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Production of a post-translationally modified protein2018-11-21T11:23:15+00:00

Project Description

Production of a post-translationally modified protein

Our client required 20mg of protein for NMR structural studies. The protein was known to be post-translationally modified with palmitic acid on a cysteine residue.

Our challenge was to obtain this amount of protein after expression in E. coli and demonstrate palmitoylation using mass spectrometry analysis. The protein was well expressed but very insoluble in initial experiments.

Our Solutions

Our solutions to this challenge were to reduce the temperature during expression far below what is normally used,  include media components to tightly regulate expression levels, and prolong expression experiments over several days rather than a few hours or overnight. Although >95% of the expressed protein was still insoluble this gave ~2-3-fold increase in soluble expression. Our next steps were to identify conditions for Ni chelate and size exclusion chromatography, which were different to standard methods, to improve purity at these stages. When all of these steps were combined together, along with a large 20 litre E.coli culture, we were able to generate 20mg of >90% pure protein. The protein was confirmed to be covalently lipid modified by mass spectrometry analysis.

The Impact

Our client had sufficient protein of the desired purity to conduct the NMR studies, when initial experiments suggested this would be impossible.

Case Study in Numbers

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Average number of years of research experience of staff
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Number of protein reagents generated in 2017
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Number of protein crystals sent for X-ray crystallography

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