In addition, it is preferable for modules to be modified with peptide tags rather than protein fusion domains (e.g., HaloTag or SNAP-tag) for minimal disruption to module function ( 22, 23). Nearly quantitative yield for each reaction is required otherwise, after a few steps the incomplete chains generate hopelessly heterogeneous products. Other important features of a system for synthesizing polyproteams are molecularly defined connections, independence from any template ( 20, 21), and simple expression of each module. Solid-phase reaction has also enabled the ligation of peptide fragments to make synthetic proteins ( 18, 19). Establishing such solid-phase chemistry for connecting amino acids underpinned the breakthroughs in the biological understanding and therapeutic use of peptides ( 14, 15), whereas the solid-phase synthesis of DNA primers underpinned the revolution in gene amplification and reengineering ( 16, 17). If the growing chain is attached to a solid phase, the reacting module can be added in large excess (driving reaction to completion), with unreacted building blocks simply washed away (so separation is unnecessary at each step). However, elongating one step at a time allows chain growth using a small number of orthogonal connections ( 13). There are a limited number of mutually unreactive (orthogonal) chemical reactions ( 12) therefore, it is impractical to link more than a few building blocks in a one-pot reaction. Even the best noncovalent linkages ( 7– 9) or reversible covalent linkages, including disulfide bonds ( 10, 11), would allow rearrangement of polyproteams, so irreversible covalent linkage is required. Expressing modules individually and then linking the modules together would overcome these challenges as well as allow independent posttranslational modification of each module. Protein units can be joined genetically into one long open reading frame, but errors in protein synthesis and misfolding soon become limiting ( 5, 6). However, clustering different kinds of proteins into programmed polyprotein teams (“polyproteams”) is an unmet challenge ( 3, 4). Clustering a single kind of protein often greatly enhances biological signals ( 1), for example in the repeating antigen structures on vaccines ( 2). This simple and modular route to programmable “polyproteams” should enable exploration of a new area of biological space.īiological events usually depend on the cooperative activity of multiple proteins. Linear, branched, and combinatorial polyproteins were synthesized, identifying optimal combinations of ligands against death receptors and growth factor receptors for cancer cell death signal activation. Solid-phase attachment followed by sequential SpyTag or SnoopTag reaction between building-blocks enabled iterative extension. By engineering the adhesin RrgA from Streptococcus pneumoniae, we developed the peptide SnoopTag, which formed a spontaneous isopeptide bond to its protein partner SnoopCatcher with >99% yield and no cross-reaction to SpyTag/Sp圜atcher. SpyTag peptide is engineered to spontaneously form an isopeptide bond with Sp圜atcher protein. Here we achieved sequence-programmed irreversible connection of protein units, forming polyprotein teams by sequential amidation and transamidation. Reacting proteins together is more complex because of the number of reactive groups and delicate stability. Programmed connection of amino acids or nucleotides into chains introduced a revolution in control of biological function.
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