Study Of Protease Triggered Conditional Protein Trans-Splicing

Intein is a protein fragment with self-splicing activity.It can spontaneously cut itself from a precursor protein and join two neighboring exteins to form a complete protein,which is called protein splicing.Among them,split intein mediated protein trans-splicing has a wide application prospect in the fields of molecular biology and protein engineering.It can be used to produce proteins with different biological functions,and can also be used to study protein structure,function and regulatory mechanism.The development of protein trans-splicing technology has also opened up a new way for conditional protein splicing(CPS),which can regulate protein function to adapt to environmental changes.CPS refers to the use of inteins as molecular switches or tools to regulate protein function,expression,or localization.The intein is engineered into molecular switches that respond to various stimulatory conditions,such as metal ions,temperature,assisted dimerization,blocking active groups,and photoactivation,through engineering techniques,through engineering techniques.However,the existing methods of CPS have some limitations.For example,the disadvantages of using photoactivation are that the harmful nature of ultraviolet rays can damage the normal function of cells,the blocking of active groups requires chemical synthesis of inteins,which is cumbersome to design,and the use of metal ions and temperature regulation has not been as widely used as expected.A new method of CPS was established by using protease to induce splicing of atypical split inteins,three atypical S11 split inteins,Ter Dna E3,Ssp Dna X,Ssp Gyr B,were chosen.By using a small ubiquitin-like modifier(SUMO)tag,which was fused to the N-terminus of the C-terminus of the S11 intein,it affected the interaction of two intein segmengts and inhibited the trans-splicing.It was found that the splicing product could be detected within 15 min by the addition of SUMO protease,and the splicing efficiency was ～ 4-fold higher than that of the control group without SUMO protease overnight.Of the three inteins described above,the SUMO protease is the best regulator of Ter Dna E3 mediated protein trans-splicing reactions.We next replaced the exteins at both ends of Ter Dna E3 and found that the use of SUMO protease was also able to trigger CPS when splicing exteins of different sizes.We also compared the difference between S0 and S11 Ter Dna E3,and found that the splicing efficiency of S0 intein was only increased by 1 fold with the addition of SUMO protease,indicating that the regulation effect of this method on S11 intein was better.Finally,we replaced the SUMO tag with a larger maltose-binding protein(MBP),which can be excised by thrombin protease,the results showed that the splicing efficiency after adding the protease was about 4 to 5-fold higher than that of the control group without the protease.Compared with SUMO tag,MBP tag had a better regulation effect on protein splicing.It is speculated that proteins with large molecular weight can produce greater steric hindrance and have a stronger inhibitory effect on protein trans-splicing.In summary,unlike previous studies on CPS,most of which focus on typical split intein,this study also achieved CPS using atypical split intein and protease,and the method is simple and feasible.Our study provides a new approach for the regulation of protein trans-splicing and a favorable tool for the control of protein structure and function in vitro.