Segmental expression and C-terminal labeling of protein ERp44 through protein trans-splicing

•Intein mediated protein trans-splicing reaction was carried out in recombinant ERp44 protein.•A complete ERp44 protein was produced through segmental expression followed by in vitro trans-splicing.•C-terminal fluorescence labeling of ERp44 was achieved through protein trans-splicing.•Atypical S1 split intein was demonstrated to be a useful alternative to S11 split intein for protein C-terminal labeling.

Endoplasmic reticulum resident protein 44 (ERp44) is a member of the protein disulfide isomerase family and functions in oxidative protein folding in the endoplasmic reticulum. A structurally flexible C-terminal tail (C-tail) of ERp44 plays critical roles in dynamically regulating ERp44’s function in protein folding quality control. The structure–function dynamics of ERp44’s C-tail may be studied further using fluorescence and other techniques, if methods are found to label the C-tail site-specifically with a fluorescent group or segmentally with other desired labels. Here we have developed such methods, employing split inteins capable of protein trans-splicing, and identifying atypical S1 split inteins able to function efficiently at a suitable split site in the ERp44 sequence. One method demonstrated segmental expression of ERp44 for segmental labeling of the C-tail, another method efficiently added a commercially available fluorescent group to the C-terminus of ERp44, and both methods may also be generally useful for studying other proteins.