| Objective:To analyse the characteristics of inverse transition cycling (ITC) of Elastin-like protein (ELP) and the application of ELP-tag in protein expression and purification.On the one hand, observe the fusion of ELP-In.On the other hand, research the factors which affect the Tt of ELP-In. In order to use ELP-tag in separation and purification of more other proteins.Methods:1. ELP and the primer, the taget gene In and the primers were obtained by Biological Inc and amplified by PCR using the obtained fragment as the template.2. Then cloned the ELP and ELP-In into the pGEM-T cloning vector.Using DNA recombinant techniques, ELP/pGEM-T easy and ELP-In/pGEM-T easy were transformed into competence E.coli DH5a. Spread plate cultivation and select single clone into LB liquid medium, after that sent it to asequence.3. Sequences in the constructed cloning vector were correct, then extracted the plasmid and were identified by restriction enzyme digestion.The product was recovery by gel extraction and inserted them into expressive vector pET28b, and transformed into E.coli DH5a for expanding culture respectively. Extracted the plasmid from the positive clones and were identified restriction enzyme digestion.Then transformed the plasmid into E.coli competence E.coli BL21(DE3), and spread plate cultivation on LK solid medium. Selected the positive clones and induced to express target protein by IPTG.4. We can observe whether the ELP/pET28b and ELP-In/pET28b gene have expressed by the Western Blot and used the method we also see the style of the expression.5. Quantify the ELP-In/pET-28b fusion protein by BCA method. Dilute the protein to1.0μM/μ, remove the liquid600fi to1.5ml EP and keep warm at the temperature of5℃,10℃,15℃,20℃,25℃,30℃,35℃and40℃respectively for about15min. Remove400μl into quartz cuvette and tested the OD350value by spectrophotometer. The reversible phase transition temperature is the temperature which corresponds to half of the Maximum absorbance.6. The plasmid ELP/pET28b and ELP-In/pET28b were successfully expressing ELP and ELP-In in the E.coli BL21(DE3). Observed the factors which affect the Tt and determines the appropriate condition, then purify the protein by ITC. Finally the protein were purified by Ni-NTA agarose affinity chromatography for further.Results1. The template and related gene were synthesed, and target genes were amplified also.2. Constructed the recombinant plasmid of ELP/pGEM-T easy, ELP-In/pGEM-T easy were successfully constructed and the gene sequence was completely correct by PCR.3. Extracted the recombinant plasmid and digested with restriction enzyme, then ligated the expression vector and transformed into E.coli DH5a.Using agarose gel electrophoresisand visuslized, the correct plasmid were transformed into E.coli BL21(DE3).It shows that the recombinant expression vectors were constructed successfully by PCR technology. 4. Induced to express by IPTG and indicated that ELP/pET28b, ELP-In/pET28b were expressed in the protein level in soluble form.5. The Tt of ELP can not be detected by spectrophotometer however the Tt of ELP-In is about30℃6. It determined that requirements of ITC are the concentration of (NH4)2SO4is0.4M, pH is6.0and Tt is25℃.In addition, ELP-In/pET28b were purified preliminary and purified by Ni-NTA agarose affinity chromatography more.ConclusionsSucceed to synthesize the fusion protein and induce the ELP/pET28b and ELP-In/pET28b soluble expression.The inverse phase transition of ELP/pET28b could not be detected, it matched the the results of others’ reports.But the ITC of target protein ELP-In/pET28b which fusions to the short ELPs was detected.Therefore, we design the ELP with very small molecular weight as the tag for the first step of big fusion protein purification, and lay the foundation for further purification of various protein. |
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