Directed Evolution Of Intein Based On Kanamycin Resistance System And Its Application

Inteins are internal protein sequences that self-excise after translation and splice the flanking sequences together.Natural and engineered inteins have been used in many practical applications.However,when placed in non-natural host proteins,inteins are usually inefficient or inactive.The requirements for the types of adjacent amino acids are strict for this,and the downstream +1amino acid is necessary for splicing reaction,usually a nucleophilic amino acid such as Cys,Ser,or Thr.However,inteins with Thr at the +1 position are relatively rare.When the +1 amino acid of a highly splicing-active intein is mutated to Thr,splicing activity is almost lost.The specific preference for the +1 amino acid position and its splicing activity in relation to the internal structure are not yet clear.Furthermore,when the downstream +2 and upstream-1 amino acids are prolines,most of the inteins have little splicing activity.In order to achieve efficient splicing,it is often necessary to retain 3-5 amino acid residues of natural extein,but these amino acids remain in the target protein and may interfere with host protein activity.Therefore,it is necessary to develop more general and highly splicing-active inteins.Based on the limited understanding of the structure of the inteins,a mutagenic gene library for intein was established,in which inteins splicing activity was associateed with kanamycin resistance.By simulating the process of natural evolution,high splicing activity inteins were obtained through screening with different concentrations of kanamycin.The main research content is as follows:1.Directed Evolution of inteins based on kanamycin resistance system.In this study,specific amino acid sequences were introduced into kanamycin resistant proteins in response to the restriction of specific amino acid residues during the use of the intein above.These amino acids provided a special extein environment for the intein.The kanamycin resistant system was used to screen inteins with high splicing activity under specific amino acid environments.First,four heterologous amino acid sequences(TCPPCP,PKSCDK,KSCDKT,KSCDKTH)were introduced into specific sites of kanamycin resistance protein as the exteins,and p KHKSCDKTH and p KH-PPCP plasmids constructed in our laboratory were selected by plate coating method as directed evolutionary vectors.Then,four inteins,including TE-3,GP41-1,TX and NE,were inserted into different insertion sites of exteins,and the inteins to be evolved were selected by Western blot.Finally,GP41-1 was selected to evolve the tolerance of exteins as proline in p KH-PPCP vector and the pre-threonine splicing activity of NE in p KH-KSCDKTH vector,namely,p KH2C-GP41-M and p KH7T-NE.Four and three rounds of directed evolution were conducted for these two inteins,and many mutants were successfully obtained,but the splicing activity was not significantly improved.In this study,the directed evolution based on kanamycin resistance system was preliminarily explored,which could be used as a vector for directed evolution,and the construction of mutant library by error-prone PCR was practiced,which laid a foundation for the subsequent evolution of high splicing activity inteins.2.Production of proteinase K via GP41-1 mediated protein trans-splicing.In addition,the application of the split GP41-1 in protein engineering was also investigated.Protease K is a powerful proteolytic enzyme,which is expressed as inclusion bodies in E.coli.The subsequent modification damages with the activity of protease K.In this study,GP41-1-S0 was used to break protease K from the intermediate GY/SSS sequence into two parts and express them as N-Protein and C-Protein in E.coli,respectively.The mixture is proportionally mixed to produce protease K by trans-splicing In vitro.In addition,solubilizing labels SUMO,GST and MBP were added to promote the soluble expression of fusion protein.Finally,two N-proteins with different fusion labels are expressed in soluble form,namely N-Protein(His-SUMO)and N-Protein(His-MBP),and C-Protein(His-MBP)with pro-solubility labels,respectively.Protease K was obtained by trans splicing GP41-1-S0.In summary,directed evolution based on the kanamycin resistance system was successfully used to construct p KH-KSCDKTH and p KH-PPCP as vectors for directed evolution.Ten clones were generated by introducing Ter Dna E-3,GP41-1,Ter Thy X,and Npu Dna E inteins into different insertion sites of the vectors.Western blot analysis was performed to select the inteins(p KH2C-GP41-1 and p KH7T-NE)for further evolution.Low,medium,and high mutation rate mutation libraries were constructed using error-prone PCR to explore the use of increased kanamycin concentration for the directed screening of highly active mutants.Additionally,a proteinase K production method regulated by GP41-1-mediated trans-splicing was developed in E.coli,which enriched the production pathways for proteinase K.