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

Molecular Biotechnology of Microbial Systems
Microbial Production of Pharmaceuticals
Pharmaceuticals

Before the advent of Recombinant DNA technology, many human protein-based therapeutics could only be produced in small quantities, making their manufacture highly expensive, while their biological Mechanisms of action were sometimes poorly understood. It was anticipated that this new technology would enable The production of the entire spectrum of such drugs in quantities sufficient for both robust testing and clinical application. These expectations have been fully realized. To date, more than 400 genes (primarily as cDNAs) for various human Proteins with therapeutic potential have been cloned. Most of these genes have already been expressed in host Cells, and their products are currently being evaluated for the Treatment of various human diseases (Table 10.1). Although more than 30 such biopharmaceuticals have received approval in the US (Table 10.2), it will still be several years before they are recommended for widespread use and enter the market; they must first undergo animal testing and rigorous clinical trials. Nevertheless, pharmaceutical companies are already showing keen interest. Experts estimate that the annual global market for human protein-based therapeutics is worth about 150 billion dollars and is steadily growing. The global market for recombinant protein-based drugs is expanding at an annual rate of 12–14% and is projected to reach approximately 20 billion dollars by 2000.

The Development of new Methods for the Prevention and treatment of many human diseases made a tremendous contribution to human well-being in the 20th century. However, this process can never be considered complete. So-called "old" diseases (such as tuberculosis) can re-emerge as soon as preventive measures are relaxed or resistant strains appear. The prospect of using specific Antibodies as therapeutic agents is highly attractive; they can be used to neutralize toxins, combat Bacteria and Viruses, and treat cancers. An antibody can be compared to a homing missile that either neutralizes the "intruder" (a foreign agent) or, if equipped with a "warhead", destroys a specific target Cell. Unfortunately, despite their promising potential, antibodies have historically been rarely used for the prevention and treatment of diseases and other pathologies. Only recently, with the development of recombinant DNA technology, the design of METHODS FOR PRODUCING Monoclonal Antibodies, and the elucidation of the Molecular Structure and function of IMMUNOGLOBULINS, has interest in using specific antibodies to treat various diseases been revitalized.

Class="center">Therapeutic Agents

Various approaches are used to isolate genes or cDNAs for human proteins. In some cases, the target protein is isolated, and the Amino Acid Sequence of a specific region of the molecule is determined. Based on this, the encoding nucleotide sequence is deduced, the corresponding oligonucleotide is synthesized, and it is used as a Hybridization probe to isolate the desired Gene or cDNA from genomic or cDNA libraries. Another approach involves generating antibodies against the purified protein and using them to screen expression libraries where specific genes are expressed. For human proteins synthesized predominantly in a single tissue, a cDNA library constructed from mRNA isolated from that tissue will be enriched for the target DNA sequence. For example, the primary protein synthesized by the Cells of the islets of Langerhans in the Pancreas is Insulin, and 70% of the mRNA isolated from these cells encodes this protein.

Table 10.1. Some human proteins produced by Introduction/32.html">Genetic Engineering METHODS

However, the cDNA enrichment principle is not applicable to human proteins that are present in extremely low Abundance or whose site of synthesis is unknown. In such cases, alternative experimental approaches may be required. Human interferons (IFNs), which include alpha, beta, and gamma interferons (IFN-α, IFN-β, IFN-γ), are naturally occurring proteins, each of which has potential therapeutic Applications (Table 10.3). To isolate their cDNAs, a novel approach had to be developed to overcome the difficulties associated with the low abundance of the corresponding mRNAs and proteins. The Procedure for isolating interferon cDNAs was as follows.

Table 10.2. Some recombinant proteins approved by the US Food and Drug Administration (FDA) for the treatment of human diseases

1. mRNA was isolated from human leukocytes and fractionated by size; reverse METABOLISM/31.html">Transcription was performed, and the resulting cDNA was inserted into the PstI site of plasmid pBR322.

2. The resulting product was used to transform Escherichia coli. The 6,000 obtained clones were divided into 12 pools of 512 clones each. Testing was performed on these pools, which accelerated the identification process.

3. Each pool of clones was hybridized with a crude preparation of IFN mRNA.

4. mRNA was recovered from the resulting hybrids containing cloned DNA and mRNA, and translated in a cell-free Protein Synthesis system.

5. The interferon antiviral activity of each Translation mixture was determined. Pools showing interferon activity contained a clone with a cDNA that hybridized to the IFN mRNA.

6. Positive pools were subdivided into 8 sub-pools of 64 clones each and retested. This subdivision process was repeated until a clone containing the full-length human IFN cDNA was identified.

To obtain large quantities of IFN, the corresponding cDNA can be subcloned into an E. coli expression vector that allows for high-level expression.

