title: Single chain antibody fragments for ocular use produced at high levels in a commercial wheat variety.
abstract: We are investigating the use of single chain antibody fragments (scFv) in eye drops for diagnosis and treatment of eye diseases. For ocular use, recombinant proteins must be free of bacterial endotoxin that causes inflammation in the eye. We required a means of generating high yields of scFvs with little endotoxin contamination. Using microprojectile bombardment we produced transgenic lines of the commercial wheat variety, Westonia, that express two scFvs that bind to CD4 or CD28 on the surface of rat thymocytes. A high level of expression of active scFv in the range 50-180 microg/g was measured by quantitative flow cytometry in crude extracts made from mature seeds. The levels of expression were stable over four generations of transgenic plants and mature seeds were stored for one year with little loss of scFv activity. Substantial purification of scFv was achieved by immobilised metal affinity chromatography. Compared to bacterial extracts, crude transgenic seed extracts contained only a small amount of endotoxin (150 EU/ml) that will be easily removed by purification. The transgenic wheat lines express functional scFv at levels comparable to production in bacteria and promise to be superior to bacteria for production of scFv pharmaceuticals for ocular use.
authors.affiliation: Department of Ophthalmology, Flinders University, Adelaide, SA 5042, Australia. helen.brereton@flinders.edu.au
authors.forename: Helen M
authors.initials: HM
authors.lastname: Brereton
authors.forename: Douglas
authors.initials: D
authors.lastname: Chamberlain
authors.forename: Rongchang
authors.initials: R
authors.lastname: Yang
authors.forename: Melinda
authors.initials: M
authors.lastname: Tea
authors.forename: Scott
authors.initials: S
authors.lastname: McNeil
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Coster
authors.forename: Keryn A
authors.initials: KA
authors.lastname: Williams
mesh_terms: D000818:Animals; D019470:Biolistics / Q000379:methods; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D005434:Flow Cytometry; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D007167:Immunotherapy / Q000379:methods*; D030821:Plants, Genetically Modified; D051381:Rats; D013950:Thymus Gland / Q000166:cytology / Q000276:immunology; D014908:Triticum / Q000235:genetics* / Q000276:immunology
chemical_list: D007128:Immunoglobulin Fragments