The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes

Citation:

Mlynek G, Lehner A, Neuhold J, Leeb S, Kostan J, Charnagalov A, Stolt-Bergner P, Djinovic-Carugo K, Pinotsis N. The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes. Amino Acids. 2014;46:1565-82.

Abstract:

Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.

Notes:

Mlynek, GeorgLehner, AnitaNeuhold, JanaLeeb, SarahKostan, JuliusCharnagalov, AlexejStolt-Bergner, PeggyDjinovic-Carugo, KristinaPinotsis, NikosengI 1593/FWF_/Austrian Science Fund FWF/AustriaI 525/FWF_/Austrian Science Fund FWF/AustriaW 1224/FWF_/Austrian Science Fund FWF/AustriaResearch Support, Non-U.S. Gov'tAustriaAmino Acids. 2014 Jun;46(6):1565-82. doi: 10.1007/s00726-014-1699-x. Epub 2014 Mar 20.

Contact Details

Biochemistry, Chemistry Department
National and Kapodistrian University of Athens
(+30) 210-727 4493
Zografou
Athens, ZipCode 157 84

Recent Publications

Ma S, Mykhaylyk V, Bowler MW, Pinotsis N, Kozielski F. High-Confidence Placement of Fragments into Electron Density Using Anomalous Diffraction-A Case Study Using Hits Targeting SARS-CoV-2 Non-Structural Protein 1. Int J Mol Sci. 2023;24.Abstract

The identification of multiple simultaneous orientations of small molecule inhibitors binding to a protein target is a common challenge. It has recently been reported that the conformational heterogeneity of ligands is widely underreported in the Protein Data Bank, which is likely to impede optimal exploitation to improve affinity of these ligands. Significantly less is even known about multiple binding orientations for fragments (<300 Da), although this information would be essential for subsequent fragment optimisation using growing, linking or merging and rational structure-based design. Here, we use recently reported fragment hits for the SARS-CoV-2 non-structural protein 1 (nsp1) N-terminal domain to propose a general procedure for unambiguously identifying binding orientations of 2-dimensional fragments containing either sulphur or chloro substituents within the wavelength range of most tunable beamlines. By measuring datasets at two energies, using a tunable beamline operating in vacuum and optimised for data collection at very low X-ray energies, we show that the anomalous signal can be used to identify multiple orientations in small fragments containing sulphur and/or chloro substituents or to verify recently reported conformations. Although in this specific case we identified the positions of sulphur and chlorine in fragments bound to their protein target, we are confident that this work can be further expanded to additional atoms or ions which often occur in fragments. Finally, our improvements in the understanding of binding orientations will also serve to improve the rational optimisation of SARS-CoV-2 nsp1 fragment hits.

Gong GQ, Bilanges B, Allsop B, Masson GR, Roberton V, Askwith T, Oxenford S, Madsen RR, Conduit SE, Bellini D, et al. A small-molecule PI3Kalpha activator for cardioprotection and neuroregeneration. Nature. 2023;618:159-168.Abstract

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development(1-5). This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kalpha isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kalpha through a distinct mechanism by enhancing multiple steps of the PI3Kalpha catalytic cycle and causes both local and global conformational changes in the PI3Kalpha structure. This compound is selective for PI3Kalpha over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kalpha signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

Le Huray KIP, Bunney TD, Pinotsis N, Kalli AC, Katan M. Characterization of the membrane interactions of phospholipase Cgamma reveals key features of the active enzyme. Sci AdvSci Adv. 2022;8:eabp9688.Abstract

PLCgamma enzymes are autoinhibited in resting cells and form key components of intracellular signaling that are also linked to disease development. Insights into physiological and aberrant activation of PLCgamma require understanding of an active, membrane-bound form, which can hydrolyze inositol-lipid substrates. Here, we demonstrate that PLCgamma1 cannot bind membranes unless the autoinhibition is disrupted. Through extensive molecular dynamics simulations and experimental evidence, we characterize membrane binding by the catalytic core domains and reveal previously unknown sites of lipid interaction. The identified sites act in synergy, overlap with autoinhibitory interfaces, and are shown to be critical for the phospholipase activity in cells. This work provides direct evidence that PLCgamma1 is inhibited through obstruction of its membrane-binding surfaces by the regulatory region and that activation must shift PLCgamma1 to a conformation competent for membrane binding. Knowledge of the critical sites of membrane interaction extends the mechanistic framework for activation, dysregulation, and therapeutic intervention.

Nikos Pinotsis

Associate Professor, Chemistry