Table 2. Average yields and purities of the studied NS4A(1–48) fusion proteins and of the resulting NS4A(1–48) target peptides obtained from 1 L of culture.

Fusion Tag	Yield
	NS4A(1–48) Fusion Protein						NS4A(1–48) Peptide
	after first purification step						after proteolytic cleavage and tag separation
	Wild Type			Mutant			Wild Type			Mutant
	amount (mg)	purity (%)	NS4A(1–48) content (mg)	amount (mg)	purity (%)	NS4A(1–48) content (mg)	amount (mg)	recovery (%)	purity (%)	amount (mg)	recovery (%)	purity (%)
GB1	15	90	6.1	20	90	8.1	3	50	<80	5	62	<80
GST-GB1	25	60	3.3	30	75	4	3	91	∼98	4	∼100	∼98
GST-GB1*	35	60	4.7	40	75	5.3	4	85	∼98	5	94	∼98

Due to the different sizes of the fusion tags (Ubi: 11.3 kDa, GST: 26.4 kDa, GB1: 7.6 kDa, GST-GB1: 34 kDa) the mass fractions of the target peptide differ significantly between the constructs (Ubi: 31.5%, GST: 16%, GB1: 40.6%, GST-GB1: 13.3%). Since Ubi and GST fusion constructs resulted in very low yields (>1 mg/l), data for these constructs have not been included. The values for GB1 and GST-GB1 have been used to calculate the theoretical NS4A(1–48) content after the first purification step for each of the constructs shown. Despite an unfavorable target peptide to fusion tag ratio highest recovery and purity values were obtained with the dual GST-GB1-NS4A(1–48) construct after proteolytic cleavage and tag removal. LB medium was used if not stated otherwise. An asterisk (*) indicates isotope ([¹³C, ¹⁵N] or [¹⁵N]) labeled minimal growth medium. Note that the overall peptide yields were higher in minimal medium compared to those in rich LB medium. Amount and purity of the fusion protein tagged peptides as well as the purity of the free peptides has been estimated from SDS PAGE analysis. Final free peptide yields were additionally calculated by measuring the concentration of the peptides at 280 nm using an extinction coefficient of 1490 M⁻¹ cm⁻¹ in water.