Multiple Post-translational Modifications Affect Heterologous Protein Synthesis

Post-translational modifications (PTMs) are required for proper folding of many proteins. The low capacity for PTMs hinders the production of heterologous proteins in the widely used prokaryotic systems of protein synthesis. Until now, a systematic and comprehensive study concerning the specific effects of individual PTMs on heterologous protein synthesis has not been presented. To address this issue, we expressed 1488 human proteins and their domains in a bacterial cell-free system, and we examined the correlation of the expression yields with the presence of multiple PTM sites bioinformatically predicted in these proteins. This approach revealed a number of previously unknown statistically significant correlations. Prediction of some PTMs, such as myristoylation, glycosylation, palmitoylation, and disulfide bond formation, was found to significantly worsen protein amenability to soluble expression. The presence of other PTMs, such as aspartyl hydroxylation, C-terminal amidation, and Tyr sulfation, did not correlate with the yield of heterologous protein expression. Surprisingly, the predicted presence of several PTMs, such as phosphorylation, ubiquitination, SUMOylation, and prenylation, was associated with the increased production of properly folded soluble proteins. The plausible rationales for the existence of the observed correlations are presented. Our findings suggest that identification of potential PTMs in polypeptide sequences can be of practical use for predicting expression success and optimizing heterologous protein synthesis. In sum, this study provides the most compelling evidence so far for the role of multiple PTMs in the stability and solubility of heterologously expressed recombinant proteins. Background: Post-translational modifications (PTMs) affect protein folding. Results: Statistically significant correlations are revealed between the yield of heterologous protein expression and the presence of multiple PTM sites bioinformatically predicted in the expressed sequences. Conclusion: Predicting potential PTMs in polypeptide sequences can help optimize heterologous protein synthesis. Significance: Correlations revealed provide insights into the role of specific PTMs in protein stability and solubility.