GP002 was created by site-directed mutagenesis of the sequence of BPN' Y217L and expressed in B. subtilisn. Enzymes were purified from fermentation broths utilizing the BioCAD 700E system (Perceptive Biosystems, Foster City, CA) using POROS HS/M resin (Perceptive Biosystems, Foster City, CA) on porous polystyrene beads, and was stored as stock solutions at 20 mg/ml in 20 mM MES, pH 5.5 with 1 mM CaCL2 at -20°C.
Enzyme activity and stability measurements
Proteolytic activity was measured by hydrolysis of the substrate succinyl-AAPF-p-nitroanilide (suc-AAPF-pNA). Serial dilutions of the proteases were made in 100 mM Tris buffer, pH 8.6, containing 10 mM CaCl2. Enzyme activity was measured at 25°C in 100 mM Tris/HCl, pH 8.6 containing 1.6 mM suc-AAPF-pNA (Bachem Bioscience, Torrance, CA), 0.0005% Tween-80 and 1% DMSO by monitoring the absorbance change at 410 nm using a spectrophotometer. Each dilution of enzyme was tested in triplicate. Three dilutions were tested for each enzyme. Activity was then determined by linear regression, and is reported as OD units/mg enzyme.
Comparative thermostability was determined for BPN' Y217L and GP002 by incubating the enzymes (0.5 ppm) at 37°C in 50 mM TES buffer, pH 7.3 containing 0.0005% Tween-80. Aliquots of the enzyme solutions were removed over time, diluted and assayed for activity using 1 mM suc-AAPF-pNA substrate in 100 mM Tris pH 8.6 containing 0.0005% Tween-80. The half-lives of the enzymes were then calculated using a linear regression.
1. BPN' Y217L, B. lentus subtilisin, and GP002 immunizations
Guinea pig immunizations were outsourced to EL Labs in Santa Cruz, CA. Female Hartley outbred guinea pigs weighing 200–600 grams were used in all studies. There were three or four dosages of the appropriate enzyme and for each dosage there were four animals immunized. Each group of four animals was immunized every two weeks, immediately after the removal of a serum sample. There were a total of six immunizations. The first immunization was in Complete Freund's Adjuvant. Incomplete Freund's Adjuvant was used for all subsequent immunizations. Each immunogen was provided as a stock solution at 20 mg/ml in 20 mM sodium acetate, 1 mM calcium chloride, and was diluted to 1,5,10, or 20 μg/ml in cold DPBS to a total volume of 1 ml. This was emulsified at a 1:1 ratio with CFA or IFA. Each guinea pig was immunized subcutaneously with a total of 500 μl of emulsion.
2. Priming study with BPN' Y217L and GP002
Guinea pig immunizations were out-sourced to EL Labs in Santa Cruz, CA. The following immunization protocol was followed: four groups of four Hartley guinea pigs were distinguished as groups A through D. All animals received 5 μg of immunogen. The first immunization was performed in CFA, then IFA for all subsequent immunizations. Each animal received a total of 500 μl of emulsion. All immunizations were performed subcutaneously using the following schedule: First immunization – Group A received BPN' Y217L, groups B-D GP002. Week two – Groups A and B received BPN' Y217L, Groups B and C received GP002. Week 4 – Groups A through C received BPN' Y217L, Group D received GP002. Serum samples were drawn at week 6.
3. T-cell epitope mapping for GP002 and BPN' Y217L
Guinea pig immunizations were outsourced to Strategic BioSolutions, Ramona, CA. Groups of 10 female Hartley strain guinea pigs were immunized every two weeks with 5 ug per dose of BPN' Y217L or GP002 in Complete Freund's adjuvant, then Incomplete Freund's as described above. Two weeks following the third immunization, animals were sacrificed and their spleens and serum samples collected.
