Affinity-induced covalent protein-protein ligation via the SpyCatcherSpyTag interaction  

作　　者：Jacob O.Fierer Omar E.Tovar-Herrera Jonathan Y.Weinstein Amaranta Kahn Sarah Moraïs Itzhak Mizrahi Edward A.Bayer 

机构地区：[1]Department of Biomolecular Sciences,the Weizmann Institute of Science,Rehovot 7610001,Israel [2]Department of Life Sciences,Ben-Gurion University of the Negev,Beer Sheva 84105,Israel [3]National Institute of Biotechnology in the Negev,Ben-Gurion University of the Negev,Be’er Sheva 84105,Israel [4]The Goldman Sonnenfeldt School of Sustainability and Climate Change,Ben-Gurion University of the Negev,Beer Sheva 84105,Israel

出　　处：《Green Carbon》2023年第1期33-42,共10页绿碳（英文）

摘　　要：Production of economically viable bioethanol is potentially an environmentally and financially worthwhile endeavor.One major source for fermentable sugars is lignocellulose.However,lignocellulosic biomass is difficult to degrade,owing to its inherent structural recalcitrance.Cellulosomes are complexes of cellulases and associated polysaccharide-degrading enzymes bound to a protein scaffold that can efficiently degrade lignocellulose.Integration of the enzyme subunits into the complex depends on intermodular cohesin-dockerin interactions,which are robust but nonetheless non-covalent.The modular architecture of these complexes can be used to assemble artificial designer cellulosomes for advanced nanotechnological applications.Pretreatments that promote lignocellulose degradation involve high temperatures and acidic or alkaline conditions that could dismember designer cellulosomes,thus requiring separation of reaction steps,thereby increasing overall process cost.To overcome these challenges,we developed a means of covalently locking cohesin-dockerin interactions by integrating the chemistry of SpyCatcher-SpyTag approach to target and secure the interaction.The resultant cohesin-conjugated dockerin complex was resistant to high temperatures,SDS,and urea while high affinity and specificity of the interacting modular components were maintained.Using this approach,a covalently locked,bivalent designer cellulosome complex was produced and demonstrated to be enzymatically active on cellulosic substrates.The combination of affinity systems with SpyCatcher-SpyTag chemistry may prove of general use for improving other types of protein ligation systems and creating unconventional,biologically active,covalently locked,affinity-based molecular architectures.

关 键 词：Synthetic Biology Protein Ligation Cellulosomes COHESIN DOCKERIN Protein Engineering Cellulose Degradation 

分 类 号：O62[理学—有机化学]