Genetic engineering

Methods for obtaining genetic material

The first step in creating recombinant molecules is the isolation or artificial synthesis of the target gene. To achieve this, genetic engineering employs several main approaches:

• Isolation of natural genes. Genetic structures (chromosomes or plasmids) are isolated intact. For precise extraction of the required region, enzymatic cleavage is used with restriction enzymes (restriction endonucleases), which cut DNA at strictly defined bonds. If RNA is the target, ribozymes (enzymes acting similarly to restriction enzymes) are used.
• 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 special device (synthesizer).
• Reverse transcription. Used when working with RNA viruses. Using the enzyme reverse transcriptase, genetic information is transcribed from viral RNA to DNA.

Vector construction and expression systems

After obtaining the target gene, it must be inserted into a delivery system — a vector. Ligation of the required gene with vector DNA is carried out by ligase enzymes (the ligation process). Plasmids, bacteriophages, and human, animal, or plant viruses are typically used as vectors.

Next, the recombinant DNA molecule is introduced into the recipient cell (host cell). The essence of the process is that after the incorporation of the hybrid gene, the cell acquires a new property and begins synthesizing a substance uncharacteristic of it.

Recipient organisms:

1. Bacteria (e.g., E. coli, B. subtilis, pseudomonads).
2. Yeasts.
3. Viruses.

When choosing a recipient, consideration is given to its ability to incorporate a foreign gene, expression level (how pronounced the synthesis will be), environmental safety, ease of mass cultivation, and the ability to secrete the product outward (into the culture medium).

Production challenges and chimeric proteins

At the stage of producing the required substance, biotechnological manufacturing faces a number of tasks. Scientists create superproducing strains capable of restructuring metabolism so that up to half of the protein they produce is the target foreign product.

However, high expression does not solve all problems. If the cell does not secrete the product into the medium, its extraction requires destruction (disintegration) of the cell itself. In addition, there is a risk to product preservation: the target protein may be destroyed by intracellular proteases or inhibitors even during the synthesis process.

To facilitate the recognition and isolation of the substance, the methodological approach of creating chimeric proteins is used. A reporter gene (e.g., the $\beta$-galactosidase gene or an interferon gene) is attached in advance to the target protein gene. As a result, the cell synthesizes a single hybrid protein that is easy to detect. After isolation, the marker part is cleaved off, yielding the pure target product.

Practical application and implementation barriers

Genetic and protein engineering makes it possible to solve a wide range of medical problems, including the therapy of congenital genetic disorders and targeted effects on the genome. Already today, hundreds of drugs have been created using genetic engineering methods:

• Vaccines: against hepatitis B.
• Hormones: insulin, growth hormones, erythropoietin.
• Immune drugs: interferons, interleukins, tumor necrosis factor (TNF), myelopeptides, thymus peptides, monoclonal antibodies.
• Hemostasis system drugs: tissue plasminogen activator, blood coagulation factors.
• Diagnostics: HIV antigens.

In the near future, recombinant vaccines against malaria, syphilis, tick-borne encephalitis, and rabies, as well as new colony-stimulating factors and neuropeptides, are expected to appear.

Obstacles to implementation (all of them are surmountable):

• Safety issues: concerns over the risk of spreading recombinant microbes (biological safety has currently been proven when working rules are followed).
• Technological complexity (processing): the need to develop complex multi-stage purification schemes for the target protein from the producer culture.
• Identity problem (bioequivalence): the need for strict evidence that the artificial protein is completely identical to the natural analogue, down to the correct folding of the molecule.

Briefly: answers to topical questions

Class and exam questions

What else to study on this topic

• Mechanism of action of restriction enzymes and DNA ligases
• Schemes of bacterial cell disintegration for product isolation
• Differences between plasmid and viral delivery systems (vectors)
• Promising developments of recombinant vaccines (HIV, malaria, syphilis)

Other topics in the «Biotechnology. Genetic Engineering» section

Prepared by WhiteCoat Pro faculty in accordance with the medical school curriculum. For educational purposes only.