Fully Automated Ancestral Sequence Reconstruction using FireProtASR
Khan, R. T., Musil, M., Stourac, J., Damborsky, J., Bednar, D.
CURRENT PROTOCOLS 1: e30 (2021)
Protein evolution and protein engineering techniques are of great interest in both basic science and industrial applications such as pharmacology, medicine, or biotechnology. Ancestral sequence reconstruction (ASR) is a powerful technique for probing evolutionary relationships and engineering robust proteins with good thermostability and broad substrate specificity. The following protocol describes the setting up and execution of an automated workflow FireProtASR using a dedicated web site or a virtual image. The service allows for inference of ancestral proteins automatically, from a single protein sequence. Once a protein sequence is submitted, the server or virtual image will build a dataset of homology sequences, perform a multiple sequence alignment, build a phylogenetic tree and reconstruct ancestral nodes. The protocol is also highly flexible and allows for multiple forms of input, advanced settings, and the ability to start jobs from: (i) a single sequence, (ii) a set of homologous sequences, (iii) a multiple sequence alignment and (iv) a phylogenetic tree. This approach automates all necessary steps and offers a way for novices with limited exposure to ASR techniques, to improve the properties of a protein of interest. The technique can even be used to introduce catalytic promiscuity to an enzyme. A web server for accessing the fully automated workflow is freely accessible at https://loschmidt.chemi.muni.cz/fireprotasr/.