Synthesis And Self-assembly Of Site-specific GFP-PNIPAM Bioconjugates

Compared with traditional amphiphilic polymer self-assembly systems,proteinpolymer self-assembly systems have received more and more attention due to their excellent biocompatibility and functionality.However,in the last few decades,various hydrophobic polymers were conjugated to hydrophilic proteins to form amphiphilic protein-polymer bioconjugates by covalent bonding between the functionalized end group of polymers and amino acid residues of the proteins.In fact,Most proteins contain numerous chemical reactive residues,which results in heterogeneous protein–polymer bioconjugates with poorly controlled stoichiometry and these will likely affect self-assembly behavior of these bioconjugates.Therefore,in this thesis,we described to synthesize site-specific and stoichiometric proteinpolymers bioconjugates GFP-PNIPAM that were utilized to explore different sites of polymers on protein for effect of bioconjugates self-assembly morphology.In this study,the green fluorescent protein(GFP)were functionalized with azide groups(at amino acid residues 35,125,133,125/205,133/194,133/194/205)by utilizing amber suppression technique using special codon TAG and orthogonal aminoacyl-tRNA synthetase/tRNA pairs to incorporate 4-Azido-L-phenylalanine into GFP.Three single site,two double sites and one three sites mutants were successfully synthesized,and the molecular weight of the targeting modified azide-based green fluorescent protein is about 28 KDa.Ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy of proteins have shown that the insertion of unnatural amino acids at these sites does not have much effect on the structure of the protein,and the protein still has good optical properties.The poly(N-isopropylacrylamide)having a molecular weight of 16000 g/mol and 9000 g/mol,respectively,were synthesized by reversible addition-fragmentation chain transfer polymerization,and subsequently modified to bear the simple strained cyclooctyne molecule,while the crucial presence of the alkyne end-group was confirmed by 1H NMR spectrum,FI-IR spectroscopy and Ultraviolet-visible absorption spectroscopy.Conjugation of the cyclooctyne-functional polymers with the azide-bearing proteins was performed in phosphate-buffered saline through copper-free strain promoted azide-alkyne click chemistry,a popular approach for biocompatible and bioorthogonal conjugation of biomolecules,to synthesize the green fluorescent protein-poly(N-isopropylacrylamide)bioconjugates that GFP was precisely modified by PNIPAM at different sites.The bioconjugates were analyzed by Ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy and the results demonstrated that the protein remained functionally active after conjugation.Finally,the self-assembly behavior of protein-polymer bioconjugates was investigated by solvent-induced method in the two-phase dissolving N,NDimethylformamide / phosphate-buffered saline system and the two-phase incompatible isooctyl alcohol / phosphate-buffered saline system and observed by confocal laser scanning microscopy.It was found that the modification sites,number and molecular weight of PNIPAM on the GFP had a great influence on the selfassembly behavior of the protein-polymer bioconjugates.