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 NcoI/PstI-pKK233-2 expression vector in Escherichia coli MV1190, 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 beta-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.
Fukazawa
C
C
Genetic Engineering Laboratory, Ministry of Agriculture, Forestry and Fisheries, Ibaraki, Japan.
Udaka
K
K
Murayama
A
A
Higuchi
W
W
Totsuka
A
A
D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D005916:Globulins / Q000096:biosynthesis* / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D030262:Soybean Proteins; D013025:Glycine max / Q000235:genetics
D005916:Globulins; D010940:Plant Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D030262:Soybean Proteins; D004247:DNA; C011474:glycinin