Identification Of Peptide Tags For Molecular Encapsulation In A Protein Capsid

Supramolecular is a complex of molecules that held together by noncovalent bonds.In nature,protein capsids are a very common of supramolecular.Protein capsids commonly act as natural molecular containers.For example,in Bacillus subtilis,the60-subunit dodecahedral capsid formed by lumazine synthase?BsLS?acts as a container for trimeric riboflavin synthase?BsRS?.The lumazine synthase and riboflavin play an important role in roboflavin biosynthesis.And in this natural encapsulation system,capsid loading occurs in the cell,and guest release can be triggered in vitro by a mild change in buffer conditions.The lumazine synthase?LS?capsid has attracted much attention as a container for bionanotechnology.Recently,much effort has done for AaLS?lumazine synthase from Aquifex aeolicus?encapsulation ability.AaLS has been engineered to encapsulate both negatively charged and positively charged cargoes through a charge complementarity strategy.Four residues per AaLS monomer were mutated to glutamate,which resulted in an expanded 180 subunit capsid?AaLS-neg?with a negatively charged interior that preferentially encapsulates cargo proteins containing a complementary positively charged peptide tag(GFP-R₁₀).The engineered AaLS encapsulation system provides a promising starting point for developing applications.But compared to the natural BsLS+BsRS encapsulation system,the engineered AaLS encapsulation system has some disadvantages.It is not very specific because encapsulation of cargoes depends only on charge-charge interactions.The encapsulation of tagged proteins by AaLS-neg is not reversible,so there is no way to release the guest proteins from the capsid.Despite a great deal of structural and functional studies on BsLS and the BsLS+BsRS complex,the three-dimensional structure of BsRS remains unknown,This lack of structural information has hampered efforts to understand how BsRS is localized within BsLS.To test whether the C-terminal sequence of BsRS is responsible for its encapsulation by BsLS,the C-terminus of the green fluorescent protein?GFP?was fused to either the last 11 or last 32 amino acids of BsRS,offering variants GFP11 and GFP32,respectively.GFP11,GFP32,and GFP lacking any BsRS fragment?GFP0?were each co-produced with BsLS,in an attempt to generate encapsulation complexes.BsLS capsids that had been co-produced with GFP11 or GFP32 are 15-fold and 6-fold more fluorescent,respectively,than BsLS co-produced with GFP lacking any BsRS fragment indicating complex formation.Analysis of size-exclusion chromatography showed the GFP variants did not change the capsid size and make the capsid that co-produce with GFP11 and GFP32 become fluorescent than the BsLS co-produce with GFP0.ELISA experiments indicate that the GFP variant in the BsLS+GFP11 complex is hidden,suggesting that GFP11 is localized within the BsLS capsid.Together,these results indicate that the C-terminal sequence of BsRS is responsible for its encapsulation by BsLS and can also target other proteins to the capsid interior.By fluorescence intensity detection and SDS-PAGE analysis we know the number of GFPs go into the capsid.Fused the last 11 amino acid of BsRS to another non-native protein Abrin A chain?Abrus precatorius?and co-produce with BsLS,Western blot indicate that the guest protein can be encapsulated by the host capsid.The encapsulation yield of Abrin A is consistent with GFP variants.As with the natural BsLS+BsRS complex,mild changes in pH and buffer identity trigger dissociation of the GFP11 guest,accompanied by substantial changes in the BsLS capsid structure.This system for protein encapsulation and release provides a novel tool for the future development of enzyme nanoreactors and protein drug delivery systems.