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Papers In Press, published online ahead of print October 21, 2002
Special Division for Human Life Technology, National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, Osaka 563-8577
Corresponding Author: hagihara-kappael{at}aist.go.jp
We have developed a screening method with which to identify stable protein mutants from a large number of sequences, using a cellular quality control system. This method was used to screen amino-acid pairs substituted for the disulfide (S–S) bond between residues 14 and 38 of bovine pancreatic trypsin inhibitor. The mutants selected could be divided into two groups: one with mutation C14G and the other with mutation C38V. Although each mutation did not fully compensate the destabilizing effect of removing the S–S bond, these mutants have midpoint temperatures of thermal unfolding 12–17 °C higher than that of the C14A/C38A mutant, indicating that these mutations are better substitutions for the S–S bond than C14A/C38A. The C14G mutants inhibited trypsin more strongly at 37 °C than did the C14A/C38A mutant, although bulky amino acids at position 14 largely diminished the inhibitory activity of the C38V mutants. Thermodynamic analysis indicated that the enthalpy of unfolding of the C14G and C38V mutant groups differed considerably, suggesting different stabilizing mechanisms in these two groups. As renaturation of S–S bonds is often difficult in the large-scale production of proteins, this method should provide a useful tool with which to increase the production of recombinant proteins by eliminating S–S bonds with minimum concomitant stability loss.
J. Biol. Chem, 10.1074/jbc.M208893200
Submitted on August 30, 2002
Revised on October 18, 2002
Accepted on October 21, 2002
Screening for stable mutants with amino-acid pairs substituted for the disulfide bond between residues 14 and 38 of bovine pancreatic trypsin inhibitor (BPTI)
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