Study On The Design Of An Automatic Protein Refolding Apparatus And The Development Of Protein Refolding Techniques

In the post genomic era, there is an explosion in the protein science research. E. coli is the most used system to produce proteins. However, a substantial hindrance to these efforts is the formation of insoluble inclusion bodies. In this study, we focused on the design of an automatic protein refolding apparatus and the development of protein refolding techniques.The most common methods reported in the literature regarding protein refolding include dilution, step-wise dialysis and on-column techniques. The recovery of active recombinant proteins from inclusion bodies is also a time consuming and laborious process, including multiple steps such as solution preparation, protein concentration, purification and trials of refolding techniques. Due to the inconsistencies and difficulties of the protein refolding process, there is a need for an automated large-scale refolding apparatus that integrated dilution, dialysis and on-column refolding, thereby reducing the need for experimenter’s time. This refolding system configuration includes4-way gradient valves, pumps, refolding reservoir, ultrafiltration membrane, valves, chromatography columns, detectors, and controllers etc. In principle, it is convenient to manipulate three refolding methods individually, and to integrate two or more refolding techniques together to develop on refolding process. Different methods were carried out to evaluate the performance of this refolding apparatus:a combination of dilution and dialysis for human stromal cell-derived factor1(SDF/CXCL12) and thioredoxin-human artemin fusion protein (Trx-ARTN); dilution refolding for thioredoxin-human insulin-like growth factor I fusion protein (Trx-IGFl) and enhanced fluorescent protein (EGFP); and on-column refolding for BSA. The refolding processes of these five proteins were preliminary optimized using a strategy of slowly descending denaturant concentration. In the protein refolding process of SDF/CXCL12, Trx-ARTN and Trx-IGFl, refolded protein solution was directly purified on line. For example,10.5mg of～92%pure SDF/CXCL12was obtained from30mg of～60%pure dentured proteins using reverse-dilution and dialysis combination refolding method, almost double that the amount using dilution and dialysis combination refolding method; in the BSA refolding process, the recovery yield in the continuous dialysis refolding method was～95%, higher than that the yield (～50%) in the on-column refolding method. This study demonstrated that the refolding apparatus can be set and maintained throughout the refolding process by adjusting the flow rates of the buffer, the denatured protein suspension, or the fluid path. What’s more, slowly descending denaturant concentration followed with on-column concentration or on-line purification performed by this refolding apparatus could be a useful tool for a preparative scale protein production.We invented a high efficient protein refolding technique, called two-step denaturing and refolding method (2DR). The first denaturing step was to thoroughly dissolve inclusion bodies, using a denaturing buffer with7M guanidine hydrochloride (GdnHCl). Subsequently, to precipitate the GdnHCl-denatured protein, the concentration of GdnHCl was rapidly diluted by10-40folds. The second denaturing step was to dissolve the protein precipitate by a denaturing buffer with8M urea. Then the refolding of the target protein was conducted on common refolding methods. We found that the general applicability of2DR was very higher than that of traditional one-step denaturing and refolding method (1DR). In view of this, the study on the development of protein refolding techniques based on the2DR is as the following. Firstly, we primarily investigated on the isolation of inclusion bodies in the2DR. Comparing the results from1DR, there was no obviously difference in the facet of protein purity, viable cell counts and the shape of inclusion bodies. Secondly, EGFP inclusion bodies were selected as a model to study the principle of2DR by means of Laman spectrum and protein unfolding/refolding equilibrium experiments. The protein precipitates in the2DR were loose and formed in-vitro under specific physical and chemical conditions; inclusion bodies were dense aggregates of mis-folded polypeptides. Although both of them were similar in the composition of secondary structures, the exposure of aromatic residues, and the exposure of cysteine residues, significant differences were monitored on the frequency and intensity of the Laman spectra such as680-800cm-1,1020～1100cm-1,1120～1220cm-1,1380～1420cm-1Thus, these differences would trigger the improvement of protein refolding efficiency in the2DR. Thirdly, based on the mechanism of chaperonin assisting protein folding, the second denaturing and conformation confinement method was performed on the optimization of protein intermediates under specific physical and chemical conditions in order to extend the application of2DR. The recovery yield of EGFP would approach to～80%in the spontaneous refolding after the conformation confinement under a buffer containing0.5M L-Arginine and300mM NaCl. Screening experiments demonstrated that protein refolding yield and recovery significantly depended on a specific secondary denaturing and conformation confinement condition. Finally, taken Schistosoma japonicum MTH1protein as an example, we integrated refolding techniques, such as two-step denaturing and refolding method, protein aggregation inhibitors screening, and continuous dialysis method to obtain15mg of～95%active MTH1from0.5L LB medium. Taken together, we preliminarily proposed a technology route based on a specific secondary denaturing and conformation confinement of protein intermidiates.In conclusion, we firstly integrated the most common refolding methods within an apparatus to improve the efficiency of protein refolding, and expanded the application of the two-step denaturing and refolding techniques. These experiments and results could prepare the foundation of techniques for improving the protein refolding efficiency.