title: Optimization of purification method and characterization of recombinant human Centrin-1.
abstract: Centrins are acidic proteins, present in all eukaryotes to perform imperative roles in centrosome positioning and segregation. Existing methods for the purification of centrins for biophysical studies involves either multiple steps or yields protein with an affinity tag, which pins additional tag-cleavage step. Therefore, we have made an attempt to develop a simple and single step method for protein purification. We have performed categorical evaluation of existing methods, and describe a one-step procedure based on cleavable Intein-tag, which can be utilized for routine preparation of any isoform of centrins. Since human Centrin-1 and Centrin-2 are devoid of Trp, we exploit this feature to assess the purity of the protein using Tyr fluorescence; an essential point ignored generally. In addition, we report important spectral and hydrodynamic characteristics of human Centrin-1, accounting that HsCentrin-1 has moderate affinity for Ca(2+). Centrin-1 does not gain structure as seen by far- and near-UV circular dichroism, rather there is a loss of ellipticity, though inconsiderable upon binding Ca(2+).
authors.affiliation: CSIR-Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad 500 007, India.
authors.forename: Regur
authors.initials: R
authors.lastname: Phanindranath
authors.affiliation: CSIR-Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad 500 007, India.
authors.forename: Digumarthi V S
authors.initials: DV
authors.lastname: Sudhakar
authors.affiliation: CSIR-Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad 500 007, India.
authors.forename: Anand Kumar
authors.initials: AK
authors.lastname: Sharma
authors.affiliation: CSIR-Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad 500 007, India.
authors.forename: Kumarasamy
authors.initials: K
authors.lastname: Thangaraj
authors.affiliation: CSIR-Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad 500 007, India. Electronic address: yogendra@ccmb.res.in.
authors.forename: Yogendra
authors.initials: Y
authors.lastname: Sharma
mesh_terms: D002118:Calcium / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: C580665:CETN1 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; D002118:Calcium
keywords: Ca(2+) binding; Centrin-1; Intein-tag; Nonspecific fluorescence; Tyrosine fluorescence