| Purification of recombinant proteins is a major task and challenge in biotechnology and medicine. Affinity tags are the most popular and highly efficient tools for purifying recombinant proteins, but these chromatography methods require expensive resins and are difficult to scale-up. In addition, the affinity tags have usually to be removed from the final protein products. This is achieved usually by inclusion of an endopeptidase cleavage site at the junction between the affinity tag and the target protein followed by digestion with the enzyme. However, such endopeptidase has to been removed from the final product, which makes the protein purification more complicated and expensive. Therefore, simple and economic purification methods are needed for recombinant protein prepartion, for animal use in particular.Elastin-like polypeptides (ELPs) and their fusion proteins undergo an inverse phase transition from soluble forms into aggregates as the temperature or the salt concentration increases, which allows easy and rapid purification of the fusion proteins by centrifugation or filtration. Self-cleaving modules such as inteins undergo temperature-, pH-and/or salt ion-induced self cleavage, and have been used for release of target proteins from their fusions. In this study, we successfully expressed and purified recombinant human β defensin (HBD3) and African swine fever virus (ASFV) K205R using ELP-Intein-based protein expression and purification system. Then, we established a novel recombinant protein expression and purification system by combining ELP with the self-processing module (SPM) of of Neisseria meningitides FrpC protein. Under the optimized conditions, three representative proteins, namely green fluorescent protein (GFP), Fc portion of porcine IgG (pFc) and HBD3were successfully expressed and purfied by using the system.ELP and△I-CM intein-mediated expression and purification of recombinant human P defensin3:The coding sequence for the mature peptide of HBD3was codon-optimized for E. coli and inserted into an ELP-△I-CM intein fusion expression vector pET-EI. The construct was transformed into BLR E. coli and expression of EI-HBD3fusion protein was induced at20℃ with IPTG. The EI-HBD3fusion protein was purified from the cell extract by ELP-mediated inverse transition cycling (ITC) and the recombinant HBD3was released from its fusion by intein-mediated self-cleavage. The antibiotical activities of the recombinant HBD3were determined using agar diffusion and minimal inhibitory concentration determination methods. The results showed that the EI-HBD3fusion protein was expressed in a soluble form. After first round of ITC, the purity of EI-HBD3fusion protein was up to85.7%, which was increased to93.1%after an additional round of ITC. After cleavage at28℃for12h, the intein-mediated self-cleavage rate was up to90.3%. The recovery rate for recombinant HBD3was up to74.8%with a purity of92.6%and yield of0.98mg/L. The endotoxin level in the purified recombinant HBD3was below0.03EU/mg. The recombinant HBD3had potent antibacterial activities against Gram’s positive Staphylococcus areus and Gram’s negative E. coli. These experimental data suggest that the ELP-and intein-based expression system could be used for large-scale preparation of recombinant HBD3.ELP and Mxe intein-mediated expression and purification of African swine fever virus recombinant protein K205R: The ELP sequence was excised from pET-EI/GFP vector inserted into the EcoRV/SacI sites of the pET-30a vector. By using pTWIN1palsmid as the template, Mxe intein (MI) sequence was amplified by PCR and N-terminally fused to the ELP sequence, resulting in MI-ELP fusion expression vector pET-MIE. African swine fever (ASFV) K205R gene was amplified by PCR and inserted into pET-MIE vector. After transformation into BLR E. coli, the conditions for IPTG-induced fusion protein expression, reverse transition cycling and intein-mediated self-cleavage were optimized. The results showed that the K205R-MIE fusion protein was expressed in a soluble form at20℃. After one cycle of reverse transition cycle at26℃, the recovery rate of the fusion protein was73.2%. After incubation at26℃for4h,91.4%of the fusion protein was self-cleaved and51.3%K205R protein was recovered with a purity of92.2%. Western blotting showed that the purified K205R protein was recognizable by anti-ASFV serum. These results showed that Mxe intein-ELP-based recombinant protein expression and purification system is useful for large-scale prepartion of recombinant proteins and the K205R protein prepared was usable for serodiagnosis of ASFV.Establishment of recombinant protein purification system based on ELP and SPM: The coding sequence for SPM of