Biotechnology - Yu.O. Sazykin 2006

General Biotechnology
Biological objects: methods of creation and improvement
Creation of biological objects via genetic engineering - Recombinant proteins as drugs

The breakthroughs in Introduction/32.html">Genetic Engineering have made it possible for over 100 human Proteins (BIOREGULATORS, Homeostasis correctors, innate and acquired Immunity factors) to retain their species Specificity. They are produced as therapeutic agents via microbiological synthesis. Moreover, Recombinant DNA technology allows for their enhancement: increasing physiological activity, reducing the likelihood of adverse post-administration reactions, etc. The primary objective in obtaining recombinant proteins is solving the raw material deficit, as obtaining them from human Tissues on an industrial scale is naturally impossible.

When selecting a microorganism (as a producer of the foreign protein for a prospective drug), it is necessary to:

✵ study The Genome as thoroughly as possible;

✵ investigate METABOLISM in detail at the species level;

✵ ensure the microorganism possesses moderate pathogenicity (ideally, complete absence thereof);

✵ ensure the microorganism is capable of growing under production conditions in non-deficient and economically accessible media.

Microorganisms chosen as prospective producers are evaluated and studied down to the level of specific strains. If necessary, strain-bioobjects (as carriers of foreign genetic material and producers of foreign protein) can be improved using Genetic engineering Methods, which helps minimize the probability of foreign protein proteolysis, foreign Messenger RNA Hydrolysis, and the "exclusion" of foreign genes from the genome. Thus, the general goal in this case is to limit The activity of Cellular Homeostasis-promoting repair systems, including Nucleases and proteases.

Sometimes, a foreign protein may accumulate within the producer Cell as protein granules (in a form inaccessible to proteases), which is characteristic of E. coli, for example.

Special attention is drawn to Structure/149.html">The problem of foreign protein secretion, since isolating the target protein in a highly purified form from the culture fluid is a much easier task than extracting it from within The Cell. As is well known, secreted proteins differ from non-secreted ones by having a so-called leader sequence of amino acid residues at the N-terminus, which facilitates their Transport Across the cell membrane into the medium. At The final stage of contact between the secreted protein and The surface of the cell that produced it, the leader sequence is cleaved from the main polypeptide chain. Accordingly, the foreign Gene (or more precisely, Operon) introduced into the microbial cell undergoes modification: either its nucleotide sequence is purposely extended so that the foreign protein incorporates a leader Amino Acid Sequence, or hybrid operons are constructed with a common promoter, comprising the foreign protein gene and the secreted protein gene, whose leader sequence extracts the foreign protein out of the cell. Subsequently, the two proteins are separated using standard chemical or enzymatic methods.

Currently, the most frequently used producers of human recombinant proteins are Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae (brewer's Yeast). The first two microorganisms are prokaryotes, while the latter is a eukaryote. These organisms are relatively safe; however, their release into the environment (as producers of a particular human protein) is undesirable for A number of reasons. Consequently, established and strictly observed safety protocols for working with recombinants are in place.

Safety must be maintained at both the genetic and physical levels. Genetic-level safety means that another modification is introduced into the genome of the foreign protein producer (In addition to the foreign genes)—certain genes are removed, such as those involved in the synthesis of an amino acid essential for the microorganism's growth. In other words, the Organism is made dependent on the presence of this amino acid in the medium. If the Cells of this microorganism happen to escape into the environment, they cannot multiply, meaning the risk of contaminating the plant area with the recombinant culture is significantly reduced.

It is worth noting that foreign protein producers do not exhibit high survival capabilities in natural conditions. More precisely, they lose The ability to produce the unnecessary foreign protein because losing this ability accelerates Cell Division; slowly dividing cells that continue to synthesize the foreign protein gradually disappear from the population.

Safety measures are also implemented at the physical level: microbiological filters are installed at all gas emission points. Upon completion of the operating cycle, the equipment is sterilized without breaking the system—a Procedure that could otherwise cause a drop in pressure across all vessels containing recombinant cells, as depressurization would draw air along with foreign cells back into the system. Lastly, manufacturing operations must fully comply with the requirements of Good Manufacturing Practice (GMP).

All of the above applies equally to The production of any recombinant proteins. The production of recombinant Insulin can serve as an example.

