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Table 1 Analysis of free thiols content in purified proteins based on mass spectrometry and Ellman’s assay

From: Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media

Protein Number of Cys Expected mw (Da) Experimental mw (Da) Free thiols per molecule
GH1 4 23,210 23,211 0.09
IL-6 4 21,894 21,894 −0.01
ScFv IgA1 4 27,342 27,342 0.03
Avidin 2 15,426 15,426 N/D
  1. The molecular weight of the purified proteins was determined after treatment with N-ethylmaleimide, which reacts with free thiol groups and adds 125 Da. Expected molecular weight assumes all cysteines are present as disulfides. No N-ethylmaleimide adducts were observed implying all cysteines were in disulfide bonds. The free thiol content of the purified proteins was determined by Ellman’s assay under denaturing conditions
  2. N/D not determined