Team:St Andrews/Demonstrate



1. Our wild type human IgG4 CH3 domain can be expressed in an E. coli expression system in any suitable biological laboratory and can be easily purified via nickel affinity chromatography.

Figure 1. Purification of the human IgG wild type using nickel affinity chromatography and TEV cleavage. The expressed protein is highly soluble and was purified well via this method. The reverse nickel purified fraction only contains one band at the expected 12 kDa mass, so pure recombinant protein was obtained.

2. The mutant human IgG4 CH3 domains can be expressed in lower quantities and these proteins are still soluble despite the high number of introduced mutations.

Figure 2. Reverse nickel affinity purification for edit 2 recombinant protein expression. The elution fraction shows a band at around the expected 15 kDa molar mass, while the concentrated reverse nickel purified fraction shows an expected band at 12 kDa. Edit 2 expressed and was successfully purified by this method, although some contaminations are still present in the final fraction, around 40 kDa and 70 kDa.

Figure 3. Mass-spectra for edit 2 showing a clear signal at the expected molecular mass at 12258.8. corresponding to a fragment with a disulphide bond. No secondary signal is present that would indicate isopeptide bond formation.

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iGEM St Andrews 2019