Construction And Screening Of Novel S1 Split Inteins For Protein Site-Specific Labeling

Intein is a kind of internal protein sequences, which can precisely excise themselves and join the flanking sequences (the N- and C- exteins) through a peptide bond during its maturation process, called protein splicing. An intein can split into two pieces as N-fragment and C-fragment of intein (IN and Ic), and express by two different open reading frame. The unique advantage of split intein is that it can be segregate expression and specific recognize each other. Thus, intein has applied a new method for protein synthesis and modification, adopted as an important tool in several crucial fields of protein engineering.In the field of protein labeling, using novel split inteins to label target protein can achieve a more in-depth study on protein structure and function. Artificial constructed split intein for site-specific labeling is a common choice, but the limitations of this method are:(1) Splicing efficiency in heterogeneous host protein may be vastly reduced or even restrained, and require conservative nature exteins for protein splicing, both of which restrict the scope of intein's application; (2) When expressing large fragment of split intein fused with target protein, it would affect the solubility of target protein or other unpredictable impacts; (3) Synthetic peptide will increased the researcher's cost, and so on.In order to overcome these limitations, we constructed six novel S1 split intei: mutated Cys- residues in C-terminal fragment to Ser- residues; changed the two sides of the nature exteins to a linker (GG-SAG); tested the splicing activity of these split inteins both in vivo and in vitro; screened suitable split inteins for further application.Based on the existed inteins in our lab, we successfully constructed six Cys-free C-terminal fragment of S1 split inteins (named CF- intein), and tested in vivo splicing activity. After analyzed the test results, we screened out three CF-inteins(CFRBS1, CFTXS1 and CFSXS1) with high splicing activity to test their in vitro splicing activity. The results showed that CFRBS1 intein can have further in vitro trans-splicing study. By optimize conditions and kinetics analysis; we know that CFRBS1 intein represents stable splicing activity, high splicing activity and fast splicing rate. And we have labeled model protein to confirm that CFRBS1 intein can be used as a tool to help label target proteins.We hope that this CF-S1 split intein can be used in protein labeling; it can attach fluorophores or tags, meanwhile, can label proteins contain ample cysteine residues with precisely side specific; and no need to modify target proteins or any potential disulfide bonds formation. We get the CFRBS1 intein with linkers on both sides instead of nature exteins, by which we can improve its compatibility and benefit the practical application of protein engineering. The research is not only a supplement based on previous studies to optimize intein's compatibility in heterogeneous proteins, but also prepare to broaden its scope of application.