Processing and localization of bovine beta-casein expressed in transgenic soybean seeds under control of a soybean lectin expression cassette

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 deve loping soybean seeds is a doublet with apparent molecular weight slightly s maller than the bovine beta -casein and expression of the protein was highe st in immature cotyledons. The casein proteins were purified from the immat ure 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 P-casein indicatin g 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 th at they are not phosphorylated. Absence of added phosphate groups is the ca use of the size difference between the soybean a-casein and native bovine P -casein protein. Immunolocalization experiments showed that the casein prot ein was found in the protein storage vacuoles (PSV) in developing and matur e soybean seeds. The precise removal of the 32 amino acid lectin amino term inal sequence from the chimeric lectin-casein fusion suggests that the lect in expression cassette can be used for production of pharmaceutical or othe r recombinant proteins of added value in the developing soybean seed. (C) 2 001 Elsevier Science Ireland Ltd. All rights reserved.