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Protein Expr Purif. 2014 Nov;103:16-22. doi: 10.1016/j.pep.2014.08.003. Epub 2014 Aug 27.

Procerain B, a cysteine protease from Calotropis procera, requires N-terminus pro-region for activity: cDNA cloning and expression with pro-sequence.

Author information

1
Department of Biotechnology, Indian Institute of Technology Guwahati, Assam 781039, India.
2
Centre for the Environment, Indian Institute of Technology Guwahati, Assam 781039, India.
3
Department of Oncology, University of Oxford, UK.
4
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.

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.

KEYWORDS:

Full length cDNA; I29 inhibitory propeptide; In vitro activation; Protease; RLM-RACE; Recombinant Procerain B

PMID:
25173974
DOI:
10.1016/j.pep.2014.08.003
[Indexed for MEDLINE]

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