Production and purification of two recombinant proteins from transgenic corn.
This study reports the production, purification, and characterization of recombinant Escherichia coli beta-glucuronidase (GUS) and chicken egg-white avidin from transgenic corn seed. The avidin and gus genes were stably integrated in the genome and expressed over seven generations. The accumulation levels of avidin and GUS in corn kernel were 5.7% and 0.7% of extractable protein, respectively. Within the kernel, avidin and GUS accumulation was mainly localized to the germ, indicating possible tissue preference of the ubiquitin promoter. The storage-stability studies demonstrated that processed transgenic seed containing GUS or avidin can be stored at 10 degrees C for at least 3 months and at 25 degrees C for up to 2 weeks without a significant loss of activity. The heat-stability experiments indicated that GUS and avidin in the whole kernels were stable at 50 degrees C for up to 1 week. The buffer composition also had an affect on the aqueous extraction of avidin and GUS from ground kernels. Avidin was purified in one step by using 2-iminobiotin agarose, whereas GUS was purified in four steps consisting of adsorption, ion-exchange, hydrophobic interaction, and size-exclusion chromatography. Biochemical properties of purified avidin and GUS were similar to those of the respective native proteins.
Kusnadi
A R
AR
Department of Food Science and Human Nutrition, Iowa State University, Ames 50011, USA.
Hood
E E
EE
Witcher
D R
DR
Howard
J A
JA
Nikolov
Z L
ZL
D000595:Amino Acid Sequence; D000818:Animals; D001360:Avidin / Q000096:biosynthesis / Q000235:genetics*; D002645:Chickens; D004355:Drug Stability; D004926:Escherichia coli; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D003313:Zea mays / Q000235:genetics*
D011994:Recombinant Proteins; D001360:Avidin; D005966:Glucuronidase