Synthesis of soluble proinsulin in a cell-free protein synthesis system.

Yuan HU; Yuchen ZHANG; Yancheng XU; Tiangang LIU

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Synthesis of soluble proinsulin in a cell-free protein synthesis system.

Author: Yuan HU ¹ ; Yuchen ZHANG ² ; Yancheng XU ¹ ; Tiangang LIU ²
Author Information

1. Department of Endocrinology, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei, China.
2. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, Hubei, China.
Publication Type:Journal Article
Keywords: cell-free protein synthesis; fusion of fluorescent protein; proinsulin; protein folding; solubility
From: Chinese Journal of Biotechnology 2017;33(3):467-477
CountryChina
Language:Chinese
Abstract: Proinsulin (Pins) is the precursor of insulin. The expression of proinsulin in Escherichia coli forms inclusion body, so that the recombinant protein should be processed with multiple steps to form active insulin. With the development in biotechnology, cell-free protein synthesis (CFPS) system is becoming a valuable tool in protein expression by decoupling the cell growth with protein production, which allows it to express proteins that would interfere with cell physiology. In this study, we synthesized soluble proinsulin in CFPS system in order to establish a new approach for both insulin expression and delivery. The soluble proinsulin was successfully expressed in CFPS system by fusing proinsulin with two types of fluorescent protein. The expression of Pins-mCherry was confirmed by Western blotting analysis, and the Pins-eGFP titer was (12.28±3.45) μg/mL in CFPS system. These results implicated that the proinsulin was expressed partially in soluble form. Here, for the first time, we successfully expressed soluble proinsulin in CFPS system by fluorescent protein fusion. These results provide useful information in developing new insulin expression and delivery method.