PROCESSING IN-VITRO OF PRONAPIN, THE 2S STORAGE-PROTEIN PRECURSOR OF BRASSICA-NAPUS PRODUCED IN A BACULOVIRUS EXPRESSION SYSTEM

The maturation of the 2S albumin, napin, in Brassica napus L. involves removal of an amino-terminal and an internal propeptide. Pulse-chase experiments with B. napus embryos showed that intermediates are detect able during the pronapin processing. Intact pronapin was expressed by baculovirus in Spodoptera frugiperda insect cells in order to obtain s ubstrate for studying the processing event. Processing of pronapin wit h a crude B. napus embryo protein extract resulted in several fragment s of similar sizes to those of napin heavy and light chains. The chara cter of the major processing activity in the B. napus extract suggeste d that it was due to an aspartic proteinase. A secondary activity indi cated an additional endoproteinase involved in the pronapin processing . Limited proteolysis of pronapin with a purified aspartic proteinase from Hordeum vulgare showed that cleavage occurred exclusively in the prosequences. The cleavage products formed in-vitro requires additiona l trimming of the propeptides in order to obtain the subunits of matur e napin.