| Objective：In this study, we had constructed a plasmid of truncated sTNFR1（soluble tumornecrosis factor receptor1） for over-expression in Escherichia coli. However, therecombinant sTNFR1protein as a new TNF inhibitor was synthesized as insoluble andinactive aggregates, which is called inclusion bodies (IBs). In order to obtain functionalsTNFR1protein, the inclusion bodies must be solubilized in vitro and refolded into anactive molecule with the correct structure conformation. By investigating the methods ofdisrupting, washing, dissolving, and refolding of recombinant sTNFR1inclusion bodiesprotein, the effective and feasible procedure of denaturation and renaturation of therecombinant sTNFR1protein should be established and provided some new ideas for otherrecombinant proteins.Methods：(1) Disrupting: The bacteria were disrupted using high pressure homogenizer, andstained with methylene blue before and after. Then the broken rate and wet weight recoveryrate were calculated to evaluate the efficiency of the condition.(2) Washing: Taking the conventional fermentation production as samples, the bestconcentrations of sodium deoxycholate and urea of the repeat-washing buffer are picked outby the factorial design experiments of two factor and five levels. In addition, a newstep-washing method was proposed and other condition was found by two experiments.Then the washing method of inclusion bodies was determined by the comparison ofrepeat-washing and step-washing in different amount of samples (3g,50g) and differentfermentation yield samples (low, high). (3) Dissolving: The urea concentration and pH of the denaturation buffer wereoptimized by two-factor analysis of variance, and then the cysteine concentration wasoptimized by one-factor analysis of variance.(4) Refolding: The refolding conditions of recombinant sTNFR1protein based on thedilution refolding method, which can be scaled up and relatively cheap, were studied deeplyfrom three aspects as follows: protein purity and refolding pattern, resin with electric charge,and MgSO4. Four refolding routs were compared with100ml refolding volume, and the bestcondition was picked out according to the product yield, protein activity, easy to operate andthe costs. Finally, the condition was amplified to1L refolding volume to evaluate.Results:(1)Disrupting: The bacteria were successfully disrupted using high pressurehomogenizer with300bar and700bar. The broken rate was over95%and the wet weightrecovery rate was about50%. The results indicated this disrupting condition was effectablefor BL21（DE3）pLysS bacteria, which were used to express recombinant sTNFR1protein.(2)Washing: The optimum repeat-washing condition was no sodium deoxycholate，1mol/L urea，repeating three times. The optimum step-washing condition was2%sodiumdeoxycholate in the first step and2%sodium deoxycholate plus2mol/L urea in the secondstep. The step-washing method was demonstrated to be better than the repeat-washingmethod in terms of the cost of time and the purity of samples. For conventionalfermentation production, purity of target protein was increased from21%to31%, and thewet weight recovery rate was about55%by step-washing.(3) Dissolving: The inclusion bodies was dissolved at room temperature in thedenaturation buffer (50mM Tris,8mol/L Urea,80mM Cys, pH9.7) with magnetic stirringabout4hours. And the quality of achieved protein was about15%of the wet weight.(4) Refolding: The higher the protein purity obtained，the better the protein refoldingwas. One-step refolding was better than gradient refolding. SPFF resin with negative chargehelped the refolding of recombinant sTNFR1protein and was suitable for high renaturationconcentration. The presence of MgSO4in refolding buffer not only significantly reduced therefolding time, but also increased refolding recovery rate. How MgSO4assisted therefolding process was not clear exactly despite our preliminary experiment results had indicated that Mg2+could help the refolding of the recombinant protein, and we guessedthere were some other unknown mechanisms. The optimum refolding condition with100mlrefolding volume was as follows: the renaturation buffer of50mM Tris,0.78mol/L Urea,10mM MgSO4, pH9.7,1:10diluting,0.7mg/ml renaturation concentration,4℃, magneticstirring. Then the refolding volume was amplified to1L, with refolding time of40hours,and recovery rate and purity of active protein were about15%and90%, respectively.Conclusions:In summary, our studies focused on three main aspects: washing, dissolving, andrefolding of recombinant sTNFR1inclusion bodies protein. The denaturation andrenaturantion procedures which were effectable and industrial-scaleable had beensuccessfully established. This work would provide the bases, both theoretical and practical,for the research and development of new potential drug PEG-modified TNFR1, and alsoprovide some valuable references for other protein production. |
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