Table 10.3. Potential therapeutic applications of selected human interferons

Genetically engineered human interferons

The first interferon gene was isolated in the early 1980s. Since then, several different interferons have been discovered. As previously mentioned, based on their chemical and biological properties, they can be classified into three groups: IFN-α, IFN-β, and IFN-γ. IFN-α and IFN-β are synthesized by cells treated with viruses or viral RNA, whereas IFN-γ is produced in response to cell-growth-stimulating agents. IFN-α is encoded by a gene family comprising at least 15 non-allelic genes, while IFN-β and IFN-γ are each encoded by a single gene. The subtypes of IFN-α exhibit different specificities. For example, when testing the efficacy of IFN-α₁ and IFN-α₂ on a virus-treated bovine cell line, these interferons show similar antiviral activity; however, in virus-treated human cells, IFN-α₂ is seven times more active than IFN-α₁. If antiviral activity is tested on mouse cells, IFN-α₂ is 30 times less effective than IFN-α₁.

Several attempts have been made to create IFNs with combined properties, exploiting the fact that members of the IFN-α family differ in the degree and Specificity of their antiviral activity. Theoretically, this can be achieved by joining PARTS OF THE gene sequences of different IFN-α subtypes. This results in a hybrid protein with properties distinct from those of either parental protein. Comparison of the cDNA sequences of IFN-α₁ and IFN-α₂ revealed that they share common restriction sites at positions 60, 92, and 150. After digesting both cDNAs at these sites and subsequently ligating the fragments, several hybrid genes were obtained (Fig. 10.1). These genes were expressed in E. coli, and the synthesized proteins were purified and evaluated for their biological Functions. Testing the protective Properties of the hybrid IFNs in mammalian cell cultures showed that some exhibited higher activity than the parental molecules. Furthermore, many hybrid IFNs induced the production of 2'–5'-oligoisoadenylate synthetase in control cells. This enzyme is involved in the synthesis of 2'–5'-linked oligonucleotides, which in turn activate a latent cellular endoribonuclease that cleaves viral mRNA. Other hybrid IFNs demonstrated greater antiproliferative activity than the parental molecules in cultures of various human cancer cells.

Fig. 10.1. Structure of IFN-α₂, IFN-α₃, and four hybrid genes. Comparison of The nucleotide sequences of the IFN-α₂ and IFN-α₃ genes reveals that they share identical restriction endonuclease sites (RE1, RE2, RE3). Restriction Digestion at these sites and ligation of the resulting fragments yield various hybrid genes. Four of these are shown at the bottom of the figure.

Genetically engineered human growth hormone

The strategy of engineering novel proteins by swapping functional domains or through Site-Directed Mutagenesis can be used to enhance or diminish the biological activity of a protein. For example, native human growth hormone (hGH) binds to both the growth hormone receptor and the prolactin receptor in various cell types.

To avoid undesirable side effects during treatment, it is necessary to prevent hGH from binding to the prolactin receptor. Since The amino acid sequence of the growth hormone region that binds to this receptor only partially overlaps with the region interacting with the prolactin receptor, it was possible to selectively reduce the hormone's binding to the latter. This was achieved using site-directed mutagenesis, which introduced specific changes into the side chains of Certain Amino acids (His-18, His-21, and Glu-174) — ligands for Zn²⁺ ions required for high-affinity binding of hGH to the prolactin receptor (Fig. 10.2). The modified growth hormone binds only to its "own" receptor. While these results are of clear interest, it remains uncertain whether modified hGHs will find clinical application.

Optimization of Gene Expression

It is not enough to create a new protein; optimizing its gene expression is crucial. First, researchers determine whether sufficient amounts of the authentic protein can be synthesized in prokaryotic or eukaryotic expression systems. Prokaryotic systems are preferred because they are more cost-effective and offer higher yields. Unfortunately, not all microorganisms synthesize functional forms of heterologous proteins with equal efficiency, making comparative quantitative assessments necessary.

When studying the expression of the human interleukin-3 gene in various host cells, Bacillus licheniformis proved to be the "best" host (Table 10.4). Although a slightly higher expression level was achieved in one of the E. coli systems, the resulting 20 kDa protein was a fusion product of interleukin-3 and a portion of E. coli β-galactosidase, rather than the mature authentic 15 kDa protein. As a rule, such a chimeric protein cannot be used as a therapeutic agent. Yeast cells of Kluyveromyces lactis and Saccharomyces cerevisiae, as well as human cells, were capable of glycosylating interleukin-3, but their expression levels were relatively low. Glycosylation does not significantly affect The activity of interleukin-3, but it leads to a noticeable difference in molecular size.

Fig. 10.2. Schematic representation of native and modified forms of human growth hormone (hGH). Using oligonucleotide-directed mutagenesis, an hGH variant was obtained that lost its ability to bind to the prolactin receptor but retained specificity for the growth hormone receptor.

Table 10.4. Expression level of the interleukin-3 gene in different host cell systems¹⁾

¹⁾ Adapted from van Leen et al., Bio/Technology 9: 47—52, 1991.

²⁾ In each case, one of the strongest promoters active in the given system was used.