Materials
DNA primers and DNA sequencing were obtained from Eurofins MWG Operon (Huntsville, AL). E. coli BL21(DE3) and 10G electrocompetent E. coli cells were purchased from Lucigen (Middleton, WI). Pfu Ultra DNA polymerase was obtained from Agilent Technologies (Santa Clara, CA). T4 DNA ligase and restriction enzymes NcoI, BamHI and SacI were purchased from New England Biolabs (Ipswich, MA). MiniElute Reaction Cleanup Kit, QIAquick Gel Extraction Kit and QIAprep Spin Miniprep kit were acquired from Qiagen (Valencia, CA). Hydrogen peroxide, 2,6-dimethoxyphenol (2,6-DMP) and 7-benzyloxy-4-(trifluoromethyl)-coumarin (BFC) were purchased from Sigma-Aldrich (St. Louis, MO). Trypsin from bovine pancreas (Type I, ~10,000 BAEE units/mg protein) was obtained from Sigma-Aldrich (St. Louis, MO).
Expression and purification of free CYPBM3 “21B3”
The plasmid pCWori encoding the heme domain of the CYPBM3 “21B3” was a kind gift from Prof. France Arnold from the California Institute of Technology (Caltech). The CYPBM3 mutant 21B3 was expressed in E. coli using the β-d-thiogalactopyranoside (IPTG)-inducible pCWori vector as previously described [21]. CYPBM3 “21B3” purification was performed by chromatography in an EconoSystem from Bio-Rad equipped with a 5 mL Ni-pre-charged HisTrap HP column (Amersham Biosciences). The equilibration buffer consisted in 50 mM NaH2PO4, 300 mM NaCl and 10 mM imidazole, pH 8. The protein mixture was loaded at 1.5 mL min−1. The CYP protein was eluted in a buffer containing 300 mM imidazole at 3 mL/min for 10 min. The colored fractions were collected, concentrated by ultrafiltration and stored at −20 °C in 50 mM Tris–HCl buffer, pH 8, containing 10 % glycerol. CYP protein concentration was determined by using the CO assay [26].
Plasmid constructs
The CYPBM3 “21B3” gene was amplified by PCR from the plasmid pCWori CYBM3 using the forward primer, 5′-AAAAATCATGCCATGGCAATTAAAGAAATGCCT-3′ and reverse primer, 5′-AAAAAAGCGGGATCCAGTGCTAGGTGAAGGAA-3′.
The amplified gene product was digested with NcoI and BamHI (underlined in primer sequences, respectively) and ligated into the previously linearized pETDuet-1 assembler vector containing the truncated scaffold protein SP141–303 and the P22 coat protein [16]. The ligation reaction was transformed into 10G electrocompetent cells and colonies were screened by colony PCR and restriction enzyme digestion. Hits were sequenced (Huntsville, AL) to confirm the correct DNA sequence. Once the correct sequence was verified, the pETDuet CYP-SP+CPP22 plasmid (AmpR) was transformed into BL21(DE3) for the simultaneous expression strategy. The fusion CYPBM3 “21B3”—scaffold protein (CYP-SP) gene was subcloned from the pETDuet CYP-SP + P22 into the pBAD plasmid. The pETDuet CYP-SP + P22 plasmid and pBAD vector were both digested with NcoI and SacI. The digested products were ligated and transformed into 10G electrocompetent cells. Colonies were screened by colony PCR and restriction enzyme digestion. Hits were sequenced (Huntsville, AL) to corroborate for the right DNA sequence. Once the correct sequence was verified, the pETDuet CYP-SP + CPP22 plasmid (AmpR) and the pRSF P22 plasmid (KmR) were transformed into BL21(DE3) for the differential expression strategy.
