Production and purification of two recombinant proteins from transgenic corn

• Published in: Biotechnology progress 1998, Vol.14 (1), p.149-155
• Main Authors: Kusnadi, A.R, Hood, E.E, Witcher, D.R, Howard, J.A, Nikolov, Z.L
• Format: Article
• Language: English
• Subjects: Adsorption; Amino Acid Sequence; Animals; AVIDIN; Avidin - biosynthesis; Avidin - genetics; AVIDINA; AVIDINE; BETA-GLUCURONIDASE; Biological and medical sciences; Biotechnology; Chickens; Coliform bacteria; Composition effects; Drug Stability; Escherichia coli; Extraction; Fundamental and applied biological sciences. Psychology; Genes; Genetic engineering; Genetic technics; GLICOSIDASAS; Glucuronidase - biosynthesis; Glucuronidase - genetics; GLYCOSIDASE; GLYCOSIDASES; Grain (agricultural product); Hot Temperature; Ion exchange; Methods. Procedures. Technologies; Modification of gene expression level; Molecular Sequence Data; Molecular Weight; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; Plants, Genetically Modified; Purification; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Size exclusion chromatography; Thermodynamic stability; TRANSGENIC PLANTS; ZEA MAYS; Zea mays - genetics
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp970138u

This study reports the production, purification, and characterization of recombinant Escherichia coli β‐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 °C for at least 3 months and at 25 °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 °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.