Micro Rings Self-assembled by Fluorescent Fusion-protein Based on Multiple Non-covalent Interactions

Rongting Hu; Jue Wang; Jing Yang; Yu Ding; Guosong Chen

doi:10.11777/j.issn1000-3304.2018.18204

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Micro Rings Self-assembled by Fluorescent Fusion-protein Based on Multiple Non-covalent Interactions

Research Article | 更新时间：2021-01-26

- Micro Rings Self-assembled by Fluorescent Fusion-protein Based on Multiple Non-covalent Interactions
  增强出版
- Acta Polymerica Sinica Vol. 50, Issue 2, Pages: 135-146(2019)
- 作者机构：
  
  1.复旦大学高分子科学系聚合物分子工程国家重点实验室　上海 200438
  2.复旦大学生命科学学院　上海 200438
- 作者简介：
- 基金信息：
- DOI：10.11777/j.issn1000-3304.2018.18204
  CLC：
- Published：2019-2，
  
  Published Online：4 January 2019，
  
  Received：23 September 2018，
  
  Revised：15 November 2018，
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Rongting Hu, Jue Wang, Jing Yang, Yu Ding, Guosong Chen. Micro Rings Self-assembled by Fluorescent Fusion-protein Based on Multiple Non-covalent Interactions. [J]. Acta Polymerica Sinica 50(2):135-146(2019)
DOI：

Rongting Hu, Jue Wang, Jing Yang, Yu Ding, Guosong Chen. Micro Rings Self-assembled by Fluorescent Fusion-protein Based on Multiple Non-covalent Interactions. [J]. Acta Polymerica Sinica 50(2):135-146(2019) DOI： 10.11777/j.issn1000-3304.2018.18204.

摘要

利用蛋白质融合技术，将绿色荧光蛋白(GFP)与可二聚化的链酶亲和素突变体(SA)融合，形成二聚化融合蛋白质GFP-SA，并以此作为生物大分子自组装的构筑基元. 同时，设计并合成了相应的配体分子RhYBio

，该配体中的2个生物素分子能特异性结合相邻融合蛋白中的SA，并将2个以上的二聚化融合蛋白GFP-SA排列成纳米线，随后该纳米线再通过配体中的罗丹明B分子二聚，进一步组装形成荧光蛋白质微米环. 采用动态光散射(DLS)对其粒径及分布进行了表征，并利用透射电子显微镜(TEM)和激光共聚焦显微镜(confocal microscope)观察了组装体的形貌，即蛋白质微米环，其形貌规整并具有较强的荧光.

Abstract

Proteins are attractive building blocks for construction of variant functional materials because of their chemical and structural diversities

and intrinsic functions. As the industry of biotechnology continues to expand

so does the expression of recombinant proteins with wide varieties. In this work

we adopted the recombinant protein technique to construct a new fusion protein

GFP-SA

as the building block of self-assemblies. The purification of GFP-SA was characterized by Superdex 75 size exclusive chromatography

SDS-PAGE

and MALDI-TOF. Then

GPC and native-PAGE were used to characterize the dimerization of GFP-SA based on the hydrogen bonds between neighboring SAs. Furthermore

ITC was employed to test the binding ability between GFP-SA and biotin

which revealed

= 0.24 μmol/L. In this study

we also designed and successfully synthesized the ligand RhYBio

which is composed of two biotin molecules and one rhodamine B molecule. The size of GFP-SA increased rapidly to 370 nm within one minute after mixing with RhYBio

We measured the particle size of GFP-SA/RhYBio

mixture every few minutes until the size stabilized at around 1300 nm 2 h later. However

size variation was barely observed for the controlled samples of SA/RhYBio2 (controlled protein) and GFP-SA/YBio (controlled ligand). We hypothesized that the two biotin molecules of RhYBio

could bind specifically with SA and align GFP-SA/RhYBio

into nanowires

which assembled further into micro rings. Their size was measured by dynamic light scattering (DLS) while the morphology was observed intuitively on a transmission electron microscope (TEM) and a confocal microscope (CM). The characteristic results from TEM and CM suggested an uneven size distribution of the micro rings prepared

which might be attributable to the flexibility of the fusion protein GFP-SA. These micro rings of GFP-SA/RhYBio

with fluorescence has great potential for biological applications.

关键词

融合蛋白绿色荧光蛋白自组装微米环

Keywords

Fusion proteinGFPSelf-assemblyMicro rings

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Related Institution

State Key Laboratory of Chemical Resource Engineering, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology

Department of Polymer Science and Engineering, University of Massachusetts, Amherst

Shanghai Key Laboratory of Advanced Polymeric Materials, School of Materials Science and Engineering, East China University of Science and Technology

College of Textile Science and Engineering, Jiangnan University

School of Materials Science and Engineering, East China University of Science and Technology

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