Comprehensive bioinformatics analysis of cell‐free protein synthesis: identification of multiple protein properties that correlate with successful expression

Abstract

High-throughput cell-free protein synthesis is being used increasingly in structural/functional genomics projects. However, the factors determining expression success are poorly understood. Here, we evaluated the expression of 3066 human proteins and their domains in a bacterial cell-free system and analyzed the correlation of protein expression with 39 physicochemical and structural properties of proteins. As a result of the bioinformatics analysis performed, we determined the 18 most influential features that affect protein amenability to cell-free expression. They include protein length; hydrophobicity; pI; content of charged, nonpolar, and aromatic residues;, cysteine content; solvent accessibility; presence of coiled coil; content of intrinsically disordered and structured (alpha-helix and beta-sheet) sequence; number of disulfide bonds and functional domains; presence of transmembrane regions; PEST motifs; and signaling sequences. This study represents the first comprehensive bioinformatics analysis of heterologous protein synthesis in a cell-free system. The rules and correlations revealed here provide a plethora of important insights into rationalization of cell-free protein production and can be of practical use for protein engineering with the aim of increasing expression success.-Kurotani, A., Takagi, T., Toyama, M., Shirouzu, M., Yokoyama, S., Fukami, Y., Tokmakov, A. A. Comprehensive bioinformatics analysis of cell-free protein synthesis: identification of multiple protein properties that correlate with successful expression.

Multiple Post-translational Modifications Affect Heterologous Protein Synthesis

“…Overall estimation of protein expression showed that the proteins of group A represented 30.9% (460); the proteins of group C represented 52.6% (782), and the proteins of group N represented 16.5% (246) of all proteins in the dataset. Similar rates of soluble expression have been reported previously for human proteins expressed in E. coli (37) and in cell-free bacterial extracts (18).…”

“…In accordance with our previous results (18), the predicted presence of disulfide bonds in proteins was found to be negatively correlated with soluble protein expression and positively correlated with the insoluble expression (Fig. 2C, curves A and C).…”

“…Protein Expression and Its Evaluation-Cell-free expression of human proteins and their domains was carried out in the E. coli S30 extracts as described previously (18). The main steps of the heterologous protein production are shown schematically in Fig.…”

“…The highest GRAVY value in the C-ranked targets (data not shown) suggests that this parameter is primarily related to protein solubility. Previously, high surface hydrophobicity has been shown to increase protein aggregation due to intermolecular hydrophobic interactions and to worsen soluble protein expression yield in bacterial expression systems (16,18,45,46). Therefore, the amino acid sequences with the predicted lipid modification sites constitute a subset of very hydrophobic proteins, which have an intrinsically low potential for soluble expression.…”

“…Computational approaches to predict protein propensity for expression and solubility have been developed (13,16,17). Most recently, a number of statistically significant correlations between the yield of heterologous cell-free protein synthesis and multiple calculated and predicted parameters of amino acid sequences have been reported (18).…”