title: Processing and localization of bovine beta-casein expressed in transgenic soybean seeds under control of a soybean lectin expression cassette.
abstract: We have examined the processing and subcellular localization of a chimeric gene consisting of the bovine milk protein, beta-casein, under the control of a soybean seed lectin promoter and its 32 amino acid signal sequence in the seeds of transgenic soybean plants. The beta-casein expressed in developing soybean seeds is a doublet with apparent molecular weight slightly smaller than the bovine beta-casein and expression of the protein was highest in immature cotyledons. The casein proteins were purified from the immature soybean seeds by immunoaffinity chromatography and were analyzed by two-dimensional gel electrophoresis, blotting, and amino terminal sequencing. The N-terminal sequences of both of the doublet soybean casein polypeptides were identical to the N-terminal sequence of the bovine beta-casein indicating that the 32 amino acid lectin signal sequence was cleaved precisely from the chimeric protein in developing soybean seeds. Analysis of the purified soybean beta-casein polypeptides by mass spectrometry (MALDI-MS) showed that they are not phosphorylated. Absence of added phosphate groups is the cause of the size difference between the soybean beta-casein and native bovine beta-casein protein. Immunolocalization experiments showed that the casein protein was found in the protein storage vacuoles (PSV) in developing and mature soybean seeds. The precise removal of the 32 amino acid lectin amino terminal sequence from the chimeric lectin-casein fusion suggests that the lectin expression cassette can be used for production of pharmaceutical or other recombinant proteins of added value in the developing soybean seed.
authors.affiliation: Gene Logic Inc., 708 Quince Orchard Road, 20878, Gaithersburg, MD, USA
authors.forename: R
authors.initials: R
authors.lastname: Philip
authors.forename: D W.
authors.initials: DW
authors.lastname: Darnowski
authors.forename: P J.
authors.initials: PJ
authors.lastname: Maughan
authors.forename: L O.
authors.initials: LO
authors.lastname: Vodkin