Table 2.

Quantification of oxidized cysteine residues in purified sdAbs and corresponding melting temperatures

Protein	Strain	Medium	% oxidized cysteine residues	T m [°C]
7C12	SHuffle® T7 Express	EnPresso	96.1 ± 0.6	64.8 ± 0.8
	BL21 (DE3)	EnPresso	84.7 ± 1.1	61.0 ± 1.0
EG2	SHuffle® T7 Express	EnPresso	97.6 ± 0.5	60.5 ± 0.5
	BL21 (DE3)	EnPresso	81.2 ± 0.7	58.9 ± 0.7

Purified sdAbs from EnPresso cultures were denatured and incubated with (S_red) or without (S_ox) tris(2-carboxyethyl)phosphine. After removal of the reducing agent by acetone precipitation, proteins were incubated with Ellman’s reagent. The formation of 2-nitro-5-thiobenzoate was quantified in a spectrophotometer by measuring the absorbance at 412 nm (A_412nm), and the percentage of oxidized cysteine residues was calculated using the formula: 100% - 100 * A_412nm[S_ox]/A_412nm[S_red]. Thermal unfolding was assessed at pH 7.4 by measuring the absorbance at 280 nm as a function of temperature.