Amino acid substitutions affecting protein solubility: high level expression of Streptomyces clavuligerus isopenicillin N synthase in Escherichia coli

Modification of specific cultivation conditions, the choice of promoters, h ost strains and temperatures used for expression have often been exploited to optimize protein folding for soluble production. However, such overexpre ssion of foreign proteins, especially in Escherichia coli, often results in inclusion body formation. Besides, when a protein's primary sequence is al tered by substitutions at certain amino acid sites, the expressed protein m ay be rendered insoluble. At present, the mechanism by which such replaceme nts affect solubility is not entirely clear. In this review, it is observed that protein insolubility is not totally dependent on parameters such as h ydrophobicity, charge and identity of the amino acid substitutions. Neither is it plainly related to the biophysical properties of the mutated protein s, such as hydropathicity scores and pi values. However, a survey of report ed data on ten proteins suggests that increasing the hydrophilicity of solv ent-exposed residues could increase solubility and vice versa. In addition, results obtained from computational analysis and expression studies of iso penicillin N synthase (IPNS) mutants indicate an apparent causal relationsh ip between secondary structure predictions and expression of soluble protei ns. Hence, specific amino acid substitutions affecting secondary structure predictions and thereby protein folding, are expected to have a greater inf luence on protein solubility than a trivial assessment of other biophysical parameters. (C) 1999 Elsevier Science B.V. All rights reserved.