The Gene Cloning And Expression Of Mung Bean Trypsin Inhibitor

Many biologically active proteins and peptides are often generated by intracellular limited proteolysis of inactive precursors. These precursor convertases (PCs) are key regulators in many physiologically and pathologically important processes. Among them, as the first found and the best studied PC in mammalia, furin is ubiquitous in all tissue and cell lines examined and closely relevant to many diseases and pathogenesis, especially the intrusion of anthrax that was once used as a tool of bioterrorist to threat the human security and infection of HIV-1 that seriously harms the human health. Accordingly, the further studies about inhibitors of PCs, especially inhibitors of furin, make it possible to explore a new way to heal those disease relevant to pathogen.So far, the majority of the known specific inhibitors of furin, except for several synthesized short peptides, are protein-derived inhibitors that had been modified by protein engineering. The mung bean trypsin inhibitor (MBTI), which belongs to Bowman-Birk family of serine protease inhibitor, has potential to be engineered to a potent inhibitor because the Lys₂₀ at P₁ position and Arg₁₇ at P₄ position in it's first Lys active domain satisfy the basic requirement of substrate of PCs. However, it's necessary for MBTI gene to be cloned and expressed firstly so as to bioengineer the MBTI by protein engineering.The thesis reviews some recent progresses and developments in the study of PCs and their inhibitors. It includes the following aspects: the structure of PCs, the tissue and cellular distribution of PCs; the relationship of PCs and disease; the relationship of PCs and pathogen, and the study on the inhibitors of PCs. In the experimental section of the thesis, the studies focus on the cloning and expression of MBTI gene.The Bowman-Birk inhibitor purified from the mung bean seeds displays strong inhibitory activity toward kexin with a Ki.of 1.53×10^-10M, but low inhibitory activity toward furin with a Ki of 1.33 × 10^-5M. Primers were designed according to the conserved regions of the determined amino acid sequence of the inhibitor. By the method of RACE (rapid amplification of cDNA ends), the cDNA of mung bean trypsin inhibitor is cloned from the total RNA of developing mung bean seeds and the gene encoding the Bowman-Birk inhibitor is identified. The ORF (open-reading frame) encodes 106 residues including a...