Limitations of Target Protein Quantities fRoM Plant sources

Typically, gram quantities of purified target protein are required for safety studies. Ideally, the protein would be isolated directly from the transgenic event to be commercialized. However, it is generally not feasible to purify the required amount of protein from transgenic plants for the following reasons. The expression of introduced proteins in edible parts of plants (e.g., grain) varies depending on the promoter governing expression of the gene, the protein's localization within the cell, and the protein's mode of action. In some cases, expression can be as low as 0.1 ppm (^g protein/g fresh weight). Depending on the level of expression, the purification of a sufficient amount of protein for the safety assessment may require hundreds of kilograms of grain. This task can be quite daunting, considering that grain accumulates high levels of storage proteins, oil, and starch. Furthermore, the numerous proteases present in grain67 that are released during the purification procedure may cause nonspecific proteolysis of the introduced protein leading to truncations on its N- or C-termini. Storage proteins present in grain might interfere with the purification of low expressed proteins, making it difficult to achieve a high level of purity of the protein of interest. Many purification methods that are based on a selective removal of seed storage proteins with alcohols and acids mixtures8 might not be applicable because of denaturation and, consequently, a loss of activity in the protein of interest.

It is feasible, however, to purify a small amount of protein from the plant source while producing a large amount of the transgenic protein in a heterologous expression system. The approach of using heterologously produced protein as a surrogate for plant-expressed protein in safety testing has been utilized for a number of proteins introduced into a variety of crops and has become a well-established and accepted strategy.1,5,9,10-15

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