Simultaneous protein expression strategy
E. coli BL21(DE3) cells harboring the expression plasmid pETDuet CYP-SP+CPP22 were grown on Terrific Broth (TB) medium, supplemented with 0.5 mM thiamine and trace elements, at 37 °C and 180 rpm in the presence of ampicillin to maintain selection for the plasmid until reaching and OD600 = 0.8. At this point, 0.5 mM of isopropyl-β-d-1-thiogalactopyranoside (IPTG) and 1 mM δ-aminolevulinic acid were added. Cell cultures were grown for additional 5 h at 30 °C and 135 rpm, then cells were harvested by centrifugation and the pellets stored at −20 °C overnight until purification.
Differential protein expression strategy
E. coli BL21(DE3) cells harboring the expression plasmids pBAD CYP-SP and pRSF P22 were grown on TB medium, supplemented with 0.5 mM thiamine and trace elements, at 35 °C and 150 rpm for 7 h in the presence of ampicillin and kanamycin to maintain selection for both plasmids. At this point, two different induction schemes were followed: (1) The CYP-SP gene was induced first with 0.2 % of l-arabinose and the culture was supplemented 1 mM δ-aminolevulinic acid. The cell culture was grown for 16 h. After this period of time, the CPP22 gene was induced with 0.5 mM of IPTG and the cultures were grown for 3 additional hours at 30 °C and 150 rpm, then cells were harvested by centrifugation and the pellets stored at −20 °C. (2) The differential expression was performed as stated above but the expression of the CYP-SP gene was induced with 0.125 % of l-arabinose and the P22 with 0.3 mM IPTG.
P22-CYP VLP purification
Cell pellets were resuspended in lysis buffer (50 mM sodium phosphate, 100 mM sodium chloride, pH 7.6) and lysed by sonication. Cell debris were removed by centrifugation at 12,000×g for 45 min at 4 °C. P22 VLPs were purified from the supernatant by ultracentrifugation over a 35 % (w/v) sucrose cushion and spun at 215,041×g on a Sorvall WX Ultra 80 ultracentrifuge (Thermo Scientific) for 50 min at 4 °C. The resulting P22 VLP pellet was resuspended in PBS (50 mM sodium phosphate, 25 mM sodium chloride, pH 7.0) and then purified over a 60 × 1.6 cm HiPrep 16/60 Sephacryl S-500 size exclusion column (GE Helathcare) using an AKTA Pharmacia FLPC. Flow rate for SEC purification was 1 mL min−1 of PBS. Fractions taken from SEC containing P22 VLPs were concentrated by ultracentrifugation at 215,041g for 50 min at 4 °C and the resulting capsid containing pellet was resuspended in 100 mM Tris–HCl pH 8 buffer. The purity of VLPs has been verified by gel electrophoresis and transmission electron microscopy (TEM). P22 VLPs concentration was determined by UV absorption at 280 nm using a molar extinction coefficients of ε280 = 44,920 M−1 cm−1 for coat protein and ε280 = 52,830 M−1 cm−1 for CYP-SP (theoretically calculated using ProtParam, [44]). The total concentration of protein in the P22 capsid with encapsulated CYP-SP (P22-CYP VLPs) was calculated using the Lambert–Beer equation, AbsT = CCP·ɛCP·l + CCYP-SP·ɛCYP-SP·l as described previously [15]. AbsT is the total absorbance of the sample measured at 280 nm.
Size exclusion chromatography with multiangle light scattering and refractive index detection (HPLC-MALS-RI)
Samples were separated through a WTC-100S5 (Wyatt Technologies) size exclusion column utilizing an Agilent 1200 HPLC. Elution was performed at flow rate of 0.7 mL min−1, for a total run time of 25 min using 50 mM phosphate pH 7.2 buffer containing 100 mM sodium chloride and 200 ppm sodium azide. Samples of 25 µL (1 mg mL−1 concentration) were loaded into the column and the total run time was 30 min. Samples were detected using a UV–Vis detector (Agilent), a Wyatt HELEOS Multi Angle Laser Light Scattering (MALS) detector, and an Optilab rEX differential refractometer (Wyatt Technology Corporation). The molecular weight of the P22-CYP VLPs, the polydispersity of the sample as well as the hydrodynamic radius of the particle was calculated with Astra 5.3.14 software (Wyatt Technology Corporation).
