title: Expression of active monomeric and dimeric nuclease A from the gram-positive Streptococcus gordonii surface protein expression system.
abstract: 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 ( approximately 18 kDa), extracellular, monomeric enzyme from Staphylococcus aureus. A deletion of amino acids 114-119 causes monomeric NucA to form homodimers. The DNA sequence encoding either wild-type or deletion mutant NucA was cloned via homologous recombination into Streptococcus gordonii. S. gordonii strains expressing either anchored or secreted, monomeric or dimeric NucA were isolated and tested for enzymatic activity using a novel fluorescence enzyme assay. We show that active monomeric and dimeric NucA enzyme can be expressed either anchored on the cell surface or secreted into the culture medium. The activity of the dimer NucA was approximately 100-fold less than the monomer. Secreted and anchored, monomeric NucA migrated on SDS-polyacrylamide gels at approximately 18 or approximately 30 kDa, respectively. In addition, similar to S. aureus NucA, the S. gordonii recombinant NucA enzyme was dependent on CaCl(2) and was heat stable. In contrast, however, the recombinant NucA activity was maximal at pH 7.0-7.5 whereas 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 anchored forms using SPEX. This further demonstrates the utility of this gram-positive surface protein expression system as a potential commensal bacterial delivery system for active, therapeutic enzymes, biopharmaceuticals, or vaccines.
authors.affiliation: SIGA Research Laboratories, Suite 230, Corvallis, Oregon 97333, USA.
authors.forename: E K
authors.initials: EK
authors.lastname: Dutton
authors.forename: S A
authors.initials: SA
authors.lastname: Ottum
authors.forename: T C
authors.initials: TC
authors.lastname: Bolken
authors.forename: C A
authors.initials: CA
authors.lastname: Franke
authors.forename: D E
authors.initials: DE
authors.lastname: Hruby
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D015153:Blotting, Western; D002122:Calcium Chloride / Q000737:chemistry; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004795:Enzyme Stability; D005453:Fluorescence; D006361:Heating; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D008836:Micrococcal Nuclease / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013291:Streptococcus / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D004247:DNA; D008836:Micrococcal Nuclease; D002122:Calcium Chloride