protein production with genetically engineered plant cell cultures

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Production of a mouse heavy chain monoclonal immunoglobulin was investigated in the suspension culture of genetically modified Nicotiana tabacum cells. A cDNA clone containing the entire coding region of a murine heavy chain antibody was inserted between CaMV 35S promoter and T-DNA gene 7 terminator using a binary Ti plasmid vector. The resulting molecule was transferred into Agrobacterium tumefaciens, which was used for the transformation of tobacco suspension cells. A separate investigation was carried out to isolate strong promoters for foreign protein production with transgenic plant cell culture. It was found that over 67% of the transformants produced a detectable level of heavy chain immunoglobulin.

The batch suspension culture showed that heavy chain immunoglobulin was most actively produced during the early part of exponential growth period. About 50% of the antibody was secreted into the culture media. The highest antibody concentration was 0.3% of the soluble protein in the medium. The effect of oxygen supply on the growth of genetically modified tobacco cell cultures and the formation of $\beta$-glucuronidase GUS) was investigated in 250-mL Erlenmeyer flasks, a 5-L stirred tank fermenter, and a 7-L air lift fermenter. Higher oxygen supply stimulated cell growth, increased oxygen consumption rate, phenolics and GUS productions.

cDNA clones were isolated from a cDNA library of exponentially growing tobacco cell suspension. The clones were present at approximate 0.5 $\sim$ 1.4% levels in the library. Northern blot studies indicated that the clones TSC29, TSC40 and TSC81 were strongly expressed in actively dividing cultured cells but weakly expressed in old cells. These clones were determined to encode ribosomal proteins and are useful for strong promoter isolation.

A strong promoter for foreign protein production was constructed by the modification of cauliflower mosaic virus CaMV) 35S promoter. Insertion of an octopine synthase gene ocs) enhancer element into the 35S promoter significantly increased the activity. The modified promoter with three copies of ocs elements increased the promoter activity by 2.6 times compared to the original 35S promoter. The modified promoter can be used to increase the yield of foreign protein production in the cultures of genetically modified plant cells.