Expression of active monomeric and dimeric nuclease A from the gram-positive Streptococcus gordonii surface protein expression system

We used the surface protein expression (SPEX) system to express an anchored and a secreted form of staphylococcal nuclease A (NucA) from gram-positive bacteria. NucA is a small (similar to 18 kDa), extracellular, monomeric en zyme from Staphylococcus aureus, A deletion of amino acids 114-119 causes m onomeric NucA to form homodimers, The DNA sequence encoding either wild-typ e or deletion mutant NucA was cloned via homologous recombination into Stre ptococcus gordonii. S. gordonii strains expressing either anchored or secre ted, monomeric or dimeric NucA were isolated and tested for enzymatic activ ity using a novel fluorescence enzyme assay, We show that active monomeric and dimeric NucA enzyme can be expressed either anchored on the cell surfac e or secreted into the culture medium, The activity of the dimer NucA was s imilar to 100-fold less than the monomer, Secreted and anchored, monomeric NucA migrated on SDS-polyacrylamide gels at similar to 18 or similar to 30 kDa, respectively. In addition, similar to S. aureus NucA, the S, gordonii recombinant NucA enzyme was dependent on CaCl2 and was heat stable. In cont rast, however, the recombinant NucA activity was maximal at pH 7.0-7.5 wher eas S. aureus NucA was maximal at pH 9.0. These results show, for the first time, expression of active enzyme and polymeric protein in secreted and an chored forms using SPEX, This further demonstrates the utility of this gram -positive surface protein expression system as a potential commensal bacter ial delivery system for active, therapeutic enzymes, biopharmaceuticals, or vaccines. (C) 2000 Academic Press.