Expression of recombinant proteins in soybean seeds under control of legumes storage proteins regulatory sequences

Soybeans supply a major portion of the world’s demand for vegetable oil and protein and have also an important part in the development of plant molecular biology. Plant seed storage proteins are of great interest for the development of regulated tissue-specific genes products of commercial interest through recombinant DNA technology. The two major storage proteins within soybean and bean seeds are multimeric and have been characterized biochemically. The 7S proteins of many legumes consist of subunit combinations yielding a molecular mass of approximately 150-220 kDa. The 7S globulins are comprised of β-conglycinin and β-phaseolin subunits. The individual protein subunits vary in size within and among species. These proteins account for approximately 50-70% of the total protein. β-conglycinin and β-phaseolin regulatory sequences are seed tissue-specific temporally regulated and expressed in both the embryonic axis and cotyledons of developing seeds. Coding sequences of the human growth hormone; human coagulation factor IX and cyanovirin (microbicide against HIV) genes were placed under control of different promoters and signal peptides. Utilizing biolistic-mediated transformation, each construct was introduced into the soybean genome and putative transgenic plants were obtained. Presence of introduced coding sequences was confirmed by molecular analysis. Biochemical assays were conducted to detect the expression and/or bioactivity of the recombinant proteins. Ultrastructural immunocytochemistry assays revealed that the recombinant proteins accumulated in the endoplasmic reticulum (ER)-derived protein storage vacuoles (PSV’s). Recombinant molecules were visualized all along the cistern lumen but were absent from the interior of seed oil bodies. Experiments have been carried out towards the utilization of a nano-UPLC fractionation and SynaptHigh Definition Mass Spectrometry (HDMS) aiming the development of a nanoESI positive mode system to conduct a characterization of the recombinant proteins in a few seeds with Identity. Exploiting the utilization of seed-specific regulatory sequences capable of directing and accumulating recombinant proteins in the PSV’s from soybean seeds, associated with recent tools in biological research, have great potential to accelerate the preliminary characterization of plant-derived biopharmaceuticals and industrial macromolecules.