Production of a Post-Translationally Modified Protein

Producing recombinant proteins for structural biology studies can be challenging, particularly when high yields, exceptional purity, and correct post-translational modifications are all required. In this project, our client required 20 mg of a lipid-modified protein for NMR structural studies. The target protein was known to undergo post-translational modification through the addition of palmitic acid to a cysteine residue, a modification that can significantly influence protein structure, stability, and biological function.

Obtaining sufficient quantities of correctly modified protein proved particularly challenging, as the protein exhibited poor solubility during expression and was difficult to purify using conventional methods.

Our Solution

To maximize soluble protein expression while preserving the required post-translational modification, we developed a tailored expression strategy in E. coli. Rather than relying on standard expression protocols, we significantly reduced the expression temperature, carefully controlled expression levels through media optimization, and extended production over several days rather than the typical overnight timeframe.

These adjustments substantially improved protein quality and resulted in a two- to three-fold increase in soluble protein expression. While more than 95% of the protein remained insoluble, the increase in soluble material provided a viable starting point for downstream purification.

We then optimized both Ni-affinity chromatography and size exclusion chromatography conditions to address protein-specific purification challenges. Standard purification workflows proved insufficient; therefore, custom purification conditions were developed to improve recovery, purity, and overall yield.

By combining this optimized expression and purification strategy with scale-up to a 20-litre bacterial culture, we successfully produced 20 mg of protein at greater than 90% purity. Importantly, mass spectrometry analysis confirmed the presence of the covalently attached palmitic acid modification, demonstrating that the desired post-translational modification had been retained throughout production and purification.

The Impact

Despite initial concerns that production of sufficient quantities of correctly modified protein would not be feasible, our integrated protein expression, purification, and analytical approach delivered the required amount of material for structural studies.

The client received 20 mg of highly purified, post-translationally modified protein suitable for NMR analysis, enabling their structural biology programme to progress with confidence. This project highlights the importance of combining customised expression strategies, robust purification workflows, and detailed analytical characterization when tackling complex recombinant protein production challenges.