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Figure 2 | BMC Structural Biology

Figure 2

From: Low-resolution structural studies of human Stanniocalcin-1

Figure 2

Large scale STC1 expression in E. coli and in insect cells and its purification. (A) Tested STC constructs (from top to bottom): an amino-6 × His tagged STC1 without the N-terminal portion which includes the signal peptide (HT-STC1ΔNterm), an amino-GST tagged C-terminal fragment of STC1 (GST-C STC1) and a full length carboxy-6 × His tagged STC1 (STC1-HT). At the right side of each construct is shown the amino acid residues from native STC1 present on that construct. (B) Expression test of HT-STC1ΔNterm. Coomassie-blue stained SDS-PAGE of soluble (S) and insoluble (I) fractions expressed in BL21DE3 non-induced (NI) or induced for indicated periods with 0.5 mM IPTG in LB at 37°C. (C) GST-C STC1 purification by affinity chromatography using glutathione sepharose beads. Coomassie-blue stained SDS-PAGE of insoluble (I), soluble (S), flow-through (FT), wash (W) and elution (E1-E3) fractions. (D) Western blot anti-GST of E2 fraction of purification shown in C. Black arrow heads at right indicate expected recombinant protein size and red arrow head indicates un-fused GST protein. (E) Expression and purification of STC1-HT from insect cells (using the baculo virus system): Coomassi-blue stained SDS-PAGE of peak-fractions after cation exchange, metal affinity and Size Exclusion chromatography. Arrow head indicates expected size of recombinant expressed protein. Invitrogen Bench Marker protein ladder (M).

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