Cleavage and purification of intein fusion proteins using the Streptococcus gordonii SPEX system

A Gram-positive bacterial expression vector using Streptococcus gordonii ha s been developed for expression and secretion, or surface anchoring of hete rologous proteins. This system, termed Surface Protein Expression system or SPEX, has been used to express a variety of surface anchored and secreted proteins. In this study, the Mycobacterium xenopi (Mxe) GyrA intein and chi tin binding domain from Bacillus circulans chitinase Al were used in conjun ction with SPEX to express a fusion protein to facilitate secretion, cleava ge, and purification. Streptococcus gordonii was transformed to express a s ecreted fusion protein consisting of a target protein with a C-terminal int ein and chitin-binding domain. Two target proteins, the C-repeat region of the Streptococcus pyogenes M6 protein (M6) and the nuclease A (NucA) enzyme of Staphylococcus aureus, were expressed and tested for intein cleavage. T he secreted fusion proteins were purified from culture medium by binding to chitin beads and subjected to reaction conditions to induce intein self-cl eavage to release the target protein. The M6 and NucA fusion proteins were shown to bind chitin beads and elute under cleavage reaction conditions. In addition, NucA demonstrated enzyme activity both before and after intein c leavage.