), biosafety, ease of large-scale cultivation, and the ability to secrete the product into the culture medium.
Production Challenges and Chimeric Proteins
During the production phase of the desired substance, biotechnological manufacturing faces several challenges. Scientists create superproducer strains capable of reprogramming metabolism such that up to half of the total protein produced is the target foreign product.
However, high expression does not solve all problems. If the cell does not secrete the product into the medium, extracting it requires cell disruption (disintegration). Additionally, there is a risk to product integrity: the target protein may be degraded by intracellular proteases or inhibitors during synthesis.
To facilitate identification and isolation, chimeric proteins are engineered. A marker or reporter gene (e.g., the $\beta$-galactosidase or an interferon gene) is attached in advance to the target protein gene. As a result, the cell synthesizes a single fusion protein that is easy to detect. After isolation, the marker portion is cleaved off, yielding the pure target product.
Practical Applications and Implementation Barriers
Genetic and protein engineering address a wide range of medical tasks, including the therapy of inherited genetic disorders and targeted modulation of the genome. Hundreds of recombinant drugs are already in clinical use:
- Vaccines: Hepatitis B.
- Hormones: Insulin, growth hormone, erythropoietin.
- Immune modifiers: Interferons, interleukins, tumor necrosis factor (TNF), myelopeptides, thymic peptides, monoclonal antibodies.
- Hemostatic agents: Tissue plasminogen activator, blood clotting factors.
- Diagnostic reagents: HIV antigens.
In the near future, recombinant vaccines against malaria, syphilis, tick-borne encephalitis, and rabies are anticipated, alongside new colony-stimulating factors and neuropeptides.
Implementation barriers (all surmountable):
- Biosafety concerns: Apprehensions regarding the spread of recombinant microbes (biological safety is currently established via strict containment protocols).
- Technological complexity (downstream processing): The need to develop complex multi-step purification schemes to isolate the target protein from the culture medium.
- Identity issues (bioequivalence): The requirement for rigorous proof that the synthetic protein is entirely identical to its natural counterpart, including correct three-dimensional folding.