Expression, Purification And Characterization Of Trucated Type Recombinant Buckwheat Trypsin Inhibitor

Protease inhibitors (PI) are small peptides or proteins which could inhibit protease activity. They are widely present in animals, plants and microorganisms in several forms of nature. Many important physiological processes rely on regulation of protease inhibitors. Up to now, hundreds of inhibitors have been isolated and characterized. According to the reactive sites of different enzymes inhibited, protease inhibitors are generally classified into five types:serine, threonine, cysteine, asparty1 and metalloprotease inhibitors. Among these, the structure, the action mechanism and the biological activity of serine protease inhibitors have been extensively studied by reserchers. TI belongs to serine protease inhibitors. The diversity and richness of TI from plants make them become a new resource of pharmacological study concerning protein drugs.Buckwheat, as a medicinal and edible crops, contains abundant of proteins, minerals and flavonoids. Our laboratory has previously obtained rBTI by genetic engineering techniques. The rBTI could inhibit trypsin specifically. Through site-directed mutagenesis, researchers defined the Arg45 as the active amino acids in active region. X-ray diffraction analysis showed that rBTI, composed of 69 amino acids, contained a a-helix structure, twoβ-fold structures and the line region. Function analysis revealed that the inhibitor could inhibit the proliferation of tumor cells.To reveal the relationship between structure and function of rBTI, recombinant plasmids (pExsecI-BTI-tl, pExsecI-BTI-t2, pExsecI-BTI-t3) were constructed by deleting VVM, TPVVM or VDTPVVM in the C-terminal region of rBTI respectively based on the amino acid sequence analysis and 3D structure of rBTI. The constructed vectors were transformed into E. coli strain BL21 (DE3). The fusion protein was purified by chromatography on Resource Q column and Superdex75. Protein in the SDS-PAGE pattern showed a single band, and its purity was above 98%. Analysis of the inhibitory activity to trypsin suggested that truncated type rBTI were similar to wild type rBTI in inhibitory activity. Probably the lack of C-terminal amino acids did not affect the active center and necessary group of rBTI, so they kept a relatively complete trypsin inhibitory activity. Analysis of physical and chemical properties revealed that truncated type rBTI showed remarkable stability to heat and pH. CD anslysis results further confirmed that the secondary structure of truncated mutants did not change significantly. In addition, anti-tumor experiments and observation of nuclear morphology showed that truncated type rBTI had a certain growth inhibition to EC9706 in a dose dependent manner.Using truncating mutant technique, this research obtained three truncated type inhibitors:rBTI-t1, rBTI-t2, rBTI-t3 through prokaryotic expression and two steps purification process. Physical and chemical properties and in vitro anti-tumor experiment results indicated that the lack of rBTI C-terminal amino acid did not cause the absence of functional part of the active region or larger changes. The result showed that amino acids in 63-69 of rBTI had no influence on combination of inhibitor and protease. This study provides a new way of thinking in-depth study of the relationship between structure and function, and mechanism of rBTI.