The number of enzymes encapsulated within a P22 VLP was calculated using the following formula
$$CYP - SP_{per capsid} = \frac{{M_{(P22 capsid + CYP - SP)} - M_{(capsid)} }}{{M_{(CYP - SP)} }}$$
(1)
where M(P22capsid+CYP-SP) is the molecular weight of the capsid with the encapsulated CYP-SP (determined experimentally with HPLC-MALS-RI), Mcapsid is 19,572 kDa (46.6 kDa × 420 subunits) and MCYP-SP is 71.5 kDa (calculated with Serial Clones 2.6, Franck Perez, Serial Basics).
Transmission electron microscopy
VLPs (10 µL, 0.1 mg mL−1) were applied to carbon-formvar coated grids and incubated for 1 min, excess sample was removed with Whatman filter paper. Grids were then washed with 10 µL of distilled water, removing away liquid shortly after addition with filter paper and stained with 5 µL 1 % uranyl acetate for 1 min. Excess stain was removed by blotting with filter paper. Samples were analyzed with a LEO 912AB transmission electron microscope operated at 100 kV.
Inductively coupled plasma-mass spectrometry (ICP-MS)
A P22-CYP VLP sample (21.6 mg) was incubated in concentrated nitric acid (HNO3) for 16 h at 70 °C. After sample mineralization, it was diluted with water to a final HNO3 concentration of 5 % in a final volume of 50 mL. Samples were sent to Energy Laboratories, Inc (Billings, MT, USA) to be analyzed. Sulfur was used as internal reference (8401 sulfur atoms per P22-CYP capsid) for determining the ratio of iron to protein.
Free CYP and P22-CYP kinetic assays
The CYP enzymatic activity was determined by the transformation of 2,6-dimetoxyphenol (2,6-DMP) and spectrometrically monitored at 468 nm (ɛ468 = 14,800 M−1 cm−1) using an Agilent 8453 UV-Vis spectrophotometer. All reactions were performed in 50 mM Tris–HCl buffer (pH 8) at room temperature in a final volume of 0.1 mL. For the determination of catalytic parameters the concentration of 2,6-DMP was fixed at 500 µM. The reaction was initiated by adding H2O2 in a range between 1 and 60 mM. Catalytic constant values were obtained by fitting the data to a Michaelis–Menten equation (GraphPad Prism 6, GraphPad Software, Inc.).
P22-CYP VLPs stability against acidic pH
The residual activity of free CYP and P22-CYP was measured at pH 5 (100 mM sodium acetate buffer) and pH 6 (100 mM potassium phosphate buffer), incubating the samples for 1 h in each buffer. Before determining enzymatic activity, samples were centrifuged for 3 min at 16,000g. The residual activity was measured in 50 mM Tris–HCl buffer (pH 8) at room temperature using 500 µM 2,6-DMP as a substrate and 5 mM H2O2 to initiate the reaction.
P22-CYP VLPs protection against protease degradation
The encapsulated and free CYP were treated with 10 U of trypsin per 1 mg of enzyme and incubated for 1 and 20 h at room temperature in 50 mM Tris–HCl buffer (pH 8). After incubation, the residual activity was determined as stated above.
Cell line and cell culture
Human cervix carcinoma cells (HeLa cells) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % Fetal Bovine Serum (FBS, BenchMark, Gemini Bio Products), 1 % Penicillin streptomycin (Sigma-Aldrich), 1 % l-glutamine and 1.5 g/l sodium bicarbonate. Cells were propagated in growth medium and maintained at 37 °C and 5 % CO2.
