Fig. 3. Sponge cystatin, a cysteine proteinase inhibitor. (A) Alignment of the sponge cystatin (CYTA_SUBDO) with the corresponding cystatins from humans, cystatin A (CYTAA_HUMAN; accession number NP_005204) and cystatin B (CYTAB_ HUMAN; NP_000091). Amino acids that are similar among all three sequences are in white type. The characteristic cystatin domain spans amino acids 11 to 66 (CysD). (B) A phylogenetic tree constructed after alignment of the above-mentioned three sequences as well as the pig leukocyte cysteine proteinase inhibitor 1 (CYSPROINH_PIG; P35479), pig stefin A8 (STEFA8; NP_999025), pig stefin A5 (CYTAA5_PIG; Q28986), cattle proteinase inhibitor stefin-C from Bos taurus (CYTAC_BOVIN; P35478), the insect putative protein CG31016 from Drosophila melanogaster (CG31016_DROME; NP_733393), the putative protein from Caenorhabditis elegans (C17H1_CAEEL; NP_493295), the yeast enzyme DNA topoisomerase III from Saccharomyces cerevisiae (DNATOPO_YEAST; NP_013335), and the plant cysteine proteinase inhibitor from Arabidopsis thaliana (CYSPROINH_ARATH; NP_850570). The protein from A. thaliana was used as an outgroup to root the tree. Scale bar indicates an evolutionary distance of 0.1 amino acid substitutions per position in the sequence. (C) The cloned cystatin cDNA from S. domuncula was used for transfection of E. coli. After treatment of the bacteria with IPTG, the protein (cystatin; lane a) was extracted and purified as described under Materials and methods. M, size markers.