Overexpression of the Soluble Form of Chicken Cystatin in Escherichia coli and Its Purification

Abstract

A cDNA encoding chicken cystatin was cloned into the pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3)pLysS expression host. An active soluble form of cystatin was expressed in the cytoplasm of E. coli induced by isopropyl beta-D-thiogalactopyranoside. The recombinant chicken cystatin was purified to electrophoretic homogeneity by a simple and rapid method involving heat treatment and Sephacryl S-100 gel filtration chromatography. The recombinant cystatin behaved as a thermal-stable protein and exhibited papain-like protease inhibition activity comparable to the natural chicken cystatin.

Purification and characterization of cysteine proteinase inhibitors from crucian carp Carassius auratus eggs

“…The molecular masses of cst-I and cst-II were found to be 11.9 and 14.4 kDa, respectively, on SDS-PAGE gels under reducing conditions, whereas they were 13.5 and 12.7 kDa, respectively, under non-reducing conditions. Both cst forms had a molecular mass similar to those of family II cystatins from other species, such as chicken [5,35], common carp [34,36], chum salmon [37], and rainbow trout [38]. The small difference in molecular mass between each cst inhibitor under reducing and non-reducing conditions could be due to the conformational change that arises with the dissociation of the native disulfide bonds.…”

“…Both cst-I and cst-II had a thermal stability similar to that of common carp cystatin [36] and Chinese sturgeon cystatin [39], but inferior to cystatins obtained from terrestrial organisms, such as chicken [24,35] and tomato leaf [40]. The broad pH stability found among pig plasma kininogen Proteinases were pre-incubated with each cst inhibitor separately at room temperature, and the remaining proteolytic activities were assayed [4], chicken recombinant cystatin [24,35], tomato leaf cystatin [40], and rabbit muscle cystatin [41] was also present in cst-I and cst-II.…”

“…The broad pH stability found among pig plasma kininogen Proteinases were pre-incubated with each cst inhibitor separately at room temperature, and the remaining proteolytic activities were assayed [4], chicken recombinant cystatin [24,35], tomato leaf cystatin [40], and rabbit muscle cystatin [41] was also present in cst-I and cst-II. A comparison of the N-terminal sequences of the two cst with those of aquatic cystatins and the well-characterized chicken egg white is aligned in Fig.…”

Expression of Chicken Cystatin for Improving Insect Resistance in Rice

“…A particular sequence, Q x v x G, was found to be totally conserved among several of the animal cystatins, as well as among phytocystatins (see Barrett, 1986). This conserved sequence had been identified at positions 76 to 80 in the chicken cystatin molecule (Auerswald et al, 1992;Chen et al, 2000;Golab et al, 2001;Janowski et al, 2001 ).…”

“…In addition, we investigated the antiviral effect of chicken cystatin. Our expectation was that the introduction of an insecticidal proteinase inhibitor gene into cereal plants might be used in a general strategy for controlling insect pests, as was suggested by Auerswald et al (1992), Chen et al (2000), and Golab et al (2001).…”