Chromosomal Integration and Environment
A major consideration for high levels and long-term stability of heterologous-protein production is the site of integration of the gene of interest into the mammalian cell genome. Expression of high levels of protein from plasmid vectors is transient and inevitably results in loss of the vector, which cannot be propagated in mammalian cells, or death of the host cell. Stable cell lines in which the target gene is integrated into a chromosome have been generated to overcome this problem. However, the site of integration can have a significant impact on the levels of target protein produced. Genomic DNA is associated with a great number of proteins, including the major histone proteins, around which the DNA is coiled, that compact (condense) the DNA so that it can fit inside the nucleus. The DNA and associated packaging proteins are known as chromatin. While much of the genome is highly condensed (heterochromatin) and contains silent genes or genes with low levels of expression, other regions are less condensed (euchromatin) and contain actively transcribed genes. For enhanced expression and stability, the target gene should be integrated into euchromatin, rather than heterochromatin. Because a larger portion of the genome is in the heterochromatin form, there is a greater chance that the target gene will be inserted into one of these regions.
Techniques to relax chromatin structure and thereby increase the expression of introduced genes include modifying host strains to express proteins that alter chromatin structure at the site of vector integration or inserting DNA elements that prevent chromosome condensation together with the target gene. One approach to alter the epigenetic environment surrounding the inserted gene is to increase histone acetylation. The extent of histone acetylation is determined by the relative activities of two host cell enzymes, histone acetyltransferase, which adds acetyl groups to lysines on histone proteins, and histone deacetylase, which removes acetyl groups from the histone. The relative influences of these two enzymes at a given promoter are determined by specific transcription factors that recruit one or the other of the enzymes to the promoter. Increased histone acetylation, which leads to increased gene transcription, can be accomplished either by increasing the expression of histone acetyltransferase or by decreasing the activity of histone deacetylase. One effective strategy to do this is to target histone acetyltransferase specifically to the site of target gene insertion to ensure that the target gene is actively and continuously transcribed. One group of researchers created a stable CHO cell line in which histone acetyltransferase was produced as a fusion protein with the LexA protein that binds to specific DNA sequences (Fig. 3.46). To test this fusion protein, the green fluorescent protein (GFP) reporter gene was employed as a target gene and was integrated into a CHO chromosome under the control of the CMV promoter with the LexA-binding sequence inserted upstream. A gene encoding resistance to the antibiotic Zeocin was coupled to the reporter gene by an IRES element and therefore was also under the control of the CMV promoter. Stable cells with an active CMV promoter were established by addition of Zeocin to the culture medium. Production of GFP, determined by measuring the emission of green fluorescence, was severalfold higher in cells that expressed the LexA–histone acetyltransferase fusion protein than in those that expressed the LexA protein alone (Fig. 3.46A). The LexA protein specifically binds to the LexA recognition site upstream of the gene encoding GFP and brings with it the fused histone acetyltransferase protein that acetylates histones associated with the promoter region and promotes a higher level of GFP transcription. Moreover, expression remained stable, although at a lower level, for at least 4 months in some of the clones.
Figure 3.46 Strategies to increase expression of recombinant proteins in mammalian cells by altering chromatin structure. Local “relaxation” of chromosome condensation, which leads to increased transcription of genes in the region, can be achieved by the addition of an acetyl group to DNA-packing proteins known as histones. Histone acetylation is catalyzed by the enzyme histone acetyltransferase (HAT). (A) To increase the expression of a recombinant protein, HAT was directed to the site of target gene (GFP gene) insertion in a mammalian chromosome. HAT was expressed as a fusion protein with the LexA protein that binds to a specific DNA sequence (LexA-BS) inserted upstream of the CMV promoter (PCMV) that directs expression of GFP. Production of the HAT-LexA fusion protein under the control of the SV40 promoter (PSV40) increased expression of GFP 6-fold compared to production of the LexA protein alone. (B) Insertion of STAR elements on both sides of the expression cassette further increased GFP expression. The gene encoding resistance to the antibiotic Zeocin was included as a selectable marker and was expressed from an IRES. The arrows above the promoter boxes indicate the direction of transcription.
To improve expression levels over a longer period, the construct was further modified to include a DNA segment known as a stabilizing and antirepressor (STAR) element on both sides of the expression cassette to block repression (Fig. 3.46B). Repression can occur when heterochromatin forms due to the association of the heterochromatin protein HP1 with methylated histones. This stimulates further histone deacetylation and methylation and, consequently, greater HP1 activity. Insertion of the relatively small (<2-kb) STAR elements was found to counteract the activity of HP1 and other heterochromatin-associated repressor proteins. Flanking the expression cassette with the antirepressor elements resulted in higher levels of GFP expression that were maintained over a longer period of time.
Other DNA elements that improve heterologous-protein expression by modifying heterochromatin structure are the ubiquitous chromatin-opening elements and matrix-associated regions. Ubiquitous chromatin-opening elements are sequences of DNA normally found near the promoters of housekeeping genes that are constitutively expressed at high levels due to enhanced histone acetylation. Inclusion of the ubiquitous chromatin-opening element from the promoter of the highly expressed CHO elongation factor 1 alpha gene in an expression vector increased recombinant protein expression in CHO cells 6- to 35-fold. Matrix-associated regions were also found to enhance the production of heterologous protein in CHO cells. These elements, found in the chromosomes of many eukaryotes, bind to protein complexes in the nucleus that arrange regions of the chromosome into loops. It is thought that these DNA loops contain transcriptionally active genes that are regulated in a coordinated fashion. Although matrix-associated regions from the human β-globin gene and the chicken lysozyme gene were found to increase expression of a target gene, not all matrix-associated regions have a positive effect on gene expression.
In sum, mammalian cell expression systems are as versatile and effective as other eukaryotic expression systems. However, industrial production of a recombinant protein with engineered mammalian cells is costly. Consequently, less expensive expression systems are favored unless authenticity of an important recombinant protein can be obtained only with mammalian cells.
Protein Engineering
The physical and chemical properties of natural proteins are sometimes not well suited to a medical, industrial, or other application. In some instances, a protein that is better suited to a particular task may be obtained by using a gene from an organism that grows in an unusual, often extreme, environment. In addition to isolating natural genes that encode proteins with useful properties, directed or random mutagenesis and selection schemes can be used to create a mutant form of a gene that encodes a protein with the desired properties. In directed mutagenesis, a specific amino acid is targeted for change by substituting nucleotides in the corresponding coding sequence in the gene. In random mutagenesis, the amino acid changes that will result in the desired properties are unknown. A large number of mutant proteins, each with a different amino acid change, are generated by randomly altering individual nucleotides within a structural gene, and then tested for the desired properties.
By using directed or random