Rational stabilization of the C-LytA affinity tag by protein engineering.
The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Owing to its affinity for choline and analogs, it is regularly used as an affinity tag for the purification of proteins in a single chromatographic step. In an attempt to build a robust variant against thermal denaturation, we have engineered several salt bridges on the protein surface. All the stabilizing mutations were pooled in a single variant, C-LytAm7, which contained seven changes: Y25K, F27K, M33E, N51K, S52K, T85K and T108K. The mutant displays a 7 degrees C thermal stabilization compared with the wild-type form, together with a complete reversibility upon heating and a higher kinetic stability. Moreover, the accumulation of intermediates in the unfolding of C-LytA is virtually abolished for C-LytAm7. The differences in stability become more evident when the proteins are bound to a DEAE-cellulose affinity column, as most of wild-type C-LytA is denatured at approximately 65 degrees C, whereas C-LytAm7 may stand temperatures up to 90 degrees C. Finally, the change in the isoelectric point of C-LytAm7 enhances its solubility at acidic pHs. Therefore, C-LytAm7 behaves as an improved affinity tag and supports the engineering of surface salt bridges as an effective approach for protein stabilization.
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Avda Universidad s/n, Elche 03202, Spain.
Víctor M
VM
Hernández-Rocamora
Beatriz
B
Maestro
Almudena
A
Mollá-Morales
Jesús M
JM
Sanz
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001709:Biotechnology; D002794:Choline / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D055550:Protein Stability; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013696:Temperature; D013816:Thermodynamics
D055767:Immobilized Proteins; D050505:Mutant Proteins; D000581:Amidohydrolases; D009238:N-Acetylmuramoyl-L-alanine Amidase; C019665:amidase; D002794:Choline