Biochemistry and Molecular Biology - Belyasova N.A. 2002

Fundamentals of Genetic Engineering
Practical Application of Genetic Engineering Methods
Production of Eukaryotic Proteins and Solutions for Heterologous Gene Expression

One of The most significant Achievements of Genetic engineering is the Cloning of Eukaryotic genes, which enables microbial Cells to synthesize Proteins of critical importance to the national economy, such as Enzymes, Hormones, immunomodulators, and others. Eukaryotic Donors of genetic material typically exhibit a much lower level of natural protein production than genetically engineered producer strains. Therefore, The production of practically valuable proteins for Structural and functional studies, as well as for industrial Applications, is more frequently carried out using hybrid microbial strains rather than plant or Animal Cell Cultures, Organs, or Tissues. This approach has successfully yielded such valuable proteins as Human and Animal growth hormones, Insulin, human epidermal growth factor, human and murine tumor necrosis factor, a-, ß-, and g-interferons, the neural mediator Somatostatin, Calcitonin, Myoglobin, hormone-like signaling interleukins, Blood Coagulation factor missing in hemophilia patients, murine leukemia virus Reverse Transcriptase, urokinase, Trypsin, certain oncoproteins, and viral Vaccines, among others.

When a Gene is transferred within a vector molecule from the cells of one Organism to those of another, challenges often arise regarding its expression—mRNA METABOLISM/31.html">Transcription and Translation may either fail entirely or occur at a very low frequency. This is due to the strict Specificity of the enzymes catalyzing transcription and translation for particular DNA (mRNA) sequences, which can be uniquely structured across different taxonomic groups. Expression problems are most commonly observed when cloning eukaryotic genes in Prokaryotic Cells, largely because the Transcription initiation signals of higher eukaryotes are not recognized by bacterial RNA polymerases.

To achieve efficient Expression of cloned genes, several approaches are employed: 1) increasing the number of gene copies within The Cell (gene Amplification); 2) inserting a strong promoter, recognizable by the host cell RNA polymerase, upstream of the foreign gene's structural region; 3) placing a regulatory element upstream of the foreign protein gene to ensure efficient Translation initiation; and 4) stabilizing the resulting mRNA and protein product. These Methods for achieving high-level Gene Expression have been most thoroughly developed for E. coli. Because such methodology is a decisive factor in engineering novel producers, the bacterium is regarded as one of the most promising organisms for this purpose.

Gene amplification. To enhance gene expression, the gene dosage within the cell can be increased. This is accomplished in two ways: by increasing the copy number of recombinant Plasmids or by increasing the number of gene copies within a single plasmid.

The first method is the simplest. As noted previously, vector construction preferentially relies on multicopy plasmids under relaxed Replication control. In such cases, the plasmid copy number ranges from 10 to 200. This figure can be elevated to several thousand by suppressing bacterial cell Protein Synthesis or by employing mutant plasmids. Utilizing such vectors significantly increases the dosage of the target gene and, consequently, the yield of the protein product. However, it should be noted that excessive plasmid amplification may reduce the viability of the producer strain due to the high toxicity of certain foreign proteins to Bacteria, as well as an extended cell generation time.

The gene copy number within vector molecules can also be increased by constructing operons containing repeating, identical cistrons of foreign genes. A combination of these techniques makes it possible to raise the intracellular gene dosage from several dozen to thousands of copies per nucleoid, thereby substantially increasing the Synthesis of the corresponding proteins (in some cases by 1 to 2 orders of magnitude).

Achieving high-level transcription of foreign genes. For eukaryotic genes to be transcribed in prokaryotic cells, they are typically placed under the control of strong prokaryotic promoters (which ensure a high frequency of transcription initiation events). The most frequently utilized options include strong E. coli promoters such as the lactose Operon promoter P_lacUV5 and the Tryptophan promoter P_trp; hybrid promoters like P_trp-lacUV5 (Pac); as well as phage promoters including λ P_R, P_L, and others (T5, T7, φХ174). Isolating these promoters and integrating them into vectors is accomplished through standard Introduction/32.html">Genetic Engineering Procedures.