ELISA protocols (whole protein IgG)
Medium binding EIA plates (Costar, Cambridge, MA) were coated with 10 μg/ml of acid-denatured enzyme in DPBS. Enzyme was coated onto the plates at 4°C overnight. Plates were then washed three times with PBS/0.25% Tween-20 (Sigma, St. Louis, MO). Plates were blocked with 1% BSA (Rockland, Gilbertsville, PA)/PBS at room temperature for 30 minutes. Ten-fold serial dilution of sera in 1% BSA/PBS were performed. The plates were covered and incubated at room temperature for two hours. Secondary biotin anti-IgG antibody (guinea pig IgG Fc specific, Jackson ImmunoResearch, West Grove, PA) was diluted 1:10,000 in 1%BSA/PBS. 100 μl of this dilution were placed per well on the plates. The plates were again covered and incubated at room temperature for 1 hour. Plates were washed (as above). Avidin-HRP (Jackson ImmunoResearch) was diluted 1:1000 in 1%BSA/PBS and 100 μl per well were added to the plates. Plates were covered and incubated at room temperature for 30 minutes. The plates were washed (as above). The ELISAs were developed with ABTS in citrate buffer with 0.03% H2O2, at room temperature for 40 minutes and then read at OD = 405 nm. Titers were found by using a log-based slope equation where the O.D. = 0.5.
1. Proliferation assays: 15-mer peptides offset by 3 amino acids describing the proteins of interest were obtained as a Pep Set from Mimotopes (San Diego, CA). Pep Sets were constructed for Bacillus lentus subtilisin (B. lentus subtilisin) and Bacillus amyloliqufaciens subtilisin (subtilisin BPN' Y217L). Peptides were resuspended in DMSO at 1 mM.
2. Linear epitopes: Biotinylated 15-mer peptides from obtained from Mimotopes (San Diego, CA). The 15-mer peptides were offset by 5 amino acids, and described the sequence of BPN' Y217L. The biotin moiety at the N-terminus was separated from the peptide sequence of interest by a 4 amino acid spacer. Biotinylated peptides were resuspended at 1 mM in DMSO.
T-cell epitope mapping of enzymes B. lentus subtilisin, BPN' Y217L and GP002
T cell epitope mapping was performed using splenocytes from immunized Hartley strain guinea pigs. Splenocytes from immunized guinea pigs were resuspended in AIM-V (Life Technologies Gaithersberg, MD) containing 100 units/ml penicillin, 100 ug/ml streptomycin, 1 mM glutamine, and either 10% autologous serum or 10% FCS (Sigma, St. Louis, MO). Using round-bottom 96 well plates (Costar, Cambridge, MA), either 5 × 105 or 1 × 106 cells/well in 200 uL of media. 5 uM of peptide were placed in each well. Each peptide was tested minimally in duplicate. DMSO was present at 0.5%. Control cultures contained 0.5% DMSO in the absence of peptide. The cultures were incubated at 37°C for 5 days. On day 5, cells were pulsed with tritiated thymidine at a concentration of 0.5 μCi/well. On day 6, cells were harvested and counted. A proliferative response was counted as positive if the average counts for a particular peptide were three times higher than the control levels.
Linear B-cell epitope mapping of enzymes BPN' Y217L and GP002
Biotinylated peptides were resuspended in 1 ml of DMSO (1 mM). A stock plate was made by diluting 10 μl of each peptide into 200 μl of PBS/ 0.025% Tween-20 (Sigma, St. Louis, MO). Streptavidin-coated plates (Pierce, Rockford, IL) were blocked with 1%BSA/PBS for 30 minutes at room temperature. Approximately 5 μM of biotinylated peptide was coated onto each well. Each peptide was tested minimally in duplicate. Plates were incubated for one hour at room temperature and then washed three times. Guinea pig serum was diluted 1:1000 in PBS/Tween-20. 100 μl of dilute sera was placed into each well and plates were incubated at room temperature for one hour and then washed three times. The secondary antibody, anti-guinea pig IgG-HRPO) (Jackson ImmunoResearch, West Grove, PA) was diluted 1:1000 in 1%BSA/PBS and 100 μl of dilute conjugate was added to each well. Plates were incubated at room temperature for 1 hour and then washed three times in PBS/Tween-20. Plates were then washed twice in PBS only. ELISAs were developed using ABTS in citrate buffer, 0.03% H2O2. Plates were then incubated at room temperature for up to 45 minutes and then read at 405 nm. OD readings for each peptide were compared to background readings with no peptide. Any peptide giving readings at least twice the background was considered a positive.