Protein Assembly Mediated By Aldehyde Modification And Protein Interaction

Molecules self-assembly which is the basis for achieving life activities and functions in organisms is widespread in nature. Self-assembly is a type of process in which a disordered system of pre-existing components forms an organized structure or pattern as a consequence of specific, local interactions among the components themselves, without external direction. This bottom-up approach has been widely used to prepare sensing and catalytic nano-materials. How to construct biomolecules, especially the catalytic proteins, into a structure similar to organisms by rational design and artificial regulation is a challenge for self-assembly nano-materials.In this study, two strategies, chemical modification and protein interaction, were utilized for protein assembly. Aldehyde tag, converted from thiol group of cysteine by specific post-translational modification of FGE, can selectively reacted with a-nucleophiles (aminooxy-or hydrazide-functionalized moieties). The chemical reaction of aldehyde tag and azido-aminooxy or cyclooctyne-aminooxy bifunctionalized linker introduced cyclooctynes or azides for targeted proteins. Protein-protein assembly was achieved by1,3-dipolar cycloaddition of cyclooctynes and azides. We explored four protein targets:EG, K107lipase, FDH, LDH. We coexpressed the tagged proteins with FGE to form formylglycine in E. coli and maximized the formylglycine formation efficiency by improving FGE expression and adding reducing agents. Aldehyde tagged protein was site-specifically modified by synthesized aminooxy functionalized PEG. So the aminooxy PEG modification rate was accordant with convertion of aldehyde tag detected by Alexa488-hydrazide. But the protein assembly by chemical modification was difficult for low conversion rate of aldehyde tag (only3.01%for aFDH).Protein assembly by protein interaction was conducted by protein-interaction domain PDZ and its ligand PDZ lig fused with LDH and FDH respectively. These proteins were co-expressed in E. coli to achieve protein assembly with ordered porous nanostructure layer. This nanostructure can be repeated used in biocatalysis, have good temperature stability and pH stability. The catalytic efficiency was improved by67%increase for the promoted transfer efficiency of substrates and coenzymes through the assembly of LDH and FDH when whole cell catalysis.Bimolecular fluorescence complementation (BiFC) system was constructed by fusing two parted non-fluorescent fragments, MN159and MC160of red fluorescent protein mCherry, with LDH-PDZ and FDH-PDZ lig respectively via a flexible peptide linker. The red fluorescence produced by BiFC system after proteins co-expression in E. coli indicated the interaction of PDZ and PDZ lig, which proved the assembly of the LDH-PDZ and FDH-PDZ lig in cell.Protein-assembly nano-materials have being applying in biological composite materials, biosensors, enzyme catalysis and biomedicine. Assembly of protein complexes play an important role in understanding of protein folding and its catalytic function, and preparing advanced nano-materials.