Design Of Protein Refolding Aids And Integrative Refolding-purification Method Of Proteins With Like-charged Particles

Protein refolding and purification process is crucial steps in the production of recombinant proteins expressed as inclusion bodies(IBs). Herein, a series of agents that can facilitate disulfide formation and inhibit protein aggregation were developed to facilitate protein refolding. Furthermore, an integrative refolding and purification method was established.Firstly, the disulfide bond formation was investigated. Based on the idea of mimicking the hydrophobic and charged patches of protein disulfide isomerase(PDI), a new peptide mimic of PDI, RKCGCFF, was designed. RKCGCFF was proved to be more effective than the traditional oxidant oxidized glutathione(GSSG) as well as its counterpart, RKCGC, in facilitating the oxidative refolding of lysozyme. More importantly, RKCGCFF could improve lysozyme refolding yield at a high concentration. The research proved that incorporation of hydrophobic and charged patches into the CGC disulfide made the oxidant more similar to PDI in structure and properties. Therefore, RKCGCFF not only accelerated the formation of disulfide bond, but also displayed a good inhibition effect for protein aggregation, and well simulated the function of PDI as molecular chaperone.Moreover, the inhibition of protein aggregation during refolding was investigated. Previous researches have proved that addition of like-charged microspheres in a refolding system can significantly inhibit protein aggregation and enhance protein refolding. And the beads of high charge density are beneficial for facilitating effect. Based on these findings, Silica nanoparticles(SNPs) were sequentially modified with poly(ethylenimine)(PEI) and 2-diethylaminoethylchloride(DEAE) to prepare a series of positively charged SNPs-PEI and SNPs-PEI-DEAE. Compared to their microsphere counterparts, the highly charged nanoparticles efficiently facilitated lysozyme refolding with significantly reduced bead utilization(nearly 96% lower than for microspheres). Hence, the polyelectrolyte-functionalized SNPs with higher specific surface area and charge density were better suited as refolding aids of like-charged proteins.In view of the purification of refolded recombiant proteins, an integrative method of protein refolding and purification was established by like-charged resin facilitated refolding and metal-chelate affinity adsorption. A metal-chelating resin was fabricated by coupling iminodiacetic acid(IDA) to agarose gel(Sepharose FF) and was used to facilitate the refolding of like-charged 6×his-EGFP IBs. It was confirmed that the negatively charged IDA-Sepharose FF not only offered an enhanced refolding yield, but also bound some opposite-charged contaminant proteins, leading to a preliminary purification effect. Afterwards, the refolded 6×his-EGFP could be recovered at high yield(85%) and purity(93%) by introducing small amounts of nickel ions, which led to the specific metal-chelate interaction between nickel ions and 6×his-EGFP. Moreover, a ―grafting-from‖ method by atom transfer radical polymerization(ATRP) was used to prepare a series of metal-chelating nanoparticles of high charge densities. These nanoparticles further improved the refolding and purification effect of 6×his-EGFP IBs as compared with IDA-Sepharose FF, proving their great superiority for applications in the integrative refolding and purification method.