Protein Engineering, Vol. 14, No. 8, 529-532,
August 2001
© 2001 Oxford University Press
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Design of the linkers which effectively separate domains of a bifunctional fusion protein
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku,Tokyo 113-8656, Japan
E-mail: nagamune{at}bio.t.u-tokyo.ac.jp
With the aim of separating the domains of a bifunctional fusion protein, the ability of several lengths of helix-forming peptides to separate two weakly interacting ß-can domains was compared with that of flexible linkers or of a three 
-helices bundle domain. We introduced helix-forming peptide linkers A(EAAAK)nA (n = 2–5) between two green fluorescent protein variants, EBFP and EGFP, and investigated their spectral properties. The fluorescence resonance energy transfer from EBFP to EGFP decreased as the length of the linkers increased. The circular dichroism spectra analysis suggested that the linkers form an -helix and the -helical contents increased as the length of the linkers increased. The results clearly suggested the ability of the helical linkers to control the distance and reduce the interference between the domains. This `linker engineering' may open a way to the rational design of linkers which maximize the multiple functions of fusion proteins or de novo multi-domain proteins.
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