Construction of a linker library with widely controllable flexibility for fusion protein design

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• 作者：Gang Li ; Ziliang Huang ; Chong Zhang ; Bo-Jun Dong…
• 关键词：Fusion protein ; Linker ; Flexibility/rigidity ; Molecular dynamics ; FRET ; DPD simulation
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2016
• 出版时间：January 2016
• 年：2016
• 卷：100
• 期：1
• 页码：215-225
• 全文大小：1,211 KB
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• 作者单位：Gang Li (1) (2)
  Ziliang Huang (1) (2)
  Chong Zhang (1) (2)
  Bo-Jun Dong (1) (2)
  Ruo-Hai Guo (1) (2)
  Hong-Wei Yue (1) (2)
  Li-Tang Yan (1) (2)
  Xin-Hui Xing (1) (2)
  
  1. Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China
  2. Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing, China
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

Flexibility or rigidity of the linker between two fused proteins is an important parameter that affects the function of fusion proteins. In this study, we constructed a linker library with five elementary units based on the combination of the flexible (GGGGS) and the rigid (EAAAK) units. Molecular dynamics (MD) simulation showed that more rigid units in the linkers lead to more helical conformation and hydrogen bonds, and less distance fluctuation between the N- and C-termini of the linker. The diversity of linker flexibility of the linker library was then studied by fluorescence resonance energy transfer (FRET) of cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) fusion proteins, which showed that there is a wide range of distribution of the FRET efficiency. Dissipative particle dynamics (DPD) simulation of CFP-YFP with different linkers also gave identical results with that of FRET efficiency analysis, and we further found that the combination manner of the linker peptide had a remarkable effect on the orientation of CFP and YFP domains. Our studies demonstrated that the construction of the linker library with the widely controllable flexibility could provide appropriate linkers with the desirable characteristics to engineer the fusion proteins with the expected functions.