title: Functional fusion expression of sunflower multicystatin in E. coli and its comparison with a single domain cystatin.
abstract: Identification of the molecular structure and novel biophysiological functions of plant cystatins or phytocystatins is of great interest in the field of molecular biology. The important requirements for these are the efficient production, purification and correctly folded forms of these proteins. We report here the cloning, easy expression and characterization of a sunflower multicystatin (SMC) as a functional fusion protein in E. coli. For the first time, the amplified cystatin coding region was expressed as a part of maltose-binding fusion protein using pMALc2X over-expression vector in TB1 strain of E. coli without affecting the recombinant bacterial growth. In comparison to the previously prepared recombinant SMC (rSMC), a high amount (-44 mg/L of bacterial cell culture) of purified fused SMC (fSMC) was obtained using single-step purification method. fSMC strongly inhibited papain activity in vitro as compared to Celosia single-domain cystatin. Purified fSMC may be used for basic biochemical, pharmacological or clinical studies without the cleavage of its fusion parts.
authors.affiliation: Research Institute for Fundamental Sciences (RIFS), Department of Molecular Biology, University of Tabriz, Tabriz, Iran. aghz_bioch@yahoo.co.in
authors.forename: Ashraf
authors.initials: A
authors.lastname: Gholizadeh
authors.forename: B Baghban
authors.initials: BB
authors.lastname: Kohnehrouz
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D015891:Cystatins / Q000235:genetics*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006368:Helianthus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D015891:Cystatins; D017931:DNA Primers; D011994:Recombinant Proteins