Spy Chemistry-enabled Directional Protein Immobilization

Site-directed protein immobilization allows the homogeneous orientation of proteins with high retention of activity,which is advantageous for many applications including biocatalysis,clinical diagnosis,and protein interaction analysis.several novel approaches have been developed in order to achieve oriented immobilization.Bioorthogonal reactions such as click chemistry require the introduction of unnatural amino acids.Approaches based on intein cleavage is limited by the need for separation of the intein,and low yields.Enzymatic modifications of particular motifs/tags require excess amounts of sortase A or FGE and have low efficiencies.Thus,these approaches for site-directed immobilization are not preferable for large-scale applications.In this study,the self-sufficient Spy Catcher-Spy Tag reaction,in which an isopeptide bond is formed,specifically and efficiently,was implemented to develop a novel approach for directional immobilization of industrial enzymes,especially directly from crude cell lysates or fermentation broth.In this scheme,Spy Catcher is first covalently immobilized onto a resin.Then,the protein of interest(POI),fused with the short Spy Tag,is allowed to ligate with the immobilized Spy Catcher.As a proof of concept,an extensively-used commercial epoxy resin LX-1000 EP was selected as the solid support and red fluorescent protein(RFP),glutaryl-7-aminocephalosporanyl acylate(GA)and formate dehydrogenase(FDH)were chosen as model proteins.Spy Catcher was immobilized on the epoxy resin LX-1000 EP,after incubation at p H 7.0,25 ? for 12 hours,with an immobilization efficiency of 89.5±2.0%.Purified Spy TagRFP/GA were immobilized through the Spy Cather-Spy Tag chemistry at mild conditions(p H 7.0,25 ?)within 2 hours with immobilization efficiencies and activity recoveries between 50% and 90%).The activity recoveries were 2 to 3 folds higher than the epoxy-based immobilization approach.We further carried out directional immobilization of target proteins(Spy TagRFP/GA/FDH)using our Spy-based approach by directly incubating crude E.coli cell lysates overexpressing the Spy Tag-fused target proteins.The immobilization efficiencies were greater than 86% for all of the tested proteins;the activity recoveries were in the range of 30-90%,which were 2 to 25 folds higher than the epoxy-based approach.In summary,the Spy-based protein directional immobilization approach established in this study requires no additional reagents,is rapid,proceeds under mild conditions,and only requires a readily available small anchoring protein Spy Catcher.Since Spy Catcher can be produced through standard fermentation processes,this immobilization method is also feasible on an industrial scale.It can directly immobilize the target protein from fermentation broth or cell lysate,with high selectivity and activity recovery rate.The method could simplify the largescale targeted immobilization process of proteins and provides a preliminary experimental basis for the preparation of high-performance immobilized proteins in the field of catalysis and analysis.