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

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

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

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
N/D not determined

ISSN: 1475-2859