Abstract
Split inteins have been widely applied in protein engineering, and the Nostoc punctiforme (Npu) DnaE split intein has been reported to have outstanding activity. To expand usage of split intein, we firstly used a circular permutation predictor (CPred) to assist discovery of new artificial split inteins. Furthermore, we customized a method based on circular permutation (CP) concept to efficiently prepare naturally occurred split intein of NpuDnaE (NpuInt102) for NMR and X-Ray study. Crystal structure of NpuInt102 was solved and represented a structural state after protein trans-splicing. The structure has high similarity to one-fragment intein. However, few structural differences occurred in the opening split site and catalytic core. To evaluate the potential in applying split intein to membrane proteins, we tested protein trans-splicing under hydrophobic environments and found that intein could well perform protein trans-splicing, except in the presence of ionic solutes. The crystal structure of NpuInt102 indicated that 6 salt-bridges distribute on the assembling interface. The ionic solutes might impede the assembling between the two fragments by reducing electrostatic force. Based on the sensitivity to ions, we employed this feature to make intein to be a great fusion tag that the cleavage could easily remove from target proteins, regulated by salt concentration.