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

被引：0
作者：
Yibing Deng
Tao Wu
Mengdi Wang
Shengchao Shi
Guodong Yuan
Xi Li
Hanchung Chong
Bin Wu
Peng Zheng
机构：
[1] Nanjing University,State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering
[2] Nanyang Technological University,School of Biological Sciences
[3] Nanyang Technological University,NTU Institute of Structural Biology
[4] EMB 06-01,undefined
来源：
Nature Communications | / 10卷
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摘要：
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.
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[1]  
Yang YJ(2017)Artificially engineered protein polymers Annu. Rev. Chem. Biomol. Eng. 8 549-575
[2]  
Holmberg AL(2016)Controlled radical polymerization as an enabling approach for the next generation of protein–polymer conjugates Acc. Chem. Res. 49 1777-1785
[3]  
Olsen BD(2018)Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology Proc. Natl Acad. Sci. USA 115 E8509-E8517
[4]  
Pelegri-O’Day EM(2006)Cysteine engineering of polyproteins for single-molecule force spectroscopy Nat. Protoc. 1 80-84
[5]  
Maynard HD(2014)Direct polymerization of proteins Acs Synth. Bio. 3 353-362
[6]  
Yang J(1997)Reversible unfolding of individual titin immunoglobulin domains by AFM Science 276 1109-1112
[7]  
Dietz H(1999)Mechanical and chemical unfolding of a single protein: a comparison Proc. Natl Acad. Sci. USA 96 3694-3699
[8]  
Albayrak C(2012)Single molecule force spectroscopy using polyproteins Chem. Soc. Rev. 41 4781-4796
[9]  
Swartz JR(2017)Engineering a catalytically efficient recombinant protein ligase J. Am. Chem. Soc. 139 5351-5358
[10]  
Rief M(2015)Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase Nat. Commun. 6 6318-6325
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