Biochemistry and Molecular Biology - Belyasova N.A. 2002

Fundamentals of Genetic Engineering
Practical Application of Genetic Engineering Methods
Production of Eukaryotic Proteins and Solution of Heterologous Gene Expression Problems

One of The most significant Achievements of Genetic engineering is the Cloning of Eukaryotic genes, which enables microbial Cells to synthesize Proteins of vital economic importance, such as Enzymes, Hormones, immunomodulators, and others. Eukaryotic donor organisms of genetic material exhibit a significantly lower level of natural protein production than engineered producer strains. Therefore, practically valuable proteins intended for The Study of their Structure and properties, as well as for commercial Applications, are more frequently obtained using hybrid microbial strains rather than plant or Animal Cell Cultures, Organs, or Tissues. This approach has yielded such valuable proteins as Human and Animal growth hormones, Insulin, human epidermal growth factor, human and mouse tumor necrosis factor, a-, ß-, and y-interferons, the neurotransmitter Somatostatin, the hormone Calcitonin, Myoglobin, hormone-like signaling proteins (interleukins), the Blood-clotting factor lacking in hemophilia patients, mouse leukemia virus Reverse Transcriptase, urokinase, Trypsin, certain oncoproteins, and Vaccines against various Viruses, among others.

When a Gene within a vector molecule is transferred from the cells of one Organism to those of another, expression issues frequently arise—mRNA METABOLISM/31.html">Transcription and Translation may either fail to occur entirely or proceed at a low frequency. This is a consequence of the Specificity of enzymes catalyzing transcription and translation toward particular DNA (mRNA) sequences, the structures of which can be unique across different taxonomic groups of organisms. 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 recognized by the host cell's RNA polymerase upstream of the structural portion of the foreign gene; 3) placing a regulatory element that ensures efficient Translation initiation upstream of the foreign protein gene; and 4) stabilizing the resulting mRNA and protein product. These Methods for achieving high-level Gene Expression have been best developed for E. coli. Because the availability of such methodology is a decisive factor in constructing novel producers, the bacterium is regarded as one of the most promising organisms for this purpose.

Gene amplification. To enhance gene expression, the number of gene copies 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, constructing vectors is preferably done using multicopy plasmids under relaxed Replication control. In this case, the plasmid copy number ranges from 10 to 200. This figure can be increased to several thousand by suppressing bacterial cell Protein Synthesis or by employing mutant plasmids. Utilizing such vectors allows for a significant increase in the dosage of the target gene and, consequently, the yield of the protein product. It should be kept in mind, however, that excessive plasmid amplification can lead to a decrease in the viability of the producer strain due to the high toxicity of certain foreign proteins to Bacteria, as well as an increase in cell generation time.

The copy number of genes within vector molecules is ensured by constructing operons with repeating identical cistrons of foreign genes. Combining these methods makes it possible to elevate the gene dosage in the cell from several dozen to thousands of copies per nucleoid and to increase the synthesis level of the corresponding proteins (in some cases by 1–2 orders of magnitude).

Achieving a high level of foreign gene transcription. To ensure that eukaryotic Genes are 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 strong promoters of E. coli most frequently used for these purposes include the lactose Operon promoter P_lacUV5 and the Tryptophan promoter P_trp; hybrid promoters such as P_trp-lacUV5 (P_tac); and phage promoters such as λ P_R, P_L, and others (T5, T7, φX174). The isolation of these promoters and their incorporation into vectors are carried out through Introduction/32.html">Genetic Engineering manipulations.

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

The use of prokaryotic regulatory elements located on multicopy plasmids makes it possible to achieve foreign gene mRNA synthesis levels of up to 25% of the total bacterial cell RNA.

Achieving a high level of foreign gene translation. Another prerequisite for the efficient expression of cloned genes is the presence of an optimal mRNA Translation initiation site upstream of the foreign protein gene. The structure of the mRNA regions responsible for ribosome and tRNA binding plays a paramount role in determining translation frequency. It has been demonstrated that the oligonucleotide sequence located at the 5'-end and immediately adjacent to the start codon provides complementary base pairing with the NUCLEOTIDES of the tRNA anticodon loop. Meanwhile, among the mRNA nucleotides involved in interacting with the ribosome, the critical role of a purine-rich region located 3–15 nucleotides away from the start codon (the Shine–Dalgarno or SD sequence) has been reliably established. Both the length of the SD sequence and its positioning relative to the start codon significantly affect ribosome binding to mRNA 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 widespread is the method of constructing "hybrid ribosome-binding sites." Its essence lies in inserting the structural part of a foreign gene (possessing its own start codon and several preceding nucleotides) between the SD sequence and the start codon of a prokaryotic gene. This method has the advantage of producing 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 translation initiation. Therefore, another approach is applied, wherein a foreign structural gene devoid of its own regulatory regions is inserted into a highly expressed bacterial gene. For this purpose, the lac promoter with its corresponding Shine–Dalgarno sequence is most frequently utilized. The insertion site must be sufficiently distant from the translation initiation site so that the new nucleotide sequence does not interfere with the efficient transcription and Translation of the bacterial gene. Expression of vectors of this type yields a fusion protein in which the N-terminal portion is represented by the Amino Acids of the prokaryotic peptide. Consequently, isolating the eukaryotic polypeptide chain requires additional chemical or enzymatic Treatment.

Finally, the third approach to constructing vectors that ensure efficient translation employs THE PRINCIPLE OF gene "overlapping." In this case, the foreign gene is integrated into the terminal region of a prokaryotic gene such that the SD sequence of the second gene is located directly within the coding region of the first. Furthermore, the Translation termination codon of the first gene forms part of the initiation codon of the second. This method was developed in 1985 in the USSR. It enables 100% translation initiation of the second gene because the Ribosomes that translated the first part of the polycistronic mRNA do not detach from it; instead, translation of the second gene is reinitiated. Thus, employing vectors with partially overlapping genes in operons makes it possible to achieve an initiation efficiency for the foreign gene that is no lower than that of the original prokaryotic Cistron.

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

A serious obstacle in obtaining overproducer strains can be the proteolysis of foreign proteins within the cell. Indeed, the Complement of cellular peptidases is specifically designed for the rapid degradation of Polypeptides with "abnormal" structures that arise, for example, As a result of translation errors. To stabilize foreign proteins, strains defective in the protein degradation system (lon-, htpR-, or deg mutants) can be used as recipients. Alternatively, one can introduce the T4 bacteriophage pin gene—which controls the synthesis of proteinase inhibitors—or other genes with similar Functions into the bacterial cell.