Inteins are protein splicing elements that mediate their excision from
precursor proteins and the joining of the flanking protein sequences
(exteins). In this study, protein splicing was controlled by splitting
precursor proteins within the Psp Pol-1 intein and expressing the res
ultant fragments in separate hosts, Reconstitution of an active intein
was achieved by in vitro assembly of precursor fragments. Both splici
ng and intein endonuclease activity were restored, Complementary fragm
ents from two of the three fragmentation positions tested were able to
splice in vitro, Fragments resulting in redundant overlaps of intein
sequences or containing affinity tags at the fragmentation sites were
able to splice, Fragment pairs resulting in a gap in the intein sequen
ce failed to splice or cleave, However, similar deletions in unfragmen
ted precursors also failed to splice or cleave, Single splice junction
cleavage was not observed with single fragments. lit vitro splicing o
f intein fragments under native conditions was achieved using mini ext
eins. Trans-splicing allows differential modification of defined regio
ns of a protein prior to extein ligation, generating partially labeled
proteins for NMR analysis or enabling the study of the effects of any
type of protein modification on a limited region of a protein.