title: Procerain B, a cysteine protease from Calotropis procera, requires N-terminus pro-region for activity: cDNA cloning and expression with pro-sequence.
abstract: We have previously reported isolation and characterization of a novel plant cysteine protease, Procerain B, from the latex of Calotropis procera. Our initial attempts for active recombinant Procerain B in Escherichiacoli expression system was not successful. The reason for inactive enzyme production was attributed to the absence of 5' pro-region in the Procerain B cDNA that may be involved in proper folding and production of mature active protein. The current manuscript reports the cloning of full length Procerain B for the production of the active protein. The complete cDNA sequence of Procerain B with pro-region sequence was obtained by using RNA ligase mediated rapid amplification of 5' cDNA ends (RLM-RACE). The N-terminus pro-sequence region consists of 127 amino acids and characterized as the member of inhibitory I29 family. Further the three dimensional structure of full length Procerain B was modelled by homology modelling using X-ray crystal structure of procaricain (PDB ID: 1PCI). N-terminus pro-sequence of full length Procerain B runs along the active site cleft. Full length Procerain B was expressed in prokaryotic system and activated in vitro at pH 4.0. This is the first study reporting the production of active recombinant cysteine protease from C.procera.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Guwahati, Assam 781039, India.
authors.forename: Vidhyadhar
authors.initials: V
authors.lastname: Nandana
authors.affiliation: Centre for the Environment, Indian Institute of Technology Guwahati, Assam 781039, India.
authors.forename: Sushant
authors.initials: S
authors.lastname: Singh
authors.affiliation: Department of Oncology, University of Oxford, UK.
authors.forename: Abhay Narayan
authors.initials: AN
authors.lastname: Singh
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Guwahati, Assam 781039, India; Centre for the Environment, Indian Institute of Technology Guwahati, Assam 781039, India. Electronic address: vdubey@iitg.ernet.in.
authors.forename: Vikash Kumar
authors.initials: VK
authors.lastname: Dubey
mesh_terms: D000595:Amino Acid Sequence; D031107:Calotropis / Q000201:enzymology*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D057056:Cysteine Proteases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D008969:Molecular Sequence Data; D011487:Protein Conformation
chemical_list: D018076:DNA, Complementary; D057056:Cysteine Proteases; D003546:Cysteine Endopeptidases; C474045:procerain
keywords: Full length cDNA; I29 inhibitory propeptide; In vitro activation; Protease; RLM-RACE; Recombinant Procerain B