Cleavage and purification of intein fusion proteins using the Streptococcus gordonii spex system.
A gram-positive bacterial expression vector using Streptococcus gordonii has been developed for expression and secretion, or surface anchoring of heterologous 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 chitin binding domain from Bacillus circulans chitinase Al were used in conjunction with SPEX to express a fusion protein to facilitate secretion, cleavage, and purification. Streptococcus gordonii was transformed to express a secreted fusion protein consisting of a target protein with a C-terminal intein 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. The secreted fusion proteins were purified from culture medium by binding to chitin beads and subjected to reaction conditions to induce intein self-cleavage 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 cleavage.
Myscofski
D M
DM
Center for Gene Research and Biotechnology, and Dept. of Microbiology, Oregon State University, Corvallis 97331-3804, USA.
Dutton
E K
EK
Cantor
E
E
Zhang
A
A
Hruby
D E
DE
D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D002686:Chitin / Q000378:metabolism; D027081:DNA Gyrase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019912:Mycobacterium xenopi / Q000201:enzymology / Q000235:genetics; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013291:Streptococcus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D002686:Chitin; D003851:Deoxyribonucleases; C029970:nuclease alpha; D027081:DNA Gyrase