Seminars

Genetic engineering of the major cereal crops creates unprecedented opportunities for the food, feed and biotech industries. Efficient transformation methods, targeted expression of products to storage organs and reliance on well-established processing infrastructures, now make it practical to introduce value-added traits such as biopharmaceuticals, nutraceuticals and industrial enzymes into the cereals. Various mother milk proteins such as human lactoferrin (hLF) and human lysozyme (hLZ) have been expressed in mature rice seeds. Expression levels, structure-function characterizaiton, process development, large-scale grow out, formulation, and product development of these proteins will be discussed. Human lactoferrin (hLF) is an 80 kDa

iron-binding glycoprotein belonging to the transferrin family. LF is found in high concentration (average 1-2 g/l) in human milk. Human LZ is one of the most abundant proteins in human milk, found at concentrations of 1?2 g/l. It is also found in other mammalian secretions, such as tears and saliva. To achieve the highest levels of expression, human LF and LZ were expressed, posttranscriptionally processed and transported to the protein storage bodies in the maturing rice gain. Appropriate selection of tissue specific promoters, signal peptide, selectable marker, and terminator produced expression levels of recombinant human lysozyme (rhLZ) and recombinant human lactoferrin (rhLF) reached 0.5 - 0.6% of the brown rice weight or up to 50% of soluble proteins. Biochemical, biophysical and functional comparisons of native and rhLF revealed correct N-terminal sequence molecular weight, pI and specific activity. Similar thermal and pH stability was observed for rhLZ. Furthermore, similar bactericidal activity was displayed towards a laboratory strain of E. coli. Like rhLZ, purified rhLF was identical to its native human counterpart with the exception its Nlinked glycosylation. Structural differences in glycan revealed that rhLF has typical plant glycan.

Transgenic rice expressing rhLZ and rhLF has been grown in USA since 1998 under permits from United States Department of Agriculture (USDA). Biotechnology Regulatory Services (BRS) of USDA conducted an “Environmental Assessment” (EA) on planting these rice lines to produce rhLZ and rhLF. Based on the scientific evidence, the USDA concluded there would be no significant effect on humans, animals, or the environment as a result of the cultivation of rhLF and rhLZ rice. Economic models indicate that high-level expression of recombinant protein in crop plants is a key requirement for the successful plant-made-harmaceuticals. Clinical data support the addition of rice derived hrLZ and rhLF improves the efficacy of oral rehydration solution (ORS) for the treatment of children with acute diarrhea. Currently, rhLF is commercially available for serum-free cell culture applications and rhLZ is commercially available for improving bioprocessing efficiency particularly for microbial

fermentation application. The use of these two recombinant proteins in human nutrition and for preventing and treatment of disease should be a reality in near future.