Title
Optimization of purification method and characterization of recombinant human Centrin-1
Author
Regur Phanindranath, Digumarthi V.S. Sudhakar, Anand Kumar Sharma, Kumarasamy Thangaraj, Yogendra Sharma
Year
2016
Journal
Protein Expression and Purification
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 Ca2+. 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 Ca2+.
Instrument
J-815
Keywords
Circular dichroism, Tertiary structure, Ligand binding, Secondary structure, Biochemistry