CaMV promoter, transgene expression

The first hurdle encountered during the development of alfalfa as a recombinant protein production system was the relative inefficiency of the available expression cassettes. A study in which a tomato proteinase inhibitor I transgene was expressed in tobacco and alfalfa under the control of the cauliflower mosaic virus (CaMV) 35S promoter showed that 3-4 times more protein accumulated in tobacco leaves compared to alfalfa leaves [5]. Despite the low efficiency of the CaMV 35S promoter in alfalfa, bio-pharmaceutical production using this system has been reported in the scientific literature. Such reports include expression of the foot and mouth disease virus antigen [6], an enzyme to improve phosphorus utilization [7] and the anti-human IgG C5-1 [8]. In this last work, the C5-1 antibody accumulated to 1% total soluble protein [8]. 

Fig. 1.1 Promoter activity in alfalfa leaves. Accumulation ofp-glucuroni-dase achieved in transgenic alfalfa leaves expressing the gusA gene under the control of CaMV 35S and alfalfa promoters. %TSP, percentage of total soluble proteins.

A. thaliana AtNrampS belongs to a family of broad specificity membrane metal transporters and can complement deficiencies in manganese and iron transport in yeast (Thomine et al., 2000). It is expressed in both the roots and the aerial parts of plants, and is induced in roots by Fe starvation. Transgenic Arabidopsis plants over-expressing the gene under the control of the CaMV 35S promoter displayed Cd hypersensitivity and increased Fe accumulation, but the accumulation of Cd was not affected. 

A functional uricase was obtained by expression in E. coli of a soybean N-35 cDNA driven by the bacterial lacZ promoter (Suzuki Verma, 1991). The uricase activity was mainly found in the cytoplasmic fraction of E. coli and had the same pH optimum and apparent Km values as in the nodules. That N-35 is able to assemble into a functional, tetrameric holoenzyme in E. coli indicates that post-translational modifications, or the presence of peroxisomes, is not essential for its proper assembly and function in this organism. However, N-35 is not active and does not accumulate to any significant levels when it is expressed in transgenic tobacco under the control of the CaMV-35S promoter (our unpublished data). 

GM oilseed rape plants containing a gene encoding the protease inhibitor OCI, under the control of the CaMV 35S promoter, had measurable quantities of this transgene product in their leaves (0.2-0.4 percent of total soluble protein) but not in their pollen [16]. This finding was confirmed by Jouanin et al. [17], who also noted that Bowman-Birk soybean trypsin inhibitor (BBI) could not be detected in the nectar or pollen of GM oilseed rape plants which had measurable expression levels in leaves (gene also on the CaMV 35S promoter). 

Figure I. T-DNA region of pIESIAI andpIJ2B6plasmids used to express human CYPl A1 and CYP2B6 in transgenic rice plants. RB, right border LB, left border NOS, nopaline synthase promoter NT, nopaline synthase terminator NPTII, neomycin phosphotransferase II 35S, cauliflower mosaic virus (CaMV) 35S promoter E7, seven-enhancer region (-290 to -90) from CaMV 35S promoter AMV-5 VTR, alfalfa mosaic virus 5 -untranslated region HPT, hygromycin B phosphotransferase.

---