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

Abstract

Modification of specific cultivation conditions, the choice of promoters, host strains and temperatures used for expression have often been exploited to optimize protein folding for soluble production. However, such overexpression of foreign proteins, especially in Escherichia coli, often results in inclusion body formation. Besides, when a protein's primary sequence is altered by substitutions at certain amino acid sites, the expressed protein may be rendered insoluble. At present, the mechanism by which such replacements affect solubility is not entirely clear. In this review, it is observed that protein insolubility is not totally dependent on parameters such as hydrophobicity, charge and identity of the amino acid substitutions. Neither is it plainly related to the biophysical properties of the mutated proteins, such as hydropathicity scores and pI values. However, a survey of reported data on ten proteins suggests that increasing the hydrophilicity of solvent-exposed residues could increase solubility and vice versa. In addition, results obtained from computational analysis and expression studies of isopenicillin N synthase (IPNS) mutants indicate an apparent causal relationship between secondary structure predictions and expression of soluble proteins. Hence, specific amino acid substitutions affecting secondary structure predictions and thereby protein folding, are expected to have a greater influence on protein solubility than a trivial assessment of other biophysical parameters.