Ranking first in production volume and product value among recombinant protein Pharmaceuticals is the well-known hormone—insulin, which controls Blood glucose levels. Commercial production of recombinant insulin was first initiated in 1982. Today, its annual turnover accounts for approximately one-third of the total turnover of all recombinant proteins used in medicine.

Insulin consists of two polypeptide chains. Chain A contains 21 amino acid residues, and chain B contains 30 amino acid residues. Chains A and B are linked together by two disulfide (—S —S —) bonds. Another such bond exists between the Cysteine residues located in the A-chain. The overall stereostructure of the molecule is maintained by these three Disulfide Bonds, and any alteration to it leads to the loss of insulin's hormonal activity.

The traditional source of Insulin is the Pancreas of agricultural animals, specifically pigs and cattle. However, not the entire gland is used, but only the tissue of the so-called "islets of Langerhans."

The Russian market consumes approximately one ton of insulin annually. It is estimated that obtaining this amount of insulin requires roughly 35 million pigs. It is also known that the number of individuals requiring regular insulin administration increases by several percentage points every year; therefore, the problem of raw material scarcity regarding animal-derived insulin persists to this day.

However, this is not the only reason for the interest in recombinant insulin obtained via microbiological synthesis. Insulin extracted from porcine pancreas differs from human insulin by a single amino acid residue. Bovine insulin differs by three. This means that when administered to a human, they receive a protein (polypeptide) of different species specificity. Consequently, There is a certain percentage of allergic reactions. Pain upon parenteral administration (especially in children) may also be observed. At the same time, clinicians face the problem of overdose, because in cases of allergy, insulin (acting as an antigen) is partially neutralized and therefore must be administered in larger quantities.

Furthermore, the precursor of insulin during its Biosynthesis in animal tissue, known as proinsulin, contains an additional polypeptide chain (the C-peptide). Later, this chain is cleaved from the mature (completed) form of the hormone; however, when extracting insulin from animal cells, it is difficult to rid the product of proinsulin impurities. It is precisely in the C-peptide that species-related differences in The amino acid sequence are much greater (compared to insulin itself), meaning that the side effects of animal-derived insulin are largely attributable to the "foreign" peptide.

Recombinant insulin synthesized in a microbial cell lacks these drawbacks because the amino acid sequence of its two chains is encoded by human genes. In principle, it is identical to insulin from human tissue. Admittedly, its Isolation and Purification require extreme care, as it is necessary to eliminate microbial Glycoproteins and Lipoproteins. Due to their toxicity, such impurities in recombinant insulin can cause undesirable side effects. However, this pertains to the quality of specific drug batches and the manufacturing standards of a given facility.

Currently, two fundamentally different technologies compete in the production of recombinant insulin. According to the first approach, host microbial cells are transfected with a plasmid containing The nucleotide sequence corresponding to proinsulin (chain A, C-peptide, chain B, followed by the leader peptide and promoter region). Subsequently, the C-peptide is cleaved. The distinct feature of the second approach is the separate production of chain A and chain B in two microbial cultures, which are subsequently combined.

The laboratories of a foreign company have developed a scheme for producing recombinant insulin based on the separate biosynthesis of its two chains; each chain is synthesized in a separate E. coli culture. Vectors were initially constructed on a basis of Plasmids (one for the A-chain gene and another for the B-chain gene). A triplet corresponding to Methionine and the NUCLEOTIDES of the inducible beta-galactosidase gene, including the operon, was attached to each sequence. Thus, a methionine triplet ended up between The nucleotide sequences of chains A, B and beta-galactosidase. This circumstance is crucial for the final stage of the work. Fermentation of the two cultures—one carrying the vector with the A-chain and the other carrying the vector with the B-chain—was carried out in a medium containing lactose (an inducer of beta-galactosidase synthesis). If glucose is not broken down, it can be utilized by the organism as an energy source. Since beta-galactosidase and chain A (or B) shared a common promoter in the vector, the accumulation of beta-galactosidase was accompanied by the accumulation of large quantities of the linked chain A (B). Upon completion of fermentation, two proteins were isolated from the two cultures: chain A plus beta-galactosidase, and chain B plus beta-galactosidase. The methionine residue linking each insulin chain to beta-galactosidase was cleaved using BrCN, thereby releasing and isolating the insulin chains.

