title: Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. revealed by de novo transcriptome analysis.
abstract: Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding.
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.
authors.forename: Chang Woo
authors.initials: CW
authors.lastname: Kwon
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.
authors.forename: Kyung-Min
authors.initials: KM
authors.lastname: Park
authors.affiliation: Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea.
authors.forename: Byoung-Cheorl
authors.initials: BC
authors.lastname: Kang
authors.affiliation: Department of Genetic Engineering and Center for Human Interface Nanotechnology, Sungkyunkwan University, Suwon, Republic of Korea.
authors.forename: Dae-Hyuk
authors.initials: DH
authors.lastname: Kweon
authors.affiliation: Department of Food Science and Biotechnology, Kangwon National University, Chuncheon, Republic of Korea.
authors.forename: Myoung-Dong
authors.initials: MD
authors.lastname: Kim
authors.affiliation: Department of Agricultural Biotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea.
authors.forename: Sang Woon
authors.initials: SW
authors.lastname: Shin
authors.affiliation: Department of Agricultural Biotechnology, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea.
authors.forename: Yeon Ho
authors.initials: YH
authors.lastname: Je
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea; Center for Food and Bioconvergence, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea.
authors.forename: Pahn-Shick
authors.initials: PS
authors.lastname: Chang
mesh_terms: D000595:Amino Acid Sequence; D031107:Calotropis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D057056:Cysteine Proteases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA
chemical_list: D012333:RNA, Messenger; D057056:Cysteine Proteases