Scan and microdissection platform for high-throughput screening based on a cell-free protein expression system (HTS-Scan)

Abstract

The aim of the project is the development of a new high-throughput screening system based on a recently developed cell-free protein expression platform to accelerate the search for new candidate therapeutic and technical proteins. The Fraunhofer Institute for Molecular Biology and Applied Ecology IME is responsible for the production of the biological components and the development of an agarose-based thin-layer in vitro transcription-translation (IVTT) system. In cooperation with LightFab, the latest developments in cell-free protein expression and laser material processing will be combined to create a new laser microdissection (LMD) platform for high-throughput screening. This platform will enable both automated and manual screening and the dissection of arbitrarily-shaped and positioned objects at rates of 100–20,000 events per second. 

© Fraunhofer IME | Matthias Buntru

Fluoreszenzmikroskopische Aufnahme einer Kapillarplatte nach Produktion eines gelben Fluoreszenzproteins (eYFP). Plasmid-DNA wurde mittels Kapillarkräfte in die Kanäle eingebracht und die Platte anschließend mit Low Melt Agarose-haltigem in vitro Transkriptions-Translations-Mix überschichtet. Die Platte wurde bei Raumtemperatur über Nacht inkubiert und anschließend mit Hilfe eines Fluoreszenzmikroskops untersucht (485/20 nm Anregungs- und 528/20 nm Emissionsfilter). Der dargestellte Ausschnitt zeigt Kapillaren mit einem Durchmesser von etwa 40 µm. Der Balken entspricht 100 µm.

Motivation/Introduction

The increasing demand for new therapeutic proteins, technical enzymes, protein engineering and functional genomics requires an efficient platform for both the production and screening of proteins. High-throughput protein screening is therefore becoming more important in the pharmaceutical, chemical and biotechnology industries. Biotechnology companies already expend much effort to discover and develop enzymes optimized for specific applications by mutation screening (e.g. the detergent industry, the food industry and the renewable energy industry). The pharmaceutical industry reinforces the use of high-throughput screening approaches to search for pharmaceutical leads (e.g. antimicrobial compounds). In addition to in vivo methods such as phage and yeast display, in vitro methods such as cell-free protein expression are becoming increasingly popular. In comparison to cell-based expression of proteins, cell-free systems have numerous advantages including a shorter processing time and the ability to express cytotoxic proteins.

Aim/Procedure

During the project “Zellfreie Bioproduktion” (FKZ 080/ZV-821969) granted by the BMBF and the Fraunhofer Society, a cell-free system based on tobacco BY-2 cell lysate (BYL) was established by Fraunhofer IME. The BYL system is superior to all commercially available cell-free systems in terms of flexibility, yield and economy. The aim of the “HTS-Scan” project is to develop a new high-throughput screening system based on the recently developed BYL system in order to accelerate the search for new therapeutic and technical protein candidates.

In the IVTT approach, the genetic information and encoded protein are kept together using a microcapillary plate and low melt agarose, which is spread on the microcapillary plate in thin layers. First, different DNA templates (or rather the corresponding mutants) are isothermally amplified in the single channels. Second, these products serve as templates for the IVTT system to synthesize proteins. Subsequent scanning and isolation of positive protein candidates by fluorescence, luminescence or absorption is achieved using a scan and LMD system. This combination of cell-free protein synthesis and LMD will create a new and more flexible high-throughput screening platform.

Project Duration

2016-2018

Project Partner

LightFab

Publications

Buntru, M., Vogel, S., Stoff, K., Spiegel, H., Schillberg, S., 2015. A versatile coupled cell-free transcription–translation system based on tobacco BY-2 cell lysates. Biotechnol. Bioeng. 112, 867–878. https://doi.org/10.1002/bit.25502

Buntru, M., Vogel, S., Spiegel, H., Schillberg, S., 2014. Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system. BMC Biotechnology 14, 37. http://doi.org/10.1186/1472-6750-14-37