Objective   Efficient method to amidate the carbon-terminal of the antimicrobial peptide MME through self-splitting intein in recombination fusion proteins expressed in E. coli with DTT in the presences of MESNa and NH₄HCO₃ was explored.

  Method   Recombinant plasmid of MME was constructed to induce the intein-mediated expression in E. coli to successively obtain fusion proteins composed of histidine, sumo label, target polypeptide, and intein. The proteins were subsequently purified in a process using a nickel column and dialysis. With MESNa and NH₄HCO₃, an intein self-split procedure was completed by DTT to amidate the carbon-terminal on MME, then cut and purify the intestinal kinase. Both MME and the amidation were verified by the standard mass spectrometry and two-stage, tandem mass spectrometry.

  Result   The resultant gene fragment of the fusion protein was determined by PCR to be 837bp long as expected. In comparison to the theoretical value of 3 057.64, the relative molecular weight of MME obtained by the standard mass spectrometry was 3 057.7. The molecular weight of the amidated carbon-terminal fragments of MME was measured by the two-stage, tandem mass spectrometry to be 1,214.728, which was close to the known theoretical value of 1 214.739 with a matching rate of 45. It appeared that the carbon-terminal of MME had been effectively amidated, and the protein obtained was soluble.

  Conclusion   The recombinant fusion proteins were successfully expressed in E. coli to enable the intein self-splitting in the presences of MESNa and NH₄HCO₃ and amidate the carbon terminal of MME. Therefore, the current simple, one-step preparation could achieve accurate, duplicatable, and meaningful results. By combining the standard mass spectrometry and two-stage, tandem mass spectrometry, the amidated carbon-terminal polypeptides could satisfactorily be detected.