At the final (purely chemical) stage of the process, chains A and B were joined into the insulin molecule (via two disulfide bonds; the third disulfide bond is formed between cysteine residues in the A-chain).

Growth Hormone (somatotropin). Another recombinant protein obtained via microbiological synthesis has been tested clinically—human growth hormone, which is secreted by the anterior Pituitary Gland and contains 191 amino acid residues. In The Human Body, this hormone is essential for bone growth. It is not required during embryonic development, but its deficiency sharply manifests in late childhood and leads to dwarfism.

The human growth hormone gene has been cloned in E. coli. The biological activity of the isolated protein was identical to that of the sample obtained from the pituitary gland. Intensive research is underway to enhance the selectivity of the growth hormone's action (by reducing its binding to the prolactin receptor).

Erythropoietin. This glycoprotein is essential for the maturation (differentiation) of erythroid precursor cells. The protein is species-specific and is produced in the Kidneys. Erythropoietin is required to treat anemia caused by renal failure, as well as anemias associated with blood transfusions, irradiation, and tumor Chemotherapy—in other words, wherever hematopoiesis may be suppressed.

In the United States, over 100,000 people receive two injections of erythropoietin per week (erythropoietin ampoules in citrate buffer at 2,000–4,000 IU also contain serum albumin). A notable and specific case is that athletes who abuse erythropoietin during the Olympic Games and other international competitions are disqualified.

The method for producing recombinant erythropoietin (it must be emphasized: a glycoprotein) has a key feature—the human erythropoietin gene is inserted not into microbial cells, but into animal cells (Chinese hamster Ovary cells), where the protein can be properly glycosylated. In this process, a monolayer culture of these cells serves as the expression system for erythropoietin.

Peptide tissue growth factors. These numerous bioregulators exhibit both species and tissue specificity. They are sometimes defined as Hormones produced outside of Endocrine glands. Their production for medical practice is only feasible through microbiological synthesis, i.e., as recombinant proteins. Currently, epidermal growth factor (EGF) and several others have been studied in detail. Research in this area is ongoing, and positive experimental results have already been achieved (such as accelerated wound healing using EGF); however, the issue of completely guaranteeing the Prevention of malignant tissue transformation remains unresolved.

Recombinant protein factors of innate immunity.

Species-specific proteins used as medications include interferons—innate immunity factors originally discovered as proteins produced by virus-infected cells. They induce local and systemic antiviral responses in neighboring cells and are consequently used as antiviral drugs. Interferons are also of potential interest as antitumor agents. They are conventionally divided into three types: a, ß, and y (derived from different cell types).

Until recently, interferons from human cells were available only in small quantities. Leukocyte interferon was used primarily as a medical preparation, sourced from blood collected in maternity hospitals. Today, the leukocyte interferon gene has been obtained via chemical synthesis. It was subsequently incorporated into plasmids, which were then introduced into Escherichia coli and yeast cells, thereby transforming them into producers of human leukocyte interferon.

The human fibroblast interferon gene has been cloned in E. coli cells. Work is also underway to engineer hybrid interferons with greater anticancer activity than their natural counterparts. This includes the generation of hybrids between a₁ and a₂ interferons.

In general, the prospect of producing and applying immunomodified recombinant proteins in medicine is attracting growing interest. Of particular note is a series of studies on the major cationic protein of neutrophils, which exhibits both bactericidal activity and the ability to neutralize gram-negative bacterial endotoxins by increasing the permeability of the outer membrane of these Bacteria (protein designation: BPI — bactericidal/permeability-increasing protein). This protein has a Molecular Weight of 55 kDa and possesses an exceptionally high affinity for lipid A (the endotoxin) in the outer membrane of gram-negative bacteria.

Using it as a chemotherapeutic agent administered externally to "assist" the body's own neutrophils holds significant promise. It is rational to use human-derived BPI to avoid allergic reactions, but this naturally raises the issue of raw material shortages. Notably, acting as an inflammatory response inhibitor at the endotoxin level, this protein competes with one of the acute-phase proteins (produced by the Liver) that stimulate anti-inflammatory responses. BPI is now produced using genetic engineering techniques, and its molecular weight in the pharmaceutical formulation is considerably smaller, as it utilizes only the N-terminal fragment of the recombinant protein with a molecular weight of 21 kDa (referred to as rBPI₂₁).