In this project, we are taking the distinct advantage of plant expression systems, which is the lack of the capacity to add sialic acids, to genetically modify plants for asialo-rhuEPO - a special glycoprotein without sialic acids. Although nearly all human glycoproteins require sialic acid residues for their in vivo functions, the one exception we could find is rhuEPO, which has both hematopoietic and tissue protective functions. When rhuEPO is used as an antiapoptotic agent, its hematopoietic activity can cause bad side effects by leading to harmful increases in red blood cell mass. This activity can be disrupted by removing sialic acids. Asialo-rhuEPO generated by total enzymatic desialylation of rhuEPO has been demonstrated to retain the cell- and tissue-protective properties of EPO without hematopoietic activity. This type of EPO derivative can be explored as the antiapoptotic agent. However, there is no expression system that can produce asialo-rhuEPO directly. It is also unlikely to use commercially available rhuEPO, which is produced only in mammalian cells, to make asialo-rhuEPO because of its limited supply. Based on available knowledge and techniques, it is likely to genetically modify plants to produce human glycoproteins without sialic acids. We have co- expressed human GalT gene and EPO gene fusing with ER-signal peptides in tobacco plants, and try to inexpensively produce asialo-rhuEPO with the potential of large scale production. We expect plant-produced asialo-rhuEPO will have similar cytoprotective function as the enzymatically produced asialo-rhuEPO. The results of this project will also provide insights into the plant glycosylation mechanism and the antiapoptotic mechanism of the plant- produced asialo-rhuEPO. In addition, the protein purification methodologies that will be established and the glycoprotein analysis methods will be very valuable for future plant glycoengineering projects.
We aim to genetically modify plants for production of asialo-rhuEPO - a special human protein. This type of biopharmaceutical protein can be explored as the neurotherapeutic and cardioprotective agent. Using a plant expression system, we expect to produce an inexpensive and functional asialo-rhuEPO to prevent cell death with the potential of large scale production.
|Kittur, Farooqahmed S; Arthur, Elena; Nguyen, Maikhanh et al. (2015) Two-step purification procedure for recombinant human asialoerythropoietin expressed in transgenic plants. Int J Biol Macromol 72:1111-6|
|Kittur, Farooqahmed S; Lalgondar, Mallikarjun; Hung, Chiu-Yueh et al. (2015) C-Terminally fused affinity Strep-tag II is removed by proteolysis from recombinant human erythropoietin expressed in transgenic tobacco plants. Plant Cell Rep 34:507-16|
|Hung, Chiu-Yueh; Umstead, Makendra L; Chen, Jianjun et al. (2014) Differential expression of a novel gene EaF82a in green and yellow sectors of variegated Epipremnum aureum leaves is related to uneven distribution of auxin. Physiol Plant 152:749-62|
|Kittur, Farooqahmed S; Bah, Mamudou; Archer-Hartmann, Stephanie et al. (2013) Cytoprotective effect of recombinant human erythropoietin produced in transgenic tobacco plants. PLoS One 8:e76468|
|Hung, Chiu-Yueh; Fan, Longjiang; Kittur, Farooqahmed S et al. (2013) Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation. Plant Physiol 161:1049-60|
|Kittur, Farooqahmed S; Hung, Chiu-Yueh; Darlington, Diane E et al. (2012) N-Glycosylation engineering of tobacco plants to produce asialoerythropoietin. Plant Cell Rep 31:1233-43|
|Hung, Chiu-Yueh; Sun, Ying-Hsuan; Chen, Jianjun et al. (2010) Identification of a Mg-protoporphyrin IX monomethyl ester cyclase homologue, EaZIP, differentially expressed in variegated Epipremnum aureum 'Golden Pothos' is achieved through a unique method of comparative study using tissue regenerated plants. J Exp Bot 61:1483-93|