COMMERCIAL PRODUCTION OF AVIDIN FROM TRANSGENIC MAIZE - CHARACTERIZATION OF TRANSFORMANT, PRODUCTION, PROCESSING, EXTRACTION AND PURIFICATION

We have produced in transgenic maize seed the glycoprotein, avidin, wh ich is native to avian, reptilian, and amphibian egg white. A transfor mant showing high-level expression of avidin was selected. Southern bl ot data revealed that four copies of the gene are present in this tran sformant. The foreign protein represents > 2% of aqueous soluble extra cted protein from populations of dry seed, a level higher than any het erologous protein previously reported for maize. In seed, greater than 55% of the extractable transgenic protein is present in the embryo, a n organ representing only 12% of the dry weight of the seed. This indi cates that the ubiquitin promoter which is generally considered to be constitutive, in this case may be showing a strong tissue preference i n the seed. The mature protein is primarily localized to the intercell ular spaces. An interesting trait of the transgenic plants expressing avidin is that the presence of the gene correlates with partial or tot al male sterility. Seed populations from transgenic plants were mainta ined by outcrossing and segregate 1:1 for the trait. In generations T2 -T4, avidin expression remained high at 2.3% (230 mg/kg seed) of extra ctable protein from seed, though it varied from 1.5 to 3.0%. However, levels of expression did not appear to depend on pollen parent or grow ing location. Cracked and flaked kernels stored at -29 degrees C or 10 CC for up to three months showed no significant loss of avidin activi ty. Commercial processing of harvested seed also generated no apparent loss of activity. The protein was purified to greater than 90% purity by affinity chromatography after extraction from ground mature maize seed. Physical characterization of purified maize-derived avidin demon strated that the N-terminal amino acid sequence and biotin binding cha racteristics are identical to the native protein with near identical m olecular weight and glycosylation. This study shows that producing avi din from maize is not only possible but has practical advantages over current methods.