Protein Engineering, Vol 7, 1495-1500, Copyright © 1994 by Oxford University Press

A single amino acid substitution can restore the solubility of aggregated colicin A
mutants in Escherichia coli

J Izard, MW Parker, M Chartier, D Duche and D Baty
Laboratoire d'Ingenierie et Dynamique des Systemes membranaires du CNRS,
UPR 9027, France.

Mutants of colicin A have been prepared in which the three tryptophan residues
(Trp86, Trp130 and Trp140), localized in the C-terminal domain of the soluble
wild-type protein, have been substituted by phenylalanine. The Trp140Phe single
mutation had the effect of decreasing the percentage of protein that is expressed as
insoluble aggregates. The created hydrophobic cavity decreased the stability of the
protein during its folding, resulting in partial aggregation in the cytoplasm of the
Escherichia coli-producing cells. Aggregation was increased when Trp140 was
substituted by Lys, Leu or Cys, or if the Trp140 mutation was combined with the
Trp86Phe and/or Trp130Phe mutations. A single mutation, Lys113Phe, however,
was able to restore the solubility of the aggregated mutants in vivo. Detailed analysis of
the 3-D structure of the C-terminal domain of colicin A suggests that filling of the
hydrophobic cavity is responsible for this effect.

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