An even more advanced methodology involves The Use of regulatable promoters, which initiate efficient transcription only under specific environmental conditions. Classic Examples of such promoters are the P_R and P_L promoters of phage λ. They are typically used in cells harboring a Temperature-sensitive cI repressor, the gene for which may reside on the vector or on a compatible plasmid. In such engineered cells, expression of the foreign gene driven by P_R or P_L occurs only after the repressor is inactivated by raising the Fermentation temperature.

The deployment of prokaryotic regulatory elements situated on multicopy plasmids makes it possible to achieve foreign mRNA synthesis levels of up to 25% of the total bacterial cellular RNA.

Achieving high-level translation of foreign genes. Another prerequisite for the efficient expression of cloned genes is the presence of an optimal mRNA Translation initiation site upstream of the foreign protein coding sequence. The frequency of translation initiation is primarily governed by The Structure of the mRNA regions responsible for ribosome and tRNA binding. It has been demonstrated that the oligonucleotide sequence located at the 5'-end and directly adjacent to the start codon facilitates complementary base-pairing with the NUCLEOTIDES of the tRNA anticodon loop. Furthermore, the critical role of a purine-rich region located 3—15 nucleotides upstream of the start codon (the Shine-Dalgarno or SD sequence) in ribosome interaction has been firmly established. Both the length of the SD sequence and its spatial positioning relative to the start codon significantly impact ribosome-mRNA binding and, consequently, the frequency of translation initiation events.

Three main approaches are known for constructing vectors that ensure the translation of foreign DNA in bacterial cells. One of the most prevalent is the construction of "hybrid ribosome-binding sites." The Essence of this method is that the structural portion of the foreign gene (along with its own start codon and a few preceding nucleotides) is inserted between the SD sequence and the start codon of a prokaryotic gene. This method has the advantage of yielding a full-length foreign protein. However, a major drawback is the difficulty of achieving an optimal distance between the start codon and the SD sequence, which, as noted, is crucial for initiation. To circumvent this, another approach is applied, wherein a foreign structural gene devoid of its own regulatory regions is inserted into a robustly expressed bacterial gene. Most commonly, the lac promoter with its corresponding Shine-Dalgarno sequence is utilized for this purpose. The insertion site must be located sufficiently far from the translation initiation point to prevent the novel nucleotide sequence from interfering with the efficient transcription and Translation of the host bacterial gene. Expression of this vector type produces a fusion protein in which the N-terminal segment consists of the prokaryotic peptide Amino Acids. Consequently, subsequent chemical or enzymatic Cleavage is required to isolate the eukaryotic polypeptide chain.

Finally, the third approach to constructing vectors that ensure efficient translation relies on THE PRINCIPLE OF gene "overlapping." In this strategy, the foreign gene is embedded into the terminal region of a prokaryotic gene such that the SD sequence of the second gene resides directly within the coding region of the first. Here, the Translation termination codon of the first gene Functions as part of the initiation codon of the second gene. This method was developed in the USSR in 1985. It achieves 100% translation initiation efficiency for the second gene because the Ribosomes translating the first part of the polycistronic mRNA do not dissociate; instead, they reinitiate translation of the downstream gene. Thus, employing vectors with partially overlapping genes within operons ensures that the translation initiation efficiency of the foreign gene is comparable to that of the native prokaryotic Cistron.

Stabilization of mRNA and foreign gene protein products. The stability of mRNA in bacterial cells can be enhanced by introducing Mutations that inactivate RNases. Additionally, polynucleotide phosphorylase (PNPase) influences mRNA stability. E. coli mutants deficient in the pnp genes are known, in which the half-life of mRNA encoding foreign proteins is increased by 1.5-fold.

A major obstacle in obtaining overproducer strains can be the intracellular proteolysis of foreign proteins. The cellular repertoire of peptidases is specifically designed for the rapid degradation of Polypeptides with "aberrant" structures, which frequently arise from translation errors. To stabilize foreign proteins, host strains defective in protein degradation pathways (such as lon, htpR, or deg mutants) can be employed as recipients. Alternatively, one can introduce the T4 bacteriophage pin gene—which controls proteinase inhibitor synthesis—or other genes with similar functions into the bacterial cell.