Controllable Regulation Of Protein Properties Via Site-specific Conjugation Of Functional Polymers

Protein bioactivity control is the critical issue for human health and disease treatment.The ability to accurately control protein structure and activity is the key issue for its biomedical application in disease therapy,molecular diagnostics and tissue engineering.Polymer conjugation for improving protein properties has received increasing attention.Well-defined protein-polymer conjugates can be achieved by site-specific conjugation of polymer to protein surface,and the polymer properties and conjugation site on protein surface play an important role in the application performance of the conjugates.The main focus of this thesis is to construct a series functional polymers conjugated protein and to investigate systematically the effect of polymer type and conjugation site on protein bioactivity.The detailed research works are introduced as follows.First,a series of model proteins with thiol group at different sites on protein surface were synthesized.Wild-type inorganic pyrophosphatase(PPase)was achieved by gene recombination.Site-directed mutagenesis was utilized to introduce an additional Cys residue into the specific sites on PPase surface,and various PPase mutants containing a thiol group at specific sites were prepared for the following site-specific polymer conjugation.The purity of the PPase and mutants was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The protein activity was assayed according to the amount of phosphate produced by the PPase enzymatic hydrolysis of sodium pyrophosphate.And the free thiol content was determined using Ellman's assay.The results suggested that purified PPase and mutants were obtained via gene mutation and protein expression and purification.The PPase mutants contain a free thiol group at different sites,and their activities were related to the mutation site and amino acid type.The change of activity has no significant influence on the further research.Next,biocompatible poly(2-Hydroxyethyl methacrylate)(p HEMA)modified PPase was prepared.The effect of p HEMA conjugation on protein activity and conformation was investigated and compared with small sulfhydryl modifier p-chloromercuribenzoate(PCMB).Pyridyl disulfide-functionalized p HEMA was synthesized via the atom transfer radical polymerization(ATRP)of HEMA initiated by pyridyl disulfide-functionalized ATRP initiator,and the polymer molecular weight and distribution were characterized by 1H-nuclear magnetic resonance(~1HNMR)and gel permeation chromatography(GPC).The functionalized p HEMA was conjugated to PPase mutant with a thiol group near the active center(PPase K148C)via the coupling of pyridine disulfide end group with thiol group.The formation of PPase-p HEMA conjugates was verified by SDS-PAGE and dynamic light scattering(DLS).The effect of PCMB and p HEMA introduction on protein activity was investigated and reversible activity control was conducted by reductive reagents treatment.Conformational changes of PPase with PCMB and p HEMA conjugation were determined by circular dichroism(CD).The results indicated that the site-specific conjugation of PCMB and p HEMA led to a significant decrease in enzymatic activity.Compared with PCMB,the p HEMA conjugation led to greater inhibitory effect on protein activity.The protein activity can be restored to different extents by the treatment with different amount of reductive reagents.Moreover,the PPase activity can be reversibly regulated by alternately repeated PCMB/p HEMA conjugation and reductant treatment.Furthermore,the effect of PCMB and p HEMA on PPase activity was caused by a tertiary structure change of protein,and the p HEMA conjugation led to greater change of the tertiary structure compared with PCMB.And the recovery of protein activity by reductive reagents was caused by the return of protein conformation,resulting from the dissociation between PPase and PCMB or p HEMA.We expected that this strategy for efficient regulating protein activity can be potentially applicable for bioassay,drug release and cell performance control.Furthermore,p H-responsive poly(2-(dimethylamino)ethyl methacrylate)(p DMAEMA)was conjugated to PPase and the influence of p DMAEMA conjugation site on protein activity and stability was investigated.p DMAEMA with different molecular weight was synthesized by reversible addition-fragmentation chain transfer polymerization(RAFT)and functionalized by pyridyl disulfide modification.The polymerization and modification were characterized by ~1HNMR and GPC.The functionalized p DMAEMA was respectively conjugated to PPase mutant with a thiol group near the active center(PPase K148C)and far from the active center(PPase N124C)via the coupling of pyridine disulfide end group with thiol group.The formation of PPase-p DMAEMA conjugates was verified by Native PAGE.The effect of p DMAEMA conjugation on PPase surface charge and aggregation state at different p Hs was characterized by Zeta potential and hydrodynamic diameter distribution.The results showed that the polymer conjugation site had different influence on protein activity.p DMAEMA conjugation close to the active center led to lower activity.In contrast,polymer conjugation far from the active center allowed retention of activity,and even caused an increase in activity at low p H.Moreover,the conjugates with polymers of higher molecular weight had a greater effect on activity.p DMAEMA attachment on PPase surface led to dependence of the surface charge and aggregation state of the protein on p H,thus preventing protein from aggregation in acidic conditions(p H 4.0-6.0),and giving a significant increase in protein activity and stability.This study provides a new strategy for improving protein activity and stability in acidic environments via site-specific polyelectrolyte conjugation.Lastly,glycopolymer p AG and p MAG were conjugated to PPase surface and the effect of glycopolymer type and molecular weight as well as conjugation site on protein activity was investigated.Glycopolymers with different molecular weight were synthesized by RAFT and functionalized at the end of polymer chains by pyridyl disulfide modification.The polymerization and modification were characterized by ~1HNMR and GPC.The pyridyl disulfide-functionalized p AG and p MAG were respectively conjugated to PPase mutant PPase K148C and PPase N124C via the coupling of pyridine disulfide end group with thiol group.The formation of protein-glycopolymer conjugates was verified by SDS-PAGE.The results demonstrated that the glycopolymer conjugation site played a significant role in protein activity.Glycopolymer attachment near the active center led to lower activity.However,the protein activity increased when the glycopolymer conjugated far from the active center.As for the same kind glycopolymer,p AG with different molecular weight showed different effect on PPase activity.Glycopolymer with largerer molecular weight showed a much higher inhibitory effect on protein activity when conjugated near active center;glycopolymer with smaller molecular weight showed a much higher promotion effect on protein activity when conjugated far away from active center.Different glycopolymers p AG and p MAG with the same molecular weight had no significant influence on PPase activity.This study provides theoretical and practical guidance for the design of protein-glycopolymer conjugate.In summary,a series of functional protein-polymer conjugates were constructed via site-specific polymer conjugation on protein surface at different sites.The effect of polymer type and molecular weight as well as conjugation site on protein properties such as activity and structure was investigated systematically and some principles have been summarized for the design of protein-polymer conjugates.Therefore,the research work of this thesis can provide both experimental evidence and theoretical guidance for the reasonable design of well-defined protein-polymer conjugates with controllable bioactivity.