RELATIONSHIP BETWEEN THERMAL-STABILITY, DEGRADATION RATE AND EXPRESSION YIELD OF BARNASE VARIANTS IN THE PERIPLASM OF ESCHERICHIA-COLI

An advantage of exporting a recombinant protein to the periplasm of Es cherichia coil is decreased proteolysis in the periplasm compared with that in the cytoplasm. However, protein degradation In the periplasm also occurs. It has been widely accepted that the thermodynamic stabil ity of a protein is an important factor for protein degradation in the cytoplasm of E.coli. To investigate the effect of the thermodynamic s tability of an exported protein on the extent of proteolysis in the pe riplasm, barnase (an extracellular ribonuclease from Bacillus amyloliq uefaciens) fused to alkaline phosphatase leader peptide was used as a model protein. A set of singly or doubly mutated barnase variants were constructed for export to the E.coli periplasm. It was found that the half-life of the barnase variants in vivo increased with their thermo dynamic stability in vitro. A dominant factor for the final yield of e xported barnase was not exportability but the turnover rate of the bar nase variant. The yield of a stabilized mutant was up to 50% higher th an that of the wild type, This suggests that exporting a protein to th e periplasm and using protein engineering to enhance the stability can be combined as a strategy to optimize the production of recombinant p roteins.