Sephacryl 100 hr hiprep 26 60 size exclusion column

Two different DNA fragments encoding for the EC1-EC2 portion of human E-cadherin, one including residues 1–213 and one lacking the first two N-terminal residues (Asp1-Trp2) (i.e. including residues 3–213) were cloned separately into two pET-3a expression vectors (Novagen) using the NdeI and BlpI restriction sites. In both cases, each fragment was fused at its N terminus to a 6His-tag, a spacer peptide (Ser-Ser-Gly-His-Ile), and the enterokinase recognition site (Asp-Asp-Asp-Asp-Lys). In both DNA constructs, the Cys9Ser mutation was also introduced. Overnight protein expression at room temperature in BL21(DE3)pLysS E.coli cells (Invitrogen) afforded large quantities of soluble protein for both constructs. Cells were lysed by sonication in TBS, pH 7.4, and 1 mM CaCl₂. The two cell lysates were purified first by Ni-affinity chromatography and then by gel filtration using a Sephacryl 100 HR HiPrep 26/60 size exclusion column (GE Healthcare). Both proteins were dialyzed in TBS buffer + 20 mM CaCl2, digested with enterokinase (New England Biolabs) at 25°C, and purified again by Ni-affinity chromatography to remove all traces of the cleaved 6His-tag and any residual uncleaved protein. The two flow-through fractions were then collected and further purified by size exclusion chromatography with TBS + 1 mM CaCl2.

A DNA fragment encoding for the EC1-EC2 portion of human E-cadherin (residues 1-213) was cloned into a pET-3a expression vector (Novagen). The protein was fused at its N-terminus to a 6His-tag, a spacer peptide (Ser-Ser-Gly-His-Ile), and the enterokinase recognition site (Asp-Asp-Asp-Asp-Lys). The protein was expressed overnight at room temperature in BL21 (DE3)pLysS E. coli cells (Invitrogen). Cells were sonicated in TBS pH 7.4 + 1 mM CaCl₂. The cell lysate was purified first by Ni-affinity chromatography and then by gel filtration using a Sephacryl 100 HR HiPrep 26/60 size exclusion column (GE Healthcare). The protein was then dialyzed in TBS pH 7.4 + 20 mM CaCl₂, digested with enterokinase (New England Biolabs) at 25°C, and further purified by Ni-affinity chromatography. The flow-through fraction was collected and purified by size exclusion chromatography in TBS pH 7.4 + 1 mM CaCl₂ for long-term storage.