EXPRESSION OF ACTIVE STREPTOLYSIN-O IN ESCHERICHIA-COLI AS A MALTOSE-BINDING-PROTEIN-STREPTOLYSIN-O FUSION PROTEIN - THE N-TERMINAL-70 AMINO-ACIDS ARE NOT REQUIRED FOR HEMOLYTIC-ACTIVITY

Streptolysin O (SLO) is the prototype of a family of cytolysins that c onsists of proteins which bind to cholesterol and form very large tran smembrane pens. Structure/function studies on the pore-forming cytolys in SLO have been complicated by the proteolytic inactivation of a subs tantial portion of recombinant SLO (rSLO) expressed in Escherichia col i. To overcome this problem, translational fusions between the E. coli maltose-binding protein (MBP) gene and SLO were constructed, using th e vectors pMAL-p2 and pMAL-c2. MBP-SLO fusion proteins were degraded i f secreted into the E. coli periplasm, but intact, soluble MBP-SLO fus ion proteins were produced at high levels in the cytoplasm. Active SLO with the expected N-terminus was separated from the MBP carrier by cl eavage with factor Xa. Cleavage with plasmin or trypsin also yielded a ctive, but slightly smaller forms of SLO. Surprisingly, uncleaved MBP- SLO was also hemolytic and cytotoxic to human fibroblasts and kerntino cytes. The MBP-SLO fusion protein displayed equal activities to SLO. S ucrose density gradient analyses showed that the fusion protein assemb led into polymers, and no difference in structure was discerned compar ed with polymers formed by native SLO. These studies show that the N-t erminal 70 residues of mature (secreted) SLO are not required for pore formation and that the N-terminus of the molecule is probably not ins erted into the bilayer, In addition, they provide a simple means for p roducing mutants for structure/function studies and highly purified SL O for use as a permeabilising reagent in cell biology research.