Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter

Abstract

The C1a isoenzyme of horseradish peroxidase (HRP) is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro) DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence) via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT) and inactive mutant (MUT) genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT) library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases.

Tandem Cell‐Free Protein Synthesis as a Tool for Rapid Screening of Optimal Molecular Chaperones

“…The conditions for expression of enzymatically active HRP in a cell‐free protein synthesis system have been studied previously . To increase the expression level of soluble HRP protein, the N‐terminus of HRP was fused to ubiquitin and its expression was examined under various conditions.…”

“…To express HRP or ubiquitinated HRP (ubi‐HRP), the S12 extract was prepared from the E. coli BL21Star(DE3) strain harboring the pK7‐DsbC plasmid that encodes bacterial disulfide isomerase. In addition, the reaction mixture was also supplemented with 16.7 μ m hemin, 2 m m calcium acetate, 100 μ m oxidized glutathione, and 1 m m reduced glutathione . In the experiment that involves in situ cleavage of ubiquitin from ubi‐HRP fusion protein, a mixture of equal volumes of the DsbC‐enriched S12 extract and ubiquitin carboxyl‐terminal hydrolase 1 (UBP1)‐enriched S12 extract was used to perform the cell‐free protein synthesis reactions.…”

Compartmentalization – A Prerequisite for Maintaining and Changing an Identity

“…Target genes can be amplified [33] on the beads and expressed via cell-free protein synthesis. [34] Several techniques exist to afterwards bind the protein to its specific DNA on the magnetic bead to generate a covalent genotype-phenotype linkage. [35] Placing the populated magnetic bead then in a compartment in an w/o or a w/o/w emulsion allows for the use of this technique for enzyme evolution as the reaction product remains in the same compartment as the information on the bead -although also alternative linkage…”

Cell‐free translational screening of an expression sequence tag library of Clonorchis sinensis for novel antigen discovery

“…In addition to the advantages of its speed and multiplexibility, the open nature of cell‐free synthesis enables a mix‐and‐match design of protein synthesis using various biological and non‐biological components. For example, a number of studies demonstrated in vitro linkage of genotype and phenotype on the surface of microbeads through cell‐free expression of bead‐immobilized template DNA . Based on a similar concept, through the use of bi‐functionalized microbeads that can capture both the template DNA and the expressed proteins, we were able to physically co‐localize the genes of C. sinensis and their translation products on microbeads .…”

“…For example, a number of studies demonstrated in vitro linkage of genotype and phenotype on the surface of microbeads through cell-free expression of bead-immobilized template DNA. [13][14][15] Based on a similar concept, through the use of bi-functionalized microbeads that can capture both the template DNA and the expressed proteins, we were able to physically co-localize the genes of C. sinensis and their translation products on microbeads. 16 The use of microbead-conjugated DNA enabled facile expression and screening of antigenic proteins.…”