The natural soybean Kunitz trypsin inhibitor (SKTI) and and synthesized based on the primary sequence of SKTI from Uniprot database with accession quantity of “type”:”entrez-protein”,”attrs”:”text”:”P01070″,”term_id”:”125020″,”term_text”:”P01070″P01070. the activity of both trypsin and chymotrypsin [1, 2]. These inhibitors have been implicated in various physiological functions, such as regulator of endogenous protease, storage proteins, and defense molecules against herb pests and pathogens [3]. In soybean seeds, SKTI is usually synthesized as a precursor of 217 amino acids that would undergo proteolytic process to remove a signal peptide of 25 amino acid residues at N terminus and a hydrophobic polypeptide of 11 amino acid residues at C terminus, yielding a mature peptide of 181 amino acids [4, 5]. The mature inhibitor is described as a low cysteine content forming two disulfide bonds. Kunitz trypsin inhibitors including SKTI have a common structure composed of 12 anti-parallel -strands separated by irregular loops [6]. In SKTI, the side chain of Arg63 residue, as an active site residue, carried positive charges, forming strong electrostatic conversation with the unfavorable charge of the side chain of Asp189 in enzyme, significantly contributing to the binding of inhibitor to the active center of trypsin. Physique ?Figure11 gives a whole view that this active residue Arg63 of SKTI combines with the active center of trypsin to form a stable enzyme-inhibitor complex. In this article, inhibition kinetics of SKTI to trypsin was investigated; molecular docking technology was adopted to give an explanation of the inhibition mechanism. According to a combination of inhibition kinetic behavior and molecular structure modeling, we concluded that the inhibition type should be an irreversible inhibition instead of a competitive one. This might provide research for understand the inhibition mechanism of such kind of Kunitz trypsin inhibitors. Open in a separate windows Fig. 1 Three-dimensional model gives a general view. a SKTI (green) and its active sites (yellow). b Showing the interactions between SKTI (yellow) and trypsin (reddish) Trypsin inhibitors are important biochemical substances. Traditionally, SKTI was extracted from soybean seeds, which limited the large-scale application in agriculture and medical center because of the high costs of preparation [7, 8]. With the development of transgenic technology, host has been widely used as a tool to produce numerous recombinant protein. Production of recombinant protein provides a suitable method for commercializing medical products [9]. Another advantage of producing recombinant proteins is better safety in comparison with sample expressed from animal cell. Perhaps considering the inhibitory ability of SKTI Rabbit Polyclonal to CREBZF to serine protease, there were few reports on recombinant expression of SKTI in prokaryote [10]. Fortunately, there have been many studies about recombinant expression of SKTI in plants to harvest the resistant plants [11C14], which provided some guidance and experience for us. Here, we reported system was SR10067 used to express rSKTI with success. In addition, the refolding conditions of rSKTI inclusion bodies were optimized. The technology would be useful for the production and study of other Kunitz trypsin inhibitors. Biochemical properties of both SKTI and rSKTI were investigated in the research, such as optimum pH and temperature, stability of pH and temperature, and inhibition kinetics behavior. Some was first studied and the results should be useful for its application. Materials and Methods Materials The synthesis and analysis of SKTI gene sequence were performed by Generay Biotechnology Corporation (Shanghai, China). The recombinant trypsin was acquired from Yaxin Biotechnology Limited Company (Shanghai, China). The natural soybean Kunitz trypsin inhibitor (SKTI) and and synthesized based on the primary sequence of SKTI from Uniprot database with accession number of “type”:”entrez-protein”,”attrs”:”text”:”P01070″,”term_id”:”125020″,”term_text”:”P01070″P01070. The gene was cloned into pET-28a (+) expression vector (Novagen) SR10067 using the (upstream) and (downstream) cloning sites and then transformed into (DE3) strains which was held in our laboratory. Expression and Refolding of rSKTI The BL21 (DE3) strains were routinely cultivated at 37?C in Luria-Bertani medium containing 50?g/mL kanamycin. When the cells reached a optical density (OD600) of 0.9 with UV spectrophotometry, the cells were induced by isopropyl–d-thiogalactopyranoside (IPTG) with a final concentration of 0.5?mM. After growing for an additional 4?h at 37?C, the cells were harvested by centrifugation at 6000?rpm for 20?min and lysed by ultrasonication. Then, inclusion bodies were separated by centrifugation at 12000?rpm for 15?min at 4?C. Triton X-100 (0.5%, v/v) was used as a detergent to purify the inclusion bodies. The inclusion bodies were washed with 20?mM Tris-HCl buffer (pH SR10067 8.0) three times to eliminate Triton X-100. Purified inclusion bodies were denatured and then diluted in refolding buffer. The final concentration of protein in the refolding buffer was 1?mg/mL. A L25(56) orthogonal experiment design.