Enterokinase

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.

The Escherichia coli strains DH5α and BL21(DE3) pLysS, as well as expression vector pET-32a (+), were purchased from Novagen (Darmstadt, Hesse, Germany). Restriction enzyme Kpn I, EcoR I and Mlu I, T4 DNA ligase, protein markers, protein HisPur^TM Ni-NTA purification kit, Fetal Bovine serum (FBS), penicillin, streptomycin, and cRNA mMESSAGE mMACHINE In Vitro Transcription Kit were purchased from Thermo Scientific (Waltham, MA, USA). enterokinase was purchased from New England Biolabs, Inc. (Ipswich, MA, USA). Vydac C18 columns (5 μm, 4.6 mm × 250 mm, 10 μm, 22 mm × 250 mm) were purchased from Grace (Deerfield, IL, USA). Acetonitrile (ACN, gradient grade for HPLC), Acetylcholine chloride (ACh) and other chemical reagents were all of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).

E. coli BL21 (DE3) was purchased from Merck (Darmstadt, Germany). The pRSET-EmGFP vector was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Enterokinase and thrombin were purchased from New England Bio Labs (Ipswich, MA, USA) and GE Healthcare (Chicago, IL, USA), respectively. Cellohexaose was obtained from Toronto Research Chemicals (Toronto, Canada). Other laboratory reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), Wako Pure Chemical Ltd. (Osaka, Japan), nacalai tesque (Kyoto, Japan), and Cambridge Isotope Laboratories (Tewksbury, MA, USA).

Human histones H2A, H2B, and H4 were bacterially produced and purified by the method described previously (Kujirai et al., 2018 (link); Machida et al., 2018 (link)). The DNA fragment encoding human histone H3 was cloned into a modified pET-15b vector containing the His₆-tag sequence and enterokinase cleavage site just upstream of the H3 sequence. The recombinant H3 protein was produced in Escherichia coli cells and purified by Ni-NTA column chromatography. To remove the His₆-tag peptide, the resulting sample was treated with enterokinase (New England Biolabs) in buffer [20 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 2 mM CaCl₂]. Subsequent H3 purification steps were the same as those described previously (Kujirai et al., 2018 (link)).

ProTx-II (UniProt ID P83476) was biosynthesized using a procedure published by Blumenthal et al. [32 (link)]. In this procedure, the ProTx-II was expressed with an N-terminal maltose-binding protein (MBP) fusion partner and an enterokinase cleavage site between them. The fusion protein was expressed in BL21(DE3) cells, and cells were lysed by sonication. The fusion protein was purified by Ni²⁺ affinity chromatography. Fractions containing fusion protein as detected by SDS-PAGE analysis were pooled and used for refolding. Before oxidative refolding, the sample was incubated with 10 mM dithiothreitol (DTT) at 4 °C for 16 h. This was followed by dialysis against 2.5 mM reduced glutathione (GSH), 50 mM TRIS pH 8.0 and 150 mM NaCl. The dialyzed solution was then supplemented with oxidized glutathione (GSSG) by dropwise addition of concentrated GSSG solution to a final concentration of 0.5 mM and incubated at 4 °C for 72 h. The resulting solution was dialyzed against 50 mM NaHCO₃ and cleaved by the addition of 10 U enterokinase (New England Biolabs, Ipswich, MA, USA). After overnight cleavage at 25 °C, the ProTx-II was purified by reverse-phase HPLC using a C-18 Zorbax column (Agilent, Santa Clara, CA, USA). The fraction containing refolded ProTx-II (confirmed by mass spectrometry) was lyophilized, and the solid peptide was stored at −20 °C until needed.