Establishment And Application Of A New ELP - SUMO System For Enhancing Highly Efficient Protein Expression In Escherichia

Recombinant proteins are used throughout biological and biomedical science. Escherichia coli is the preferred host for rapid and low-cost production of recombinant proteins and peptides for biochemical analysis, therapeutics or structural studies. E. coli heterologous protein production has challenges including protein misfolding and insolubility. Several approaches, including protein fusions, chaperone co-expression, and promoter alterations, have been used to overcome these problems. Although alteration of expression conditions can sometimes solve the problem, the best available tools to date have been fusion tags that enhance the solubility of expressed proteins. As such, protein fusions have generally been used to improve the expression and solubility of recombinant proteins. No single fusion tag can increase the expression and solubility of all target proteins. However, some fusion tags have been more successful than others in increasing the solubility of many proteins. SUMO tags are emerging as a viable alternative for increasing both the expression and solubility of otherwise hard-to-express proteins.In the present paper, we are focus on developing a new expression system can increase the solubility of target proteins in E.coli. The new system will be used in soluble expression and purification of target protein. Two tags were choosed for the study of soluble ablity, removal of the tag. Firstly, we tested human SUMO as fusion tags for recombinant proteins. Three candidate proteins, hAPRIL、gelonin and hHAPO were expressed as fusion proteins with His6-SUMO. These constructs were expressed in E. coli and evaluated for expression and solubility. Finally, the SUMO-hHAPO fusion protein expressed in a soluble form, cleavage and purification of the fusion protein using the human SUMO expression system is easily accomplished by using a SUMO protease to cleave the fusion tag followed by purification of the hHAPO to homogeneity by Ni-Sepharose column. Mass spectrometry analysis and Cell proliferation assays showed that the recombinant hHAPO with high activity has the same MW with the natural protein. The soluble expression form of the other two fusion construct of SUMO-hAPRIL and SUMO-gelonin can be partly obtained in these expression conditions:ArcticExpress (DE3) strain, low induxtion temperature and low concentration of IPTG. The fusion protein can be complete digested by SUMO protease, the target protein was obtained by affinity chromatography. These results showed thay the SUMO modification is effective.The ELP expression system is developed by WOOD. An expression system is present for non-chromatographic purification of recombinant proteins expressed in Escherichia coli. In our previous experiment, all the target proteins, expressed in a soluble form when fused to ELP. Next, we evaluated the ELP system for expression and solubility of hAPRIL and gelonin. All the proteins, expressed as soluble fusions, were isolated from the protein debirs by the method called inverse transition cycling (ITC); but the efficiency of self-cleavage is low, and the target can not be separated form the fusion protein. The most widely viewed hypothesis for this result is that the seemingly soluble proteins are actually existing as ’soluble aggregates’, held in solution by interactions with the solubility partner, but not in their true, native, soluble form.The solubility and cleavage of the fusion protein using the ELP-SUMO co-expression system is easily accomplished. Using two fusion partner co-expresses with target protein maybe a new expression method in the future. The two proteins of hAPRIL and gelonin were also used in the analysis of ELP-SUMO co-expression system. The ELP-SUMO fusion protein were expressed soluble and with high purity by ITC. The fusion partner can be self-cleavage well in the cleavage buffer showed that the fusion protein folding well that expressed with ELP tag. Finally, we showed that cleavage and purification of the target protein using the ELP-SUMO expression system is easily accomplished by using a SUMO protease to cleave the fusion tag (ELP-SUMO) followed by purification of the target protein to homogeneity by Ni-Sepharose column. Mass spectrometry analysis and Cell proliferation assays showed that the recombinant hHAPO with high activity has the same MW with the natural protein.The ELP-SUMO expression system was also used to purify the LTVGel protein, a LTVSPWY peptide at the N terminal of gelonin. The MTT analysis showed a significant increase in cytotoxic SKOV3 cell by LTVGel compared with Gel. LTVGel kill SKOV3 cell by the mechanism:induction of apoptosis through caspase activation, and then cause cell death.