P22-CYP VLPs transfection
Cell culture Petri dishes coated with Poly-d-lysine (MatTek P35GC1.5-10C) were used to seed 250,000 HeLa cells in DMEM media and incubated overnight at 37 °C and 5 % CO2. Transfection of P22-CYP nanoparticles was achieved using Lipofectamine 2000 reagent (Life technologies), according to [45] with few modifications to the manufacture’s protocol. Briefly, 3 µl of Lipofectamine 2000 was diluted in 100 µl of DMEM media without antibiotic and FBS (DMEM-SF) and 3.14 × 1011 P22-CYP nanoparticles were mixed with 100 µl of DMEM-SF media, both preparations were pre-incubated for 15 min at room temperature (RT). Afterwards, both samples were mixed for 30 min at RT. Prior to the addition of this transfection mixture to HeLa cell culture; cells were rinsed twice with sterile PBS buffer. The transfection mixture was added slowly on the top of the cell culture and let stand for 30 min at RT, then 1.8 ml of DMEM-SF media was added into HeLa cells culture and incubated for 4 h at 37 °C and 5 % CO2. After incubation, cell media was removed and the culture was rinsed once with PBS and 2 ml of complete DMEM media was added. HeLa cells transfected with VLP-CYP nanoparticles were incubated overnight at 37 °C and 5 % CO2.
P22-CYP VLP enzyme activity in vitro assay
CYP enzyme activity was assayed in HeLa cells (endogenous CYP activity) and in HeLa cells transfected with P22-CYP VLPs. The CYP activity was estimated by the transformation of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) in the fluorescent product 7-hydroxy-4-[trifluoromethyl]-coumarin (HFC) according to [46] with some modifications. Briefly, cell culture media was discarded and 15 µl of 20 mM BFC diluted in 150 µl of complete DMEM media was added to each culture plate and incubated in darkness for 10 min at RT. Complete DMEM media was added up to 1.5 ml to each plate and further incubated for 30 min at 37 °C and 5 % CO2. Then, 4.5 µl of 1 mM of hydrogen peroxide was added to each culture and incubated for 10 min at 37 °C and 5 % CO2. Cell culture plates were rinsed three times with PBS before the addition of 2 mL of complete DMEM media was added to each plate and incubated for 2 h at 37 °C and 5 % CO2 for further imaging analysis.
Confocal microscopy cell imaging
HeLa cell cultures treated with BFC reagent were fixed with 4 % formaldehyde-PBS solution at 4 °C for 15 min. After fixation, cells were permeabilized with 0.5 % Triton X/PBS for 15 min at 4 °C. Nuclear staining was achieved by incubated the cells with DAPI at 0.5 ng/ml in darkness for 10 min at RT, followed by five washes with PBS. Nuclear staining with DAPI was also visualized with an inverted laser-scanning microscope Olympus FluoView FV1000 (Japan) using an argon ion laser for excitation at 405 nm wavelength and filters for emission of DAPI. BFC transformation into the fluorescent reagent HFC was detected using the GFP filter channel (excitation at 488 nm and emission at 515–530 nm). Cells were visualized with a 63 × (DIC), 1.4 N.A. planapochromatic oil immersion objective. The imaging parameters used produced no detectable background signal from any source other than from BFC and DAPI. Confocal images were captured using MetaMorph software for Olympus.
CYP activity quantification in HeLa cells transfected with P22-CYP VLPs
The enzymatic activity of CYP in non-transfected and transfected HeLa cells with P22-CYP VLPs was measured spectrofluorimetrically. The fluorescence intensity originated by CYP-catalyzed transformation of BFC into HFC was monitored. After transfection, media from P22-CYP transfected cells and non-transfected HeLa cells (control cells) was replaced by 1.5 ml of complete DMEM media containing 15 µl of 20 mM BFC and incubated under darkness for 30 min at 37 °C and 5 % CO2. Then, cell culture plates were rinsed three times with PBS before the addition of 4.5 µl of 1 M of hydrogen peroxide and incubated for 10 min at 37 °C and 5 % CO2. After incubation, cells were rinsed with PBS and harvested with trypsin/EDTA treatment. Pelleted cells were counted and diluted in PBS to obtain 200,000 cells. Fluorescence intensity from transfected and non-transfected HeLa cells was measured in a fluorescence spectrophotomerer (Hitahchi F-7000), using an excitation source at 254 nm and emission measurement at 510 nm.