RESUMEN
Formation of protein-polymer conjugates (PPCs) is critical for many studies in chemical biology, biomedicine, and enzymatic catalysis. Polymers with coordinated physicochemical properties confer synergistic functions to PPCs that overcome the inherent limitation of proteins. However, application of PPCs has been synthetically restricted by the limited modification sites and polymer grafting method. Here, we present a versatile strategy for site-selective PPC synthesis. The initiator was specifically tethered to the preoxidized glycan moieties through oxime chemistry. Polymer brushes were grown in situ from the glycan by atom-transfer radical polymerization to generate well-controlled PPCs. Notably, the modification is site-specific, multivalent, and alterable depending on protein glycosylation. Additionally, we demonstrated that the cytocompatible method enabled the growth of polymer chains from the surface of living yeast cells. These results verified a facile technology for surface modification of biomacromolecules by desired polymers for various biomedical applications.
Asunto(s)
Polímeros , Polisacáridos , Glicoproteínas , Polimerizacion , Propiedades de SuperficieRESUMEN
A recoverable and thermoresponsive polymer-protein bioconjugate is synthesized and employed in the purification of protein with free sulfhydryl groups. Initiator with disulphide was modified on the cysteine residue of the target protein. Poly(N-isopropylacrylamide) exhibiting a lower critical solution temperature was grown from the protein. The resulting protein-polymer conjugate was successfully thermoprecipitated and separated from other proteins. The approach was demonstrated with bovine serum albumin with the recycling yield of 76.4%. Enzyme activity test with papain verified the reversible polymer modification protected protein under extreme environments without affecting the functionality of the protein. This study implies the favorable potential of chemo-selective enriching and purification of proteins.
Asunto(s)
Precipitación Química , Polímeros/química , Proteínas/aislamiento & purificación , Temperatura , Bromelaínas/metabolismo , Electroforesis en Gel de Poliacrilamida , Papaína/metabolismoRESUMEN
In view of the biological significance and micro-heterogeneity of protein glycosylation for human health, specific enrichment of N-glycosylated proteins/peptides from complex biological samples is a prerequisite for the discovery of disease biomarkers and clinical diagnosis. In this work, we propose a "grafting-from" N-glycoprotein enriching method based on the in-situ growth of thermoresponsive polymer brushes from the N-glycosylated site of proteins. The initiator was first attached to the pre-oxidized glycan moieties by hydrazide chemistry, from which the thermoresponsive polymers can be grown to form giant protein-polymer conjugates (PPC). The thermosensitive PPC can be precipitated and separated by raising the temperature to above its lower critical solubility temperature (LCST). Mass spectrometry verified 210 N-glycopeptides corresponding to 136 N-glycoproteins in the rabbit serum. These results demonstrate the capability of the tandem thermoprecipitation strategy to enrich and separate N-glycoprotein/glycopeptide. Due to its simplicity and efficiency specifically, this method holds the potential for identifying biomarkers from biological samples in N-glycoproteome analysis.