Improvement Of Protein Stability Via Site-Specific Conjugation Of Functional Polymers

Proteins, as the essential component of human body, are not only specific and efficient catalysts, but also therapeutic drugs for disease diagnosis and clinical treatment. However, in spite of wide spread application, there are still some practical limitations due to its structural complexity and vulnerability, exhibiting unstable and poor pharmacokinetics in complicated conditions. Therefore, it is critical to improve biological activity and stability of proteins. While polymers modified proteins to improve protein properties get more and more from researchers.Hence, in this thesis, different kinds and molecular weight of polymers with pyridine disulfide ester group were synthesized using reversible addition-fragmentation chain transfer(RAFT) polymerization and synthetic chemistry. The polymers were subsequently conjugated with the side-directed mutant of inorganic pyrophosphatase(PPase) which the cysteine residue was introduced in the specific site of PPase. The influence of activity and stability in different conditions were investigated systematically. The conjugates were applied in the enzymatic reaction and clinical treatment. The detail are introduced as follows.Firstly, site-directed mutagenesis was used to generate a mutant(K148C) with a cysteine residue in position 148 to replace the original lysine residue near the active center. Subsequently, the thiol group of cys148 was used to conjugate to poly(Nisopropylacrylamide)(PNIPAM), which is known as thermal-sensitive polymer. As compared to the free enzyme, the optimum temperature of the conjugate was shown to increase from 45 to 60 °C. For the conjugate, about 77% enzyme activity was retained after incubation at 60 °C for 3 h, representing a 6.8-fold increase as compared to the unconjugated enzyme, exhibiting excellent thermal-stability at elevated temperature. Polymer conjugation may also provide a strategy to improve the heat resistance of proteins more generally.Next, the obtained conjugate was applied in the enzymatic reaction——polymerase chain reaction(PCR), which is a powerful method for nucleic acid amplification. The normal PCR is inhibited in its yield due to its byproduct, pyrophosphate(PPi), a byproduct of the reaction; the yield is thereby limited. The conventional method for hydrolysis of PPi by PPase is not well adapted for operation at elevated temperatures over long times as required during the PCR. Therefore, after the PCR using the conjugate, the yield was 1.5-fold greater than using the unconjugated enzyme without any specificity loss. As well as improving the yield of the PCR(and possibly other biological reactions) at elevated temperature, this strategy should be favorable for applications in biological reactions, enzyme engineering, and other related fields.Finally, same strategy was used to prepare glycoprotein mimics. Here, an ideal and facile one-step method by using N-acetyl-D-glucosamine(Glc NAc), a common unit of universal biological polysaccharide, was proposed for successful preparing glycopolymer-protein conjugates having a high catalytic activity and improved stability in a variety of adverse conditions. A series of well-defined and controllable molecular weight glycopolymers, Poly(2-methacrylamido glucopyranose)(PMAG), were prepared via RAFT polymerization from 2-methacrylamido glucopyranose(MAG), a Glc NAc analogue monomer. PMAG was conjugated with PPase through one-step thioldisulfide exchange reaction to produce PMAG-PPase conjugates. The comprehensive study focused on their capability of enhancing enzymatic activity and maintaining stability under several adverse conditions is further developed. The results showed that the PMAG-PPase conjugate with a polymer chain of comparatively lower molecular weight(8.0 k Da) exhibited excellent ability to improve enzymatic activity by about 50%; it can maintain the enzymatic activities at higher levels at extreme p H and high salt concentration; and the glycopolymer-protein conjugates also exhibited excellent resistance to both proteases and glycosidases. Moreover, the glycopolymer-protein conjugates could efficiently catalyze the hydrolysis reaction of calcium pyrophosphate(CPP) and exhibit great potentials in the treatment of the CPP deposition disease(CPDD). It can be predicted that, these kinds of glycoprotein mimics will be favorable and valuable for applications in biological detection and clinical treatment.In conclusion, a series of different kinds and chain length of polymer-protein conjugates were fabricated via specific-site conjugation. The influence of activity and stability in various conditions were investigated systematically to summarize some experience and principles. Furthermore, the obtained conjugates were preliminary applied in the biological reaction and clinical treatment. The functional polymers conjugated proteins via specific-site modification not only enhance the protein activity and stability, but also have a favorable prospect in the biological engineering and clinical diagnosis.