Pcep4 vector

His tag-modified recombinant fibromodulin (human, amino acids 19–376) and its fragments (FMOD90, amino acids 31–376; FMODNT, amino acids 19–95) were expressed in 293T cells using the pCEP4 vector (Invitrogen). Proteins were purified on Ni-NTA-Sepharose (Qiagen) and further on Superose 6 (GE Healthcare) in PBS. Bacterial His-tagged FMOD was expressed in BL21 Escherichia coli using the pET27(b) vector (Novagen) and purified as follows. Cells were lysed in 8 m urea in 100 mm NaH₂PO₄ and 100 mm Tris (pH 8.0) (lysis buffer). The lysate was cleared by sonication and centrifugation, and the supernatant was incubated with Ni-NTA-Sepharose. The Sepharose was then washed in lysis buffer (pH 6.3), and fibromodulin was eluted in lysis buffer (pH 8.0) containing 250 mm imidazole. Fibromodulin was then dialyzed against PBS with 10% glycerol and gradually decreasing urea concentrations (from 8 to 0 m). In the final step, the protein was purified on size exclusion chromatography using PBS. All steps were performed at 4 °C. All protein identities were confirmed by mass spectrometry.

Affinities and selectivity of AKDS001 for human EP were evaluated using human embryonic kidney (HEK)-293 cells stably expressing human EP, as described in a previous report (Suzawa et al., 2000 (link)). In brief, EP cDNAs subcloned into a pCEP4 vector (Invitrogen, San Diego, CA, United States) were transfected into HEK cells. HEK cells expressing the cDNA together with the hygromycin resistance gene were selected and expanded into clonal cell lines. Membranes were prepared by centrifugation following lysis of the cells by nitrogen cavitation. Assays were performed in a final incubation volume of 0.2 ml in 10 mM MES/KOH containing 1 mM EDTA, 10 mM MgCl₂, and [3H] PGE2 (181 Ci/mmol) (1.5 nM for EP1, 3 nM for EP2, and 0.5 nM for EP3 and EP4). The reaction was initiated by the addition of membranes expressing human EP (30 μg for EP1, 20 μg for EP2, 2 μg for EP3, and 10 μg for EP4) and AKDS001. Non-specific binding was determined in the presence of non-radioactive PGE2 (10 μM for EP1, EP2, and EP4, and 1 μM for EP3). Incubations were conducted for 120 min at room temperature and the assays were terminated by filtration through a 96-well Unifilter GF/C. The filters were washed and dried, and the residual radioactivity was determined by liquid scintillation counting.

The bicistronic vector for VKORC1 and FIX coexpression was constructed by cloning FIX cDNA [GenBank: NM_000133] into a pCEP4 vector (Invitrogen, Karlsruhe, Germany) using 5′ Nhe I and 3′ Xho I restriction sites and inserting VKORC1 cDNA [GenBank: NM_024006] together with an IRES sequence (internal ribosome entry site) downstream from the FIX cDNA using 5′ Sfi I and 3′ Bgl II sites.
Site-directed mutagenesis was performed using the QuikChange mutagenesis kit (Stratagene, Amsterdam, NL) following the manufacturer’s instructions. Mutagenic primer pairs were about 30 base pairs in length and flanked the desired mutation position (primer sequences available on request).

cDNAs encoding either full-length mouse Gas6/Pros1 or Gas6/Pros1 lacking the Gla domain were sub-cloned into pCep4 vector (Invitrogen, Carlsbad, CA). PCR primers introduced a C-terminal His₆ tag for purification and the constructs were transfected into HEK293 cells. Cells were grown to confluency in DMEM supplemented with 10% FBS, 0.25 mg/ml G418, 100 µg/ml hygromycin, and Pen/Strep and subsequently switched to serum-free Pro CDMa medium (Lonza, Walkersville, MD) supplemented with 10 µM vitamin K2 (Sigma, St. Louis, MO) and Pen/Strep for 72 hr. Conditioned medium was passed through a 0.22 µm filter, affinity purified through a nickel-NTA resin, and further purified on an anion exchange column (Mono Q 5/5; GE Healthcare, Pittsburgh, PA) in 20 mM Tris–HCl, pH 8.0 using a sodium chloride gradient.

Fragments encoding CM, Rh, AGM, and Hu TRIM5αs C-terminally tagged with hemagglutinin (HA) were amplified by PCR using the oligonucleotide primers 5’-GGCAGCTGGCCACCATGGCTTCTGG-3’ and 5’- CCGCGGCCGCTCAAGCAGCATAATCAG-3’ from the cDNA clones described previously [10 (link),20 (link)]. These DNA fragments were cloned separately into the PvuII/NotI-sites of the pCEP4 vector (Invitrogen), which carries the hygromycin B resistance gene. GH123-Nh is a mutant of the GH123 proviral clone in which an NheI restriction enzyme cleavage site at position 6183 was blunted and re-ligated, introducing frame-shift mutations in the env gene. ASA-Nh is a mutant of GH123-Nh in which the CA-encoding gene has been mutated to encode a capsid protein with substitutions of alanine, serine, and alanine for the proline residues at positions 120, 160, and 179, respectively. The HIV-2 GH123 ASA CA-encoding fragment derived from ASA-Nh [19 (link)] was inserted into the PmlI/Bsu36I-sites of the green fluorescent protein (GFP)-encoding HIV-2 construct GH123-GFP, which was a pROD-env(-)-GFP derivative carrying the gene encoding GH123 CA [18 (link)].

A DNA fragment encoding the RBD(N501Y) was prepared by site-directed mutagenesis and cloned into the pCEP4 vector (Invitrogen). The expression and purification procedures were virtually identical to those used for the preparation of D27LEY-Fab.

The encoding genes for full-length human AGT and prorenin including signal peptide were amplified from I.M.A.G.E clones (Source BioScience, catalog no. 4213559 for human AGT and 5188566 for human prorenin) and inserted into the pCEP4 vector (Invitrogen). The coding sequence of spent AGT was inserted into a modified pCEP4 vector with the antitrypsin signal peptide at its N terminus for secretion. All of the constructs included a His₆ tag at the C terminus of the gene and were verified by DNA sequencing (Source BioScience) before being transfected transiently into HEK293 EBNA cells using polyethyleneimine (linear, molecular weight 25,000, Polysciences, Inc.). Recombinant proteins were purified from concentrated serum-free expression medium by nickel-affinity (HisTrap, GE Healthcare Life Sciences) and anion-exchange chromatography (Hitrap Q, GE Healthcare) consecutively.