Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level

The recent development of chemical and bio-conjugation techniques allows for the engineering of various protein polymers. However, most of the polymerization process is difficult to control. To meet this challenge, we develop an enzymatic procedure to build polyprotein using the combination of a str...

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Main Authors: Deng, Yibing, Wu, Tao, Wang, Mengdi, Shi, Shengchao, Yuan, Guodong, Li, Xi, Chong, Hanchung, Wu, Bin, Zheng, Peng
Other Authors: School of Biological Sciences
Format: Article
Language:English
Published: 2019
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Online Access:https://hdl.handle.net/10356/107449
http://hdl.handle.net/10220/49733
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Institution: Nanyang Technological University
Language: English
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spelling sg-ntu-dr.10356-1074492023-02-28T17:06:48Z Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level Deng, Yibing Wu, Tao Wang, Mengdi Shi, Shengchao Yuan, Guodong Li, Xi Chong, Hanchung Wu, Bin Zheng, Peng School of Biological Sciences NTU Institute of Structural Biology Atomic Force Microscopy Peptides Science::Biological sciences The recent development of chemical and bio-conjugation techniques allows for the engineering of various protein polymers. However, most of the polymerization process is difficult to control. To meet this challenge, we develop an enzymatic procedure to build polyprotein using the combination of a strict protein ligase OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases 1) and a protease TEV (tobacco etch virus). We firstly demonstrate the use of OaAEP1-alone to build a sequence-uncontrolled ubiquitin polyprotein and covalently immobilize the coupled protein on the surface. Then, we construct a poly-metalloprotein, rubredoxin, from the purified monomer. Lastly, we show the feasibility of synthesizing protein polymers with rationally-controlled sequences by the synergy of the ligase and protease, which are verified by protein unfolding using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides a strategy for polyprotein engineering and immobilization. MOE (Min. of Education, S’pore) Published version 2019-08-21T07:16:17Z 2019-12-06T22:31:22Z 2019-08-21T07:16:17Z 2019-12-06T22:31:22Z 2019 Journal Article Deng, Y., Wu, T., Wang, M., Shi, S., Yuan, G., Li, X., . . . Zheng, P. (2019). Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level. Nature Communications, 10(1), 2775-. doi:10.1038/s41467-019-10696-x https://hdl.handle.net/10356/107449 http://hdl.handle.net/10220/49733 10.1038/s41467-019-10696-x en Nature Communications © 2019 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 11 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Atomic Force Microscopy
Peptides
Science::Biological sciences
spellingShingle Atomic Force Microscopy
Peptides
Science::Biological sciences
Deng, Yibing
Wu, Tao
Wang, Mengdi
Shi, Shengchao
Yuan, Guodong
Li, Xi
Chong, Hanchung
Wu, Bin
Zheng, Peng
Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level
description The recent development of chemical and bio-conjugation techniques allows for the engineering of various protein polymers. However, most of the polymerization process is difficult to control. To meet this challenge, we develop an enzymatic procedure to build polyprotein using the combination of a strict protein ligase OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases 1) and a protease TEV (tobacco etch virus). We firstly demonstrate the use of OaAEP1-alone to build a sequence-uncontrolled ubiquitin polyprotein and covalently immobilize the coupled protein on the surface. Then, we construct a poly-metalloprotein, rubredoxin, from the purified monomer. Lastly, we show the feasibility of synthesizing protein polymers with rationally-controlled sequences by the synergy of the ligase and protease, which are verified by protein unfolding using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides a strategy for polyprotein engineering and immobilization.
author2 School of Biological Sciences
author_facet School of Biological Sciences
Deng, Yibing
Wu, Tao
Wang, Mengdi
Shi, Shengchao
Yuan, Guodong
Li, Xi
Chong, Hanchung
Wu, Bin
Zheng, Peng
format Article
author Deng, Yibing
Wu, Tao
Wang, Mengdi
Shi, Shengchao
Yuan, Guodong
Li, Xi
Chong, Hanchung
Wu, Bin
Zheng, Peng
author_sort Deng, Yibing
title Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level
title_short Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level
title_full Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level
title_fullStr Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level
title_full_unstemmed Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level
title_sort enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level
publishDate 2019
url https://hdl.handle.net/10356/107449
http://hdl.handle.net/10220/49733
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