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 Escherichia coli 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 stability of a protein is an important factor for protein degradation in the cytoplasm of E.coli. To investigate the effect of the thermodynamic stability of an exported protein on the extent of proteolysis in the periplasm, barnase (an extracellular ribonuclease from Bacillus amyloliquefaciens) 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 thermodynamic stability in vitro. A dominant factor for the final yield of exported barnase was not exportability but the turnover rate of the barnase variant. The yield of a stabilized mutant was up to 50% higher than that of the wild type. This suggests that exporting a protein to the periplasm and using protein engineering to enhance the stability can be combined as a strategy to optimize the production of recombinant proteins.
Kwon
W S
WS
Department of Chemical and Biochemical Engineering, University of California, Irvine 92717, USA.
Da Silva
N A
NA
Kellis
J T
JT
D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006207:Half-Life; D006358:Hot Temperature; D009154:Mutation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000096:biosynthesis* / Q000235:genetics*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease