Large-scale experimental studies show unexpected amino acid effects on protein expression and solubility in vivo in E. coli

Overexpression of recombinant proteins is a central method in contemporary biochemistry, structural biology, and biotechnology. Unfortunately, it is often a significant experimental challenge [1,2]. Many recombinant proteins express at low levels or not at all, and those proteins which express at high levels but in an insoluble form cannot be used without applying technically challenging refolding procedures [3]. Despite significant experimental [1,2,4-7] and computational [8-12] progress, the physical mechanisms underlying this variability remain poorly understood. The goal of the work reported in this paper is to increase understanding of the sequence parameters and physicochemical mechanisms influencing protein overexpression and solubility in vivo.Most existing experimental techniques to improve yield of soluble protein focus on optimization of factors extrinsic to the target protein itself. Experimental techniques for increasing expression have been reviewed elsewhere [1,2]; they include co-expression of fusion partners (including MBP [13] and smt [14] ), codon enhancement [15], or optimization [16,17] (including removal of 5' RNA secondary structure [18]), and the use of protease-deficient strains [19]. Techniques that have been developed specifically to improve solubility of recombinant proteins include chaperone co-expression [3], fusion to solubility-enhancing tags or protein domains [13,4], heat shock [20] or expression at lower temperature [1], expression in a different growth medium [1], reduction of protein expression level (e.g., by using less inducer or a weaker promoter [21]), directed evolution [22,23], and rational mutagenesis [24]. Of these, only the last relies on understanding the properties of the protein itself, rather than on modifying an external factor. Intrinsic biophysical features influencing protein expression and solubility in vivo have received comparatively little systematic study, at least in part because of the complexity of the ph