Enzymatic Biosynthesis and Immobilization of Polyprotein Verified at the Single-molecule Level

Overview

Journal Nat Commun

Specialty Biology

Date 2019 Jun 26

PMID 31235796

Citations 32

Authors

Yibing Deng

Tao Wu

Mengdi Wang

Shengchao Shi

Guodong Yuan

Xi Li

Hanchung Chong

Bin Wu

Peng Zheng

Affiliations

Soon will be listed here.

Abstract

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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