Cloning and cell culture
The gene for recombinant human renin protein (accession No. AAA60363, 406 amino acid residues) was cloned by PCR from a kidney cDNA library. The polymerase chain reaction conditions were optimized using Elongase polymerase mixture (Invitrogen, Carlsbad, CA) and the following primers: huRenin-fwd-5'-CGT CTA AGC TTG CCA CCA TGG ATG GAT GGA GAA GGA TCG-3' and huRenin-rev-5'-CGG TCA ATT CTA GAC TTC AGC GGG CCA AGG C-3'. The 5'-sense primer introduced a consensus Kozak translation initiation sequence [24] and a Hind III restriction site. The 3' antisense primer introduced an Xba I restriction site and a stop codon for native protein expression. The PCR product was digested to completion with Hind III and Xba I (NEB, Beverly, MA) and cloned into the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). The complete renin sequence was confirmed by DNA sequencing performed by BigDye terminator reaction chemistry for sequence analysis on the ABI Prism 377 (Kimmel Center Nucleic Acid Facility, Thomas Jefferson University, Philadelphia, PA). Human embryonic kidney cells, HEK 293 (ATCC, Manassas, VA) were cultured in Eagle's Minimum Essential Medium, supplemented with 10% fetal bovine serum (Hyclone, South Logan, UT), 2 mM L-glutamine, and 100 μg/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA). Transfection experiments with pcDNA3.1-huRenin were conducted using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer's protocols. Stable transfectants were obtained by selection in media supplemented with Geneticin (800 μg/ml), and single clones were obtained by limiting dilution. A single clone was scaled up and introduced into a hollow fiber bioreactor using a medium sized cartridge with a 5 kD molecular weight cut-off (Fibercell Systems Inc, Frederick, MD) to trap the secreted enzyme in the extra-fiber space. Once the cell line was established in the bioreactor it was adapted incrementally to a serum-free media for HEK-293, SFM4HEK293 (Hyclone, South Logan, UT) which would aid purification of the protein. Protein was harvested from the extra-fiber space daily and the cell line was maintained in the bioreactor for over 6 months with no observable decrease in protein production.
Protein purification
The clarified conditioned medium (typically 250 – 300 ml), was loaded by gravity at 4°C to a 30 ml Concanavalin A (Con A) column (Pharmacia/GE Healthcare, Piscataway, NJ), which had been equilibrated with Buffer A (25 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.1 mM CaCl2 and 0.1 mM MnCl2). The column was then connected to the FPLC system (GE Pharmacia/GE Healthcare, Piscataway, NJ) and washed with 5 volumes of Buffer A or until the baseline was stabilized. The prorenin protein was eluted in Buffer A supplemented with 150 mM D-methyl-mannopyranoside (Sigma-aldrich, St. Louis, MO) at slow flow rate (0.5 ml/min) in a "soak/elute" fashion. The propeptide was removed by trypsin digestion (1:100 by weight, trypsin:prorenin) at 4°C for 60 minutes. The reaction was quenched by the addition of trypsin-inhibitor (from soybean) beads (T0637, Sigma-aldrich, St. Louis, MO). The activated renin was dialyzed overnight at 4°C against Buffer B (10 mM sodium acetate, pH 5, and 10 mM NaCl). This solution was chromatographed on the 50 ml SP Sepharose Fast Flow column (Pharmacia/GE Healthcare, Piscataway, NJ) using a NaCl gradient (10 mM – 500 mM). Homogeneous renin was obtained after the gel filtration column Superdex 75 HR 10/30 (Pharmacia/GE Healthcare, Piscataway, NJ). The purified renin in a buffer containing 20 mM Tris-HCl, pH 7.0 was concentrated to 5 mg/ml, by ultrafiltration using an Amicon stirred cell (Millipore, MA) with a membrane of 10,000 MWCO (YM10), and stored at a -80°C freezer until future use. No deglycosylation was performed.
Protein samples were analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and by Edman's N-terminal sequencing (the Wistar Institute, University of Pennsylvania, Philadelphia, PA).
SDS-PAGE and protein assays
The protein samples were analyzed using precast NuPage Novex 4–12% Bis-Tris gels with MES running buffer (Invitrogen, MD). Proteins on the gel became visible after staining with 0.01% Coomassie Blue R in a solution containing 10% glacial acid and 20% methanol. Protein concentrations were measured using Bio-Rad Assay Kit (a Bradford-based dye binding method [25]), with bovine serum albumin (BSA) as standard.
Functional assay
The fluorescence resonance energy transfer (FRET) assay for renin activity was performed essentially as described in literature [11]. Recombinant human renin (0.3 nM) was incubated with 1 μM FRET substrate DABCYL-γAbu-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS (AnaSpec, San Jose, CA) in the 50 mM BES buffer, pH 7.0 containing 0.5 mg/ml BSA, 150 mM NaCl and 2% DMSO. Incubation was performed at room temperature in a final volume of 200 μl in white opaque 96-well optiplates (PerkinElmer, Wellesley, MA). Fluorescence (λex= 336 nm, λem= 495 nm) was measured repeatedly over extended period of time (1–2 hrs) on either Victor V or Fusion plate reader (PerkinElmer, Wellesley, MA), and the intensity of fluorescence was regressed against reaction time to derive velocities. The reaction rates were used for calculating percent inhibition values using uninhibited renin as a positive control and either no enzyme or renin inactivated with 2 μM SR 42128 (Isovaleryl-Phe-Nle-Sta-Ala-Sta) (Bachem, King of Prussia, PA) as a negative control. IC50 values were calculated by fitting the percent inhibition values vs. inhibitor concentration into a four-parametric model using XLFit software (IDBS, Guildford, UK).
Sample preparation and crystallization
The hanging-drop, vapor-diffusion method was used for renin crystallization. Renin was first mixed with 2 mM VTP24631 and incubated on ice for 30 minutes. For each hanging drop, 1 ul of the protein solution was combined with 0.6 ul of precipitant (0.1 M Tris-HCl, pH 7–8.2, 0.2 mM (NH4)2SO4 and 18–26% PEG 3550). Crystals were grown at 4°C for 5–7 days and streak seeding was required for crystals to reach their maximal sizes (200–500 μm).