Cloning, Functional Expression, and Characterization of Cystatin in Sesame Seed

Abstract

A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds. The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli. The recombinant cystatins were found in the soluble fraction of cell extract and were demonstrated to be functionally active in a reverse zymographic assay. The corresponding endogenous 22 kDa cystatin of low abundance in mature seeds was purified to homogeneity via a papain-coupling affinity column and confirmed by western blotting with antibodies against the recombinant cystatin. Both endogenous and recombinant cystatin proteins showed effective inhibitory activities against papain with K(i) values of 7.89 x 10(-8) M and 2.77 x 10(-8) M, respectively. Immunodetection indicated that cystatin was specifically expressed in maturing seeds and rapidly degraded in germination. Accordingly, zymographic and inhibition analyses showed that sesame cystatin could not inhibit the de novo synthesized proteases in germinating seeds. It is suggested that sesame cystatin may play a role in the regulation of endogenous cysteine proteases during seed maturation and germination.

A cold inducible multidomain cystatin from winter wheat inhibits growth of the snow mold fungus, Microdochium nivale

“…The mTaMDC1 constant was lower than the Ki value reported for WC5 (10 À6 M; Corre-Menguy et al 2002), but higher than those of rice OC I (3.0·10 À8 M; Kondo et al 1990) and maize CC I (2.3·10 À8 M; Abe et al 1994). Comparison of papain inhibition activity of 23 kDa cystatins showed that the Ki of mTaMDC1 was in the same order as soybean cystatin (1.9·10 À7 M; Misaka et al 1996), but higher than cystatins from sesame (2.7·10 À8 M; Shyu et al 2004) and strawberry (1.9·10 À9 M; Martinez et al 2005a).…”

“…2). Comparison of TaMDC1 with some of a non cereal 23 kDa cystatins characterized so far showed 65, 64, 62, 62, 61, 59% identity with cystatin from cabbage (Lim et al 1996), sesame (Shyu et al 2004), apple (Ryan et al 2003), soybean (Misaka et al 1996), taro (Yang and Yeh 2005), and strawberry (Martinez et al 2005a), respectively (Fig. 2).…”

“…Most of them have a molecular mass of 12-16 kDa, show significant amino acid sequence homology and contain no disulfide bonds. Several phytocystatins with molecular mass about 23 kDa (Lim et al 1996;Misaka et al 1996;Shyu et al 2004;Yang and Ye 2005;Martinez et al 2005a;Masonneau et al 2005) and higher (Waldron et al 1993;Kouzuma et al 2000;Wu and Haard 2004) due to multiplication of cystatin or cystatin-like domains have also been identified and characterized.…”

Theobroma cacao cystatins impair Moniliophthora perniciosa mycelial growth and are involved in postponing cell death symptoms

“…Several members of this family have been characterized in various plant species, and homology with animal cystatins has been described (Margis et al 1998). Most of phytocystatins are small proteins, ranging in size from 12 to 16 kDa, with a distinct group having a higher molecular weight (*23 kDa) due to a C-terminal extension (Shyu et al 2004;Margis-Pinheiro et al 2008). Several multicystatins with up to eight cystatin domains have been reported, particularly in tomato (Wu and Haard 2004).…”

Processing of soybean (Glycine max) extracts in aqueous two‐phase systems as a first step for the potential recovery of recombinant proteins

“…11,12 The use of seeds as host organ for recombinant protein production can be advantageous as they are natural storage organs with high expression levels, but this can be a challenge for downstream processing when the protein of interest has to be purified from the seed extract. 8 Soybean seeds contain more protein than any other commercial crop (up to 48%) consisting of a mixture of proteins (α-, β-, and γ -conglycinin, glycinin and other globulins) ranging in molecular weight from 140 to 300 kDa with different physicochemical properties allowing fractionation and differentiation.…”