Screening for stable mutants with amino acid pairs substituted for the disulfide bond between residues 14 and 38 of bovine pancreatic trypsin inhibitor (BPTI).
We have developed a screening method 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 for the destabilizing effect of removal of the S-S bond, these mutants have midpoint temperatures of thermal unfolding that are 12-17 degrees C higher than that of the C14A/C38A mutant. This fact indicates that these mutations are better substitutions for the S-S bond than C14A/C38A. The C14G mutants inhibited trypsin more strongly at 37 degrees 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, which suggests different stabilizing mechanisms in these two groups. Because 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.
Hagihara
Yoshihisa
Y
Special Division for Human Life Technology, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan. hagihara-kappael@aist.go.jp
Shiraki
Kentaro
K
Nakamura
Tsutomu
T
Uegaki
Koichi
K
Takagi
Masahiro
M
Imanaka
Tadayuki
T
Yumoto
Noboru
N
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D007611:Aprotinin / Q000737:chemistry* / Q000235:genetics*; D002417:Cattle; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry; D019277:Entropy; D015723:Gene Library; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D013816:Thermodynamics
D004220:Disulfides; D007611:Aprotinin