Design of the linkers which effectively separate domains of a bifunctional fusion protein
Design of the linkers which effectively separate domains of a bifunctional fusion protein
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DOI：
10.1093/protein/14.8.529
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发表时间：
2001-08-01
期刊：
影响因子：
--
通讯作者：
Nagamune, T
中科院分区：
文献类型：
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作者：
Arai, R;Ueda, H;Nagamune, T
为了分离双功能融合蛋白的结构域，比较了几个长度的螺旋形成肽分离两个弱相互作用的β-CaN结构域的能力，与柔性连接子或三个α-螺旋束域的能力进行了比较。我们在两个绿色荧光蛋白变异体EBFP和EGFP之间引入了形成螺旋的多肽连接物A(EAAAK)(N)A(n=2-5)，并研究了它们的光谱性质。EBFP到EGFP的荧光共振能量转移随着连接体长度的增加而减小。圆二色谱分析表明，连接体形成α-螺旋，且随着连接体长度的增加，α-螺旋的含量增加。结果清楚地表明螺旋连接体能够控制结构域之间的距离和减少干扰。这种“连接体工程”可能为连接体的合理设计开辟一条道路，从而最大限度地发挥融合蛋白或从头开始的多结构域蛋白的多种功能。
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 beta -can domains was compared with that of flexible linkers or of a three alpha -helices bundle domain. We introduced helix-forming peptide linkers A(EAAAK)(n)A (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 alpha -helix and the alpha -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.