proteins of therapeutic value, such as antibodies and interferon, are secreted. However, the high levels of these proteins that are desirable from a commercial standpoint can quickly overwhelm the capacity of the cell secretory system. Thus, protein processing is a major limiting step in the achievement of high target protein yields. Although high levels of recombinant protein production have been found to increase the levels of proteins associated with proper protein folding and secretion in the endoplasmic reticulum, the levels are usually not sufficient for optimal protein processing. Researchers have therefore devised methods to increase the capacity for protein secretion by engineering cell lines with enhanced production of components of the secretion apparatus. In this regard, an effective strategy may be to simultaneously overexpress several, if not all, of the proteins that make up the secretory mechanism. This can be achieved through the enhanced production of the transcription factor X box protein 1 (Xbp-1), a key regulator of the secretory pathway. Normally, full-length, unspliced xbp-1 mRNA is found in nonstressed cells and is not translated into a stable, functional protein (Fig. 3.45A). However, when unfolded or misfolded proteins accumulate in the endoplasmic reticulum, a ribonuclease is activated that specifically cleaves xbp-1 mRNA (Fig. 3.45B). This results in the production of a functional transcription factor that activates the expression of a number of proteins of the secretion apparatus. A truncated xbp-1 gene that encodes an actively translated form of xbp-1 mRNA (Fig. 3.45C) was overexpressed under the control of the CMV promoter in recombinant CHO cell lines that were previously constructed to express human erythropoietin, human γ-interferon, and human monoclonal antibodies either stably or transiently. Expression of the genes encoding proteins of the secretion apparatus that are controlled by Xbp-1 was found to increase in response to elevated levels of Xbp-1. Although overexpression of Xbp-1 did not increase the production of recombinant proteins in stable cell lines in which the target gene is inserted in a chromosome, a significant increase was observed in cell lines engineered to express the target proteins transiently from a plasmid-encoded gene.
Figure 3.45 Strategy to increase yields of secreted recombinant proteins from mammalian cells by simultaneously upregulating the expression of several proteins in the secretion apparatus. The expression of chaperones and other proteins of the secretion apparatus is controlled by the transcription factor Xbp-1. (A) In unstressed cells, the intron (green box) is not cleaved from the xbp-1 transcript, and therefore, functional Xbp-1 transcription factor is not produced. (B) However, in stressed cells that have accumulated misfolded proteins, an endoribonuclease cleaves the transcript to yield mature xbp-1 mRNA (the red and blue boxes represent exons) that is translated into a stable, functional transcription factor. (C) Recombinant CHO cells were transfected with a truncated gene including only the xbp-1 exons and overproduced a functional Xbp-1 transcription factor that directed the production of high levels of proteins required for protein secretion.
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 (
