Directed Evolution Of Mini Intein And Novel Split Intein

Intein is the protein equivalent of intron; it is an internal protein sequence in host protein. The flanking peptide sequence on it’s N and C-terminal are called extein. Inteins have been divided into3classes based on structure:canonical intein, mini-inteins, and split inteins. Intein able to catalyze self-splicing from the precursor protein and the exteins were ligated with a peptide bond. This post-translation process was termed protein splicing. Protein splicing provides brand new solution for protein modifications at protein level, thus this technology has a broad perspectives in protein engineeringBased on present research, most inteins didn’t show high-activities as it used to De in the natural host proteins which is the biggest obstacle of intein applications. Although the crystal structure of some inteins like Ssp DnaB, Ssp DnaE, Mxe GyrA were obtained, information about the relationship between intein structure and function was not enough for improving intein splicing activities though rational design.In this study, the intein splicing activity was combined with the kanamycin resistance though inserting the intein coding sequence into the kanamycin resistance protein gene. The kanamycin resistance-depended screening system is based on the strategy above. The coding sequences of Ter ThyX and Ter DnaE-3mini, SI and SI1inteins were inserted into different sites of kanamycin resistance gene. The result shows the TX mini-intein exhibited splicing activity in several site while TX Sl-intein exhibited activity in just one site. The splicing activity of TX S11-intein can not be observed at all sites. The TE3intein was much worse, TE3mini-intein shows activity in one site. Neither S1nor S11-intein shows any activity at all sites. Though error-prone PCR, a mutation library process over106colonies was set. After two rounds screening,9evolved inteins were obtained on the kanamycin plate. The western blot result shows evolution improves the splicing activity40%(from20%to60%). Combined with the structure information all the mutations were analyzed, E71D (Glu to Asp) was noticed located in the contact zone between intein and N-terminal extein, the side chain of D is shorter than one of E, this shorter side chain could provide larger space for the N-terminal extein which can makes the N-terminal extein more flexible. That’s the possible reason for the improved activity. The TX S1and S11-intein were also evolved though directed evolution, but unfortunately, no positive colony was obtained.This study successfully improved the splicing activity of intein, It will provide more abundant materials in the application of inteins. Combined with the structure information, the analysis of intein mutants can provide large amount of information about the relationship between the intein structure and function. With more and more study in the intein structure and function, intein will be used to more appreciable extent.