In vivo-seulontamenetelmän kehittäminen peptidi-proteiini-parien ligaation havainnointiin

Catcher-proteiinit ja Tag-peptidit ovat muokattu halkaisemalla grampositiivisten bakteereiden pintaproteiinien CnaB-domeeneista. CnaB domeenit järjestäytyvät molekyylin sisäisellä isopeptidisidoksella, joka muodostuu lysiini- ja aspargiini-sivuketjujen spontaanissa reaktiossa. Multikomponenttifuusioproteiinien muodostamiseen hyödynnettäviä Catcher/Tag -pareja, joissa reaktio tapahtuu lähellä diffuusionopeutta, ja joiden vastakkaiset Catcher- ja Tag-osat eivät reagoi ristiin, on kuitenkin löydetty vain rajallinen määrä. Näin ollen uusi Catcher/Tag -pari – LplCatcher/LplTag – kehitettiin tutkimusryhmässämme Lactobacillus plantarum -bakteerin CnaB domeenista. Sovellusmahdollisuuksien lisäämiseksi ligaationopeutta tulisi kuitenkin parantaa. Näin ollen tarvitaan uusia menetelmiä mutanttikirjastojen tehokkaaseen massaseulontaan, jotta voidaan löytää uusia peptidi-proteiini-pareja, joissa kovalenttinen sidos muodostuu tehostetusti. Tässä työssä kehitettiin uusi in vivo-massaseulontamenetelmä visualisoimaan Catcher/Tag-parien interaktiota. SplitFAST-reportteriproteiinia hyödynnettiin fenotyypin määrittämisessä ja virtaussytometriaa (FACS) solujen erottelussa fluoresenssin intensiteettiä hyödyntäen. SplitFAST on kehitetty halkaisemalla fluoresenssia aktivoiva ja absorboiva FAST-proteiini CFAST- ja NFAST-osiin. Menetelmää voidaan hyödyntää proteiini-interaktioiden visualisoinnissa, sillä CFAST- ja NFAST-osien assosioituessa muodostuu kokonainen FAST-proteiini, joka on fluorogeenin läsnäolleessa fluoresoiva. Tässä työssä CFAST fuusioitiin Catcher-proteiinin kanssa ja NFAST Tag-peptidin kanssa, ja tutkittiin, onko Catcher-Tag-ligaatiota mahdollista visualisoida splitFAST-systeemiä hyödyntäen. Kun interaktion visualisoiminen vaikutti toimivan, seulontamenetelmä kehitettiin ligaationopeudeltaan tehokkaampien Catcher-varianttien löytämiseksi. Menetelmä osoittautui hyvin lupaavaksi ja sitä voitaisiin soveltaa uusien Catcher/Tag -parien kehittämisessä, sillä menetelmä mahdollistaa proteiiniligaation havainnoinnin sekä suurten mutanttikirjastojen analyysit.Catcher-protein and Tag-peptide originate from split CnaB domains of Gram-positive bacteria surface proteins, which are stabilized by spontaneous intramolecular isopeptide bonds formed between lysine and asparagine residues. However, there is a limited number of non-cross-reacting Catcher and Tag pairs available where the reaction occurs close to the diffusion limit, and which can be used in multiple fragment ligation to construct recombinant fusion proteins. Therefore, a new Catcher/Tag system – LplCatcher/LplTag – was developed in our group from CnaB domain of Lactobacillus plantarum. However, the ligation efficiency of this pair needs to be improved to expand the application possibilities. Therefore, there is a need for efficient library screening method, which allows to detect improved protein-peptide pairs where the covalent interaction takes place rapidly. In this study a new high-throughput in-vivo screening system was developed for visualizing the ligation of Catcher/Tag fusion proteins using splitFAST fluorogenic reporter system for detecting the phenotype, and Fluorescence-activated cell sorting (FACS) for separating the variants at single cell level based on fluorescence intensity. splitFAST is a system engineered by splitting a fluorescent protein named Fluorescence-Activating and absorption-Shifting Tag (FAST) into CFAST and NFAST. The system can be utilized in visualizing the protein interactions because once NFAST and CFAST associate, in the presence of a fluorogen, they form the active and highly fluorescent FAST protein. Herein, Catcher-protein was fused with CFAST and Tag-peptide with NFAST, which allowed detecting the Catcher-Tag ligation ratio based on fluorescence with splitFAST system. Next, a screening system was developed for detecting Catcher variants with improved ligation efficiency. The developed high-throughput screening system showed high potential since visualizing the protein ligation was possible, and hence the system could help in expanding the Catcher/Tag toolbox by allowing large mutant library analyzes

Biomolecular Click Reactions Using a Minimal pH‐Activated Catcher/Tag Pair for Producing Native‐Sized Spider‐Silk Proteins**

A type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple-fragment ligation. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We show that a newly engineered pair, called SilkCatcher/Tag enables efficient pH-inducible protein ligation in addition to being compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90 % purity, which is not possible by traditional recombinant production methods

Biomolecular Click Reactions Using a Minimal pH-Activated Catcher/Tag Pair for Producing Native-Sized Spider-Silk Proteins

Funding Information: We thank Eva Crosas for her help with the early phases of the structure refinement. Protein crystallization was performed at SPC facility at EMBL Hamburg and the CD spectroscopy and mass spectrometry of the crystallized proteins at the Center for Structural Systems Biology (CSSB, Deutsches Elektronen‐Synchrotron DESY). We acknowledge technical support by the SPC facility at EMBL Hamburg. The synchrotron data was collected at beamline operated by EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany). This work was supported by the Academy of Finland through its Centres of Excellence Programme Life‐Inspired Hybrid Materials (LIBER, 2022–2029) under project no 346105 and Academy of Finland projects nos. 317395, 308772, and 333238. We are grateful for the support by the FinnCERES Materials Bioeconomy Ecosystem and use of the Bioeconomy Infrastructure at the Aalto University. Publisher Copyright: © 2023 Wiley-VCH GmbH.A type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple-fragment ligation. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We show that a newly engineered pair, called SilkCatcher/Tag enables efficient pH-inducible protein ligation in addition to being compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90 % purity, which is not possible by traditional recombinant production methods.Peer reviewe