Chikafusa Fukazawa

Expression of soybean glycinin subunit precursor cDNAs in Escherichia coli

As the cDNAs encoding A1aB1b and A2B1a subunit precursors of the glycinin A2 subfamily contain a unique NcoI site sequence, (A)CCATGG, occurring at their translation initiation sites, plasmids were constructed to direct the synthesis of those precursor proteins by inserting NcoI/PstI fragments derived from those cDNA clones into the Nco/PstI‐pKK233‐2 expression vector in Escherichia coli MV 1190, respectively. The resultant plasmids directed the expression of 57‐kDa protein components that have molecular masses in agreement with those of the in vitro translation products directed by glycinin A2 subfamily mRNAs, by the addition of isopropyl β‐D‐thiogalactoside. These proteins, which comprised as much as 1% of the total bacterial protein, are immunoprecipitable with rabbit antibodies specific for glycinin subunits. This procedure makes glycinin subunits available as a model for studying structure‐function relationships in seed proteins using site‐directed mutagenesis. This is the first expression of glycinin‐like storage protein in E. coli.