BIOTECHNOLOGY - V. H. Gerasymenko - 2006

Part II. Specialized Biotechnologies

Chapter 12. BIOTECHNOLOGY OF HORMONE PRODUCTION

12.1 METHODS FOR HORMONE PRODUCTION

Biotechnology drives The Development of innovative medical approaches for obtaining valuable BIOLOGICALLY ACTIVE SUBSTANCES, such as hormonal preparations. Human and Animal organisms produce dozens of different compounds involved in the Hormonal Regulation of growth, development, and METABOLISM. Remarkable progress has been achieved in the synthesis of Peptide Hormones, which consist of a relatively small number of building blocks—ranging from a few amino acid residues to several dozen. These include hypothalamic factors, certain Pituitary Hormones, THYROID HORMONES, Pancreatic and Intestinal hormones, and Neuropeptides. In endocrine Cells, hormones of this group are synthesized from significantly larger precursors through the specific Enzymatic Cleavage of strictly defined peptide bonds.

Growth hormones and prolactin consist of a larger number of amino acid residues (approximately 190–195) and are formed by cleaving an N-terminal peptide, known as the "signal peptide" and about 25 amino acid residues in length, from their precursors (prohormones).

Until recently, medical-grade peptide hormones were extracted primarily from animal and human Organs and Tissues (donor Blood, surgically removed organs, cadaveric material, post-slaughter animal organs, etc.). Hormones applied in cases where the hormone lacks pronounced species Specificity were obtained from animal organs. Cadaveric material served as the sole source for obtaining hormones with highly pronounced species specificity, such as human somatotropin (Growth Hormone). Obtaining even a small amount of a hormonal preparation required a vast amount of source material (raw biomass).

The early successes of Introduction/32.html">Genetic Engineering instilled hope that microbial cells could eventually be utilized to produce any protein-based product by introducing genes encoding these Proteins into their genomes and establishing conditions for their expression. In developing novel technologies for hormone production using Plasmids, the primary objective was to construct recombinant DNA molecules containing nucleotide sequences programmed to synthesize specific hormones, introduce them into Bacteria, and induce The production of these hormones.

The technology for producing hormones using recombinant DNA involves the following stages: 1) isolation of genetic material (genes); 2) introduction of the genetic material into the bacterial Cell's genetic apparatus and creation of conditions for its expression.

Isolation of Genes. The required genetic material (a Gene or group of genes) for subsequent Amplification via Genetic engineering Methods aimed at synthesizing the biologically active proteins encoded by these genes can be obtained through three distinct methods: 1) isolation directly from DNA; 2) chemo-enzymatic synthesis; 3) enzymatic or template-directed synthesis based on Messenger RNA (mRNA) isolated from cells.

The first method involves "excising" the desired gene from natural genetic material (DNA) using appropriate Enzymes (Restriction Endonucleases). This approach has significant drawbacks. First, it is difficult to optimize enzyme action to excise precisely the required gene from the DNA. Typically, superfluous nucleotide sequences remain "flanking" the gene, hindering its subsequent application, or conversely, the enzymes excise part of the gene, rendering it functionally defective. Second, eukaryotic genes possess a complex "mosaic" (exon-intron) Structure, which may subsequently impede their normal functioning when replicated in microorganisms, as bacteria lack the machinery to remove intervening sequences (introns). Consequently, this gene isolation technique works best for Viruses and bacteria rather than eukaryotes. Third, if the gene constitutes a negligible fraction of the DNA from which it is isolated, severe difficulties may arise regarding its isolation and identification.

The second method involves the chemo-enzymatic synthesis of a gene, provided the Primary Structure of the protein or polypeptide encoded by the synthesized gene is known. It serves as a vital alternative to excising genes from native DNA using restriction enzymes. The method comprises the Chemical synthesis of short (8–16 nucleotide) single-stranded DNA fragments via the stepwise formation of ester bonds between NUCLEOTIDES, followed by the ligation of oligonucleotides using DNA ligase to form double-stranded polynucleotides.

Chemo-enzymatic synthesis enables the precise recreation of the minimally required nucleotide sequence while avoiding Problems associated with eliminating superfluous nucleotide sequences in DNA fragments, including introns. However, this method is labor-intensive and costly. Chemo-enzymatic synthesis has been successfully employed to obtain the genes for Somatostatin, the A and B chains of Insulin, proinsulin, the E. coli lac operator, etc.

The third method of gene generation—template-directed synthesis—is the most widespread and serves as the primary source of genes subsequently amplified in the form of recombinant DNA within unicellular and occasionally Multicellular Organisms. The Essence of the method lies in obtaining genes through enzymatic synthesis and can be briefly summarized as follows. First, messenger RNAs (mRNAs) are isolated from cells, among which is the mRNA encoded by the target gene to be isolated. Next, under specialized conditions, RNA-directed single-stranded DNA Synthesis is performed, catalyzed by the enzyme Reverse Transcriptase (revertase). Upon completion of the reaction, the synthesized single-stranded DNA (referred to as complementary DNA, or cDNA) is purified and used as a template for the second reaction—DNA-dependent Synthesis of the second DNA strand. This became possible due to the discovery in 1970 (USA) of the enzyme reverse transcriptase, whose primary property is The ability to perform synthesis reverse to that occurring during Transcription: synthesizing mRNA on a DNA template. This marked a scientific sensation and refuted the "central dogma" of molecular biology, which asserted that Genetic information flows in only one direction: DNA → RNA → protein.

The efficiency of mRNA copying into DNA—that is, the completeness of the synthesis of one of the strands of the target gene—depends on the absence of contaminants in the mRNA and enzyme preparations that could degrade the mRNA or the DNA product of synthesis. Therefore, the success of the synthesis reaction relies on the flawless purification of all components.

Introduction of the Gene into the Bacterial Cell. Subsequently, Gene cloning proceeds According to the following scheme. A circular vector molecule (most commonly an E. coli plasmid) is cleaved by restriction enzymes at a specific site in both DNA strands, converting it from a circular to a linear form. The gene (or genes) is "stitched" into the linear DNA using the enzyme DNA ligase (ligation), and the molecule is then re-circularized using the same DNA ligase. The resulting recombinant DNA is introduced into an E. coli cell, which, upon Replication, forms a clone in which all cells contain the recombinant DNA (plasmid) and, consequently, the foreign gene. The latter, now cloned within the bacterial cells, induces The Biosynthesis of the corresponding protein product.