Abstract
A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.
References
Abe M, Abe K, Kuroda M, Arai S: Corn kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin. Eur J Biochem 209: 933–937 (1992).
Abe K, Emori Y, Kondo H, Suzuki K, Arai S: Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). J Biol Chem 262: 16793–16797 (1987).
Abe K, Emori Y, Kondo H, Arai S, Suzuki K: The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily. J Biol Chem 263: 7655–7659 (1988).
Abrahamson M, Ritonja A, Brown MA, Grubb A, Machleidt W, Barrett AJ: Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin. J Biol Chem 262: 9688–9494 (1987).
Auerswald A, Genenger G, Assfalg-Maschleidt I, Machleidt W, Engh RA, Fritz H: Recombinant chicken egg white cystatin variants of the QLVSG region. Eur J Biochem 209: 837–845 (1992).
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Short Protocols in Molecular biology: A Compendium of Methods from Current Protocols in Molecular Biology. John Wiley, New York (1992).
Barrett AJ, Rawlings ND, Davies ME, Macheleidt W, Salvesen G, Turk V: Cysteine proteinase inhibitor of the cystatin superfamily. In: Barrett AJ, Salvesen G (eds), Proteinase Inhibitors, pp. 515–569. Elsevier, Amsterdam (1986).
Bode W, Engh R, Musil D, Thiele U, Huber R, Karshikov A, Rrzin J, Kos J, Turk V: The 2.0 Å X-ray crystal structure of chicken egg white cystatin and its possible mode of Interaction with cysteine proteinases. EMBO J 7: 2593–2599 (1988).
Brizin J, Ritonja A, Popovic T, Turk V: Low molecular mass protein inhibitor of cysteine proteinase from soybean. Hoppe Seyler J Biol Chem 371: 167–170 (1990).
Fernandes KVS, Sabelli PA, Barratt DHP, Richardson M, Xavier-Filho J, Shewry PR: The resistance of cowpea seeds to bruchid beetles is not related to levels of cysteine proteinase inhibitors. Plant Mol Biol 23: 215–219 (1993).
Hildmann T, Ebneth M, Pena-Cortes H, Sanchez-Serrano JJ, Willmitzer L, Prat S: General roles of abscisic acid and jasmonic acids in gene activation as a result of mechanical wounding. Plant Cell 4: 1157–1170 (1992).
Kondo H, Abe K, Emori Y, Arai S: Gene organization of oryzacystatin-II, a new cystatin superfamily member of plant origin, is closely related to that of oryzacystatin-I but different from those of animal cystatins. FEBS Lett 278: 87–90 (1991).
Kondo H, Abe K, Nishimure I, Watanabe H, Emori Y, Arai S: Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. J Biol Chem 265: 15832–15837 (1990).
Kondo H, Emori Y, Abe K, Suzuki K, Arai S: Cloning and sequence analysis of the genomic DNA fragment encoding oryzacystatin. Gene 81: 259–265 (1989).
Laemmli UK: Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 227: 680–685 (1970).
Liang C, Brookhart G, Feng GH, Reeck GR, Kramer KJ: Inhibition of digestive proteinases of stored grain coleoptera by oryzacystatin, a cysteine proteinase inhibitor from rice seed. FEBS Lett 2: 139–142 (1991).
Lütcke HA, Chew KC, Mickel FS, Moss KA, Kern HF, Scheele GA: Selection of AUG initiation codons differs in plants in plants and animals. EMBO J 6: 43–48 (1987).
Newman T, de Bruijn FJ, Green P, Keegstra K, Kende H, McIntosh L, Ohlrogge J, Raikhen N, Somerville S, Thomashow M, Retzel E, Somerville C: Genes Galore: A Summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol 106: 1241–1255 (1994).
Park YS, Kwak JM, Kwon O, Kim YS, Lee DS, Cho MJ, Lee HH, Nam HG: Generation of expressed sequence tags of random root cDNA clones of Brassica napus by single-run partial sequencing. Plant Physiol 103: 359–370 (1993).
Saitoh E, Kim HS, Smithies O, Maeda N: Human cysteine-proteinase inhibitors: nucleotide sequence analysis of three members of the cystatin gene family. Gene 61: 329–338 (1987).
Stanley KK, Herz J, Haymerle H: constructing expression cDNA libraries using unphosphorylated adaptors. In: Walker JM (eds) Methods in Molecular Biology, New Nucleic Acid Techniques, pp. 319–328, Humana Press Clifton, New Jersey (1988).
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW: Use of T7 RNA polymerase to direct expression of cloned genes. Meth Enzymol 185: 60–89 (1990).
Turk V, Bode W: The cystatins: Protein inhibitors of cysteine proteinases. FEBS Lett 285: 213–219 (1991).
Waldron C, Wegrich LM, Merlo PAO, Walsh TA: Characterization of a genomic sequence coding for potato multicystatin, an eight-domain cysteine proteinase inhibitor. Plant Mol Biol 23: 801–812 (1993).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lim, C.O., Lee, S.I., Chung, W.S. et al. Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds. Plant Mol Biol 30, 373–379 (1996). https://doi.org/10.1007/BF00020124
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00020124