ARTICLE Received 10 Jul 2013 | Accepted 8 Nov 2013 | Published 19 Dec 2013 DOI: 10.1038/ncomms3901 Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell Ignacio Asial1, Yue Xiang Cheng1, Henrik Engman1, Maria Dollhopf2, Binghuang Wu1, Pa¨r Nordlund1,2 & Tobias Cornvik1 Protein stability is often a limiting factor in the development of commercial proteins and biopharmaceuticals, as well as for biochemical and structural studies. Unfortunately, identifying stabilizing mutations is not trivial since most are neutral or deleterious. Here we describe a high-throughput colony-based stability screen, which is a direct and biophysical read-out of intrinsic protein stability in contrast to traditional indirect activity-based methods. By combining the method with a random mutagenesis procedure, we successfully identify thermostable variants from 10 diverse and challenging proteins, including several bio- technologically important proteins such as a single-chain antibody, a commercial enzyme and an FDA-approved protein drug. We also show that thermostabilization of a protein drug using our approach translates into dramatic improvements in long-term stability. As the method is generic and activity independent, it can easily be applied to a wide range of proteins. 1 School of Biological Sciences, Nanyang Technological University, 639798 Singapore, Singapore. 2 Division of Biophysics, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles va¨g 2, SE-171 11 Stockholm, Sweden. Correspondence and requests for materials should be addressed to P.N. (email: [email protected]) or to T.C. (email: [email protected]). NATURE COMMUNICATIONS | 4:2901 | DOI: 10.1038/ncomms3901 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3901 ethodologies to improve protein stability are of great Here we develop a broadly applicable, high-throughput and importance for the biopharmaceutical industry. More activity-independent method to study and increase protein Mefficient strategies of stabilizing proteins could poten- thermostability. The method is an extension and modification tially reduce efforts in protein manufacturing and formulation, as of our previously published colony filtration (CoFi) blot well as improve the safety of protein drugs. Biopharmaceuticals method26. It circumvents most of the problems linked to that have reached the clinic are generally highly thermostable1–4. current stability selection methods while expanding its appli- In contrast, many protein drug candidates in development are cability to virtually any protein target that can be expressed suffering from poor stability5,6, which will affect efficacy and can recombinantly in a colony-forming microorganism. We show lead to increased immunogenicity of the biopharmaceutical, that the method can be used to accurately determine the intrinsic inhibiting their advancement to the clinic. In several cases, these protein-melting temperatures in cells and is an effective screening effects have been shown to arise from protein aggregation, often technology to identify more stable variants of a target protein in induced by exposure to ambient temperatures during protein directed evolution experiments. Importantly, we present a clear, handling or storage7,8. streamlined process that allows studying and evolving several Similarly, biocatalysts used in a wide range of applications, targets in parallel with no optimizations required. The wide such as in agriculture, paper, textile, the bio-energy industry or applicability and potential of this method is demonstrated on a household care, are often highly stable at the conditions where broad range of targets representing protein drugs, biotechnology they are applied. Unfortunately, in most cases the original enzyme tools, enzymes and challenging structural biology targets. is not directly suitable for the desired application because of the ‘hostile’ conditions at which the reactions are performed. For Results instance, thermal stability of biocatalysts is of major importance Thermal stress leads to protein aggregation in vivo. Thermal for industrial processes, since performing enzymatic reactions at stress of a purified protein above a certain critical temperature high temperature has many advantages9,10. Therefore, during usually leads to irreversible protein unfolding, followed by rapid development, considerable engineering efforts are carried out to aggregation27. We hypothesized that a similar scenario might be stabilize enzymes to increased temperatures11–14. in effect for recombinant proteins overexpressed in Escherichia Protein stability is also of great importance in basic research, as coli, and that this could be utilized to select more stable proteins. stable proteins are more amenable to biochemical and structural When cells are subjected to increased temperatures, the proteins studies. Thermal stability has, for instance, been shown to might unfold and aggregate in a similar way as purified proteins. increase the likelihood of crystallization of both soluble and We selected a set of nine proteins with a midpoint of unfolding membrane proteins. This concept is now one of the most (melting temperature; T ) of the purified protein ranging from promising avenues on the path to obtain crystal structures of m 15,16 40 °Cto68°C, which were cloned into expression plasmids, challenging proteins, such as G protein coupled receptors and transformed into E. coli and plated. The colonies were then protein kinases17. transferred to a filter membrane that previously has been shown In addition, in the field of directed evolution and protein to let soluble proteins pass through while retaining aggregates26. engineering improved thermostability of the target is usually After expression, the colonies were incubated at a wide range of desirable since it correlates with the mutational robustness of the temperatures after which they were subjected to concurrent lysis target: destabilizing mutations, that are necessary for a new and CoFi26 (Fig. 1a,b). In this step, the filter membrane retains activity, are more easily tolerated18. aggregates, while soluble proteins diffuse from the lysed colonies For all the applications mentioned above, understanding and onto a nitrocellulose membrane. The protein is then detected improving protein stability through directed evolution is a key using affinity reagents against the target itself or against a strategy. Current approaches rely on an indirect read-out of detection tag fused to the target. A high signal indicates the protein stability, typically a functional assay after a stability presence of soluble protein, while signal loss accounts for protein challenge. For enzymes, for example, a heating step is typically aggregation in the cell. For all proteins tested, there was a good performed, followed by a read-out of residual activity in multi- 10 correlation between signal loss and the Tm determined by well plates . For proteins able to bind with relatively strong differential scanning fluorimetry (DSF) that measures the affinity to a target, such as antibodies, a denaturation step can be 28 19,20 unfolding of the purified proteins (Fig. 1c) . This correlation followed by a read-out on residual binding . There are many strongly indicates that it is the unfolding and subsequent successful examples in the literature of this type of approaches; aggregation of the protein inside the cell that we are measuring. however, their customized nature limits the number of targets We therefore consider our screen to be a biophysical screen, that can be evolved in parallel. Directed evolution is therefore analogous to differential static light scattering29, setting it apart usually restricted to specialized research teams working on a from previously published thermostability screens used for small number of proteins. Moreover, as the number of sequenced directed evolution of stability. Interestingly, the midpoint of the genomes is expanding rapidly, many research fields have cellular aggregation curve (named here Tcagg) was generally a few streamlined their research structure to be able to study as many B degrees lower than the Tm obtained by DSF ( 5 °C lower on proteins as possible in a high-throughput manner, for example, average). This difference might reflect the influence of the protein production, purification and structural biology21,22. The macromolecular crowding of the cell on protein in vivo thermal study and improvement of protein stability through directed stability. The wide temperature range accessible by the method evolution approaches has not made that transition yet, as there covers the Tm of most mesophilic proteins, making the method are no generic, streamlined techniques for this purpose. widely applicable. There have been attempts to develop generic (thermo)stability screens that are independent of protein function, that allow the study of many different targets in parallel and have enough Evolution of protein thermostability. The efficiency of our throughput to screen libraries of significant size: these include method (hereafter referred to as Hot-CoFi) to assess protein- Proside23, THR24 and the tripartite fusion system25. However, melting temperatures in cells encouraged us to implement it as a these methods are handicapped by indirect read-outs of screen for improvement of thermostability using directed evolu- thermostability, which increases