Neisseria meningitides FrpC protein was codon-adapted to E. coli using a JAVA Codon Adaptation Tool. The chemically synthesized sequence, with NdeI, EcoRI and SalI sites introduced at the5’-end and a VspI site at the3’-end, was N-terminally fused to the ELP coding sequence in pET-30a (+) vector, resulting in an ELP and SPM fusion expression vector pSPM-ELP. The GFP sequence was amplified by PCR and was inserted into the pSPM-ELP vector. The resultant recombinant vector pGFP-SPM-ELP was transformed into BLR E. coli and induced for expression at20℃with0.5mM IPTG. The results showed that the GFP-SPM-ELP fusion protein was expressed in a soluble form. The transition temperature (Tt) of the fusion protein GFP-SPM-ELP was determined to be28℃. After first round of ITC, the purity of GFP-SPM-ELP fusion protein was75.1%, which was increased to89.2%after an additional round of ITC. The optimal conditions for SPM-mediated cleavage were determined to be24℃for12h with5mM Ca2+and10mM DTT. After an additional round of ITC, the purified recombinant GFP exhibited bright green fluorescence when exposed to UV light. The endotoxin level in the purified recombinant GFP was below0.03EU/mg. The yield of the recombinant GFP was36mg/L with a recovery rate of90%and a purity of100%. These results showed that the SPM-ELP-based fusion system could be used for recombinant protein expression and purification.ELP and SPM-mediated expression and purification of pFc: The coding sequence for the Fc portion of porcine IgGl (pFc) was amplified from pShuttle-Fc vector with an EcoRI site introduced at the5’-end and a SalI site at the3’-end. After subcloning into the pSPM-ELP vector, the resultant vector pFc-SPM-ELP was transformed into BLR E. coli and induced for expression at20℃for24h with0.5mM IPTG The result of SDS-PAGE indicated that the Fc-SPM-ELP fusion protein was in a good solubility. The Tt of the Fc-SPM-ELP fusion protein was calculated to be26℃. After first round of ITC, the purity of GFP-SPM-ELP fusion protein was77.9%, which was increased to90.4%after an additional round of ITC. Under the optimized conditions for SPM-mediated self-cleavage and another round of ITC, the endotoxin level in the purified recombinant Fc was below0.03EU/mg. The yield of the recombinant Fc was11.5mg/L with a recovery rate of64%and a purity of90%. The purified Fc protein was recognizable by positive serum against porcine IgG. These results further confirmed that the SPM-ELP-based fusion system could be used for recombinant protein expression and purification.ELP and SPM-mediated expression and purification of HBD3:The coding sequence for the mature peptide of HBD3was codon-adapted to E. coli and was cloned into NdeI/SalI site of pSPM-ELP vector, resulting in the fusion expression vector pHBD3-SPM-ELP. The pHBD3-SPM-ELP construct was transformed into BLR E. coli and induced for the fusion protein expression at20℃for24h with0.5mM IPTG. The results showed that the HBD3-SPM-ELP fusion protein was expressed in a soluble form. The Tt of the fusion protein was28℃. After first round of ITC, the purity of HBD3-SPM-ELP fusion protein was36.0%, which was increased to42.6%after an additional round of ITC. Under the optimized conditions for SPM-mediated self-cleavage and another round of ITC, the endotoxin level in the purified recombinant HBD3was below0.03EU/mg. The yield of the recombinant Fc was1.1mg/L with a recovery rate of89%and a purity of94%. The purified recombinant HBD3exhibited antibacterial activities against all tested S. aureus and E. coli strains, with MIC values12.5μg/mL,100μg/mL,25μg/mL and50μg/mL against S. aureus (ATCC26003), S. aureus (ATCC6538), E. coli (K12-MG1655) and E. coli (ATCC44102), respectively. These results confirmed that the SPM-ELP-based fusion system could be used for the expression and purification of HBD3, and the purified HBD3maintained it natural antibacterial activity.In conclusion, we confirmed that the ELP-and△I-CM intein-mediated expression system could be used for the expression and purification of recombinant proteins like HBD3. We successfully constructed the fusion expression vector based on ELP and Mxe intein, and purified the ASFV K205R recombinant protein. We firstly constructed a novel recombinant protein purification system based on ELP with SPM. The conditions for fusion protein purification and self-cleaving were systematically optimized. Using this new system, we finally purified three target proteins with different molecular weight and structure, including GFP, Fc and HBD3. Our data indicated the applicability of the SPM-ELP fusion system for purification of recombinant proteins, for animal use in particular. |
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