Team:Tianjin/Experiment/CFPS

Experiment of CFPS

Overview

The ability to produce a functional protein in the test tube, rather than in cells, is the essence of cell-free protein synthesis (CFPS)[1]. .

                  Fig.1. The basic process of CFPS system

The lack of high-throughput approaches for expression and screening of large enzyme libraries remains a major bottleneck for current enzyme engineering efforts. To address this need, some researchers[2] have developed a high-throughput, fluorescence-based approach for rapid one-pot, microscale expression, and screening of different kinds of enzymes. To go further, we try to make our effert to achieve integration of cell-free protein expression with activity screening of enzymes( site-directed mutations of PETase). .

                  Fig.2.One-pot approach for integrated expression and activity screening of enzymes

Background

The diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications, such as screeening of enzymes activities. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high-throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology.

Experiment Design

Basically, we utilized the cell-free system to express the enzymes which had been modified in 22 different sites. Besides, we added a fluorescet protein, CFP, before the enzyme. And there is a flexible linker, GGGGSGGGGS , between them. So that we could detect the expression of enzymes by detecting expression of the fluorescent protein with a fluorescence readout instrument, for example, a microplate reader. We conceived that with this method we could acquire the best modifications by screening them in a high-throughput way. Then we used the proteins we got to degrade PET.

                  Fig.3.The expression vector in CFPS system

How to characterize the degradation velocity is the main problem in our scheme. We analyzed the experiment consequences in two ways. For the first one, we rendered the enzymes degrade pNPa, a general substituent for the detection of PET. Then we measured the absorbance of pNP in the optical density of 400 nanometers, which is the degrading product of pNPa. For the second one, we detected the absorbance of MHET in the optical density of 260 nanometers, which is the product in the first step of PET degradation.

                  Fig.4.Steps for integrated expression and activity screening of enzymes

Reference

[1]Gabriel Rosenblum and Barry S. Cooperman. Engine out of the Chassis: Cell-Free Protein Synthesis and its Uses. FEBS Lett. 2014 January 21; 588(2): 261–268. doi:10.1016/j.febslet.2013.10.016.

[2]Aarthi Chandrasekaran and Anup K. Singh. One-Pot, Microscale Cell-Free Enzyme Expression and Screening. DOI 10.1007/978-1-62703-782-2 Springer New York Heidelberg Dordrecht London.