Plasmid constructs for expression of PfBCCP
The expression vector pGEXT was constructed from pGEX-4T-3 (GE Healthcare) by replacing the sequence encoding the thrombin cleavage site (beginning at base 918) with that of the tobacco etch virus (TEV) protease. This was done with the QuikChange site-directed mutagenesis kit (Stratagene), using primers TEV1, 5'-GGTGGCGACCATCCTCCAAAATCGGATgaaaacctgtattttcagggcGGATCCCCGAATTCCCGGGTC-3' and TEV2, 5'-GACCCGGGAATTCGGGGATCCgccctgaaaatacaggttttcATCCGATTTTGGAGGATGGTCGCCACC-3' (TEV cleavage site shown in lower case). The resulting vector, pGEXT, produces a glutathione S-transferase (GST) fusion protein which can be cleaved by TEV protease.
The biotin carboxy carrier protein (BCCP) domain (residues 1156-1369) of the P. falciparum acetyl-CoA carboxylase (ACC) gene [GenBank:XM_001348802; PlasmoDB:PF14_0664] was amplified by PCR from cDNA of P. falciparum 3D7 erythrocyte stages, using primers BCCP1, 5'-GGTGGTGGATCCTTATGTGCTACCATATTTAAACTATTAATATATTTTATG-3' and BCCP2, 5'-GGTGGTCTCGAGTTATTCTATTATTCCTAATAAATCTCCTATTTTAATAATTG-3'. The PCR product was digested with BamHI and XhoI (underlined) and ligated into the pGEXT vector digested with the same endonucleases, generating plasmid pMSp038. The resulting fusion protein (PfBCCP) contained the entire BCCP domain with an N-terminal GST tag. In order to test for solubility of the fusion protein, 100 ml cultures were grown to an OD600 of 0.6, protein expression was induced with IPTG, and the cultures were incubated for an additional three hours at 37°C. Cells were lysed, and whole cell lysate was centrifuged to separate the soluble and insoluble fractions. The PfBCCP fusion protein was found in the insoluble fraction. A second construct was designed (PfBCCP-79) based on the biotinylation domain found in the crystal structure of the E. coli BCCP [PDB:1BDO]. Nucleotides encoding ACC residues 1291-1369 were amplified from pMSp038 using primers BCCP3, 5'-GGTGGTGGATCCGATAATATTTTCATACCTAATGTTAGGAATCC-3' and BCCP2, digested with BamHI and XhoI and ligated into the pGEXT vector, generating plasmid pTDe003. Expression tests using pTDe003 indicated that the resulting GST fusion protein was soluble. PfBCCP-79 was purified from E. coli (see below) and used for the generation of specific antisera in rabbits.
Purification of PfBCCP-79 for generation of rabbit antisera
Plasmid pTDe003, encoding GST-tagged PfBCCP-79, was transformed into BL21-Star(DE3) cells (Invitrogen) previously transformed with the pRIL plasmid isolated from BL21-CodonPlus(DE3) cells (Stratagene). pRIL encodes rare tRNAs that aid in the expression of P. falciparum proteins in E. coli. Cells were grown to an OD600 of 0.8 in LB medium at 37°C, and then protein expression was induced with 0.4 mM IPTG and the cultures were maintained at 20°C for 10 hrs. Cells were harvested by centrifugation at 4,000 g for 20 min at 4°C followed by resuspension in 20 mL of lysis buffer (phosphate buffered saline (PBS) solution pH 7.5, 1 mg/mL lysozyme, 2.5 μg/mL DNAse I, 10 mM PMSF, 10 mM DTT) per liter of cell culture. The resuspended cell mixture was sonicated, and then cleared by centrifugation at 30,000 g for 20 min at 4°C. Cleared supernatant was loaded on a GSTrap™ Fast-Flow chromatography column (GE Healthcare) equilibrated in PBS pH 7.5. After washing with 5 column volumes of PBS, GST fusion protein was eluted with 5 mM reduced glutathione in equilibration buffer. Fractions containing fusion protein were pooled and digested with 10 μg/ml TEV protease for 6 days at 4°C. The protein sample was dialyzed to remove glutathione and then re-applied to the GSTrap™ FF column to remove the liberated GST. The purified protein was concentrated to 1.185 g/L with a 5000 MW cutoff concentrator (Vivascience) and sent to Cocalico Biologicals, Inc. for production of specific antisera in rabbits.
Polyclonal antibodies were raised against PfBCCP-79 according to the standard immunization protocol specified by Cocalico Biologicals, Inc. Prebleeds from four rabbits were screened for cross-reactivity to P. falciparum and human red blood cell antigens by western blot analysis. The two rabbits that were least reactive were selected for inoculation with 100 μg purified PfBCCP-79. The rabbits were boosted with 50 μg antigen on days 14, 21 and 49 after initial inoculation. Test bleeds were performed on days 35 and 56, and were tested for reactivity to the PfBCCP-79 antigen by western blot analysis. Production bleeds were performed on days 63 and 84, and the rabbits were exsanguinated on day 91. These antisera were used without further purification for western blot analysis, as described below.
Plasmid constructs for expression of biotinylated PfBCCP-79
For labeling with 35S, the GST fusion protein is undesirable due to the presence of multiple cysteine residues that will incorporate the label. To address this problem, we constructed a plasmid expressing PfBCCP-79 with a six-histidine tag. The PfBCCP-79 gene sequence was amplified from pTDe003 using primers BCCP4, 5'-GGTGGTCATATGGATAATATTTTCATACCTAATGTTAGGAATCC-3' and BCCP5, 5'-GGTGGTGAATTCTTATTCTATTATTCCTAATAAATCTCCTATTTTAATAATTG-3', digested with NdeI and EcoRI (underlined) and ligated into the pET28a vector (Invitrogen), generating plasmid pTDe010. This plasmid contains a kanamycin resistance cassette and a pMB1 origin of replication, and produces His-tagged PfBCCP-79 under control of a T7 promoter.
Plasmid pDB1282 was a gift from Dennis Dean at Virginia Polytechnic Institute and State University (Virginia Tech). Plasmid pDB1282 contains the Isc (Iron-Sulfur Cluster) operon from Azotobacter vinelandii, which is required for iron-sulfur cluster biogenesis in this organism. The Azotobacter vinelandii Isc operon is composed of the genes iscR, iscS, iscU, iscA, hscB, hscA, fdx and iscX. The essential genes of this cluster (iscS, iscU, iscA, hscB, hscA and fdx) were cloned into a variant of the pARA13 expression vector to form pDB1282. Sequencing of the 5' and 3' splice sites suggests that the Isc operon was restricted with BspHI and ligated into the unique NcoI site of pAra13. Interestingly, pDB1282 contains the seven complete genes from iscS to iscX as well as about 200 nucleotides of a downstream nucleoside-diphosphate kinase (ndk). This plasmid contains an ampicillin resistance cassette and produces the Isc genes under control of an arabinose-inducible promoter.
Plasmid pCY216 [GenBank:AAD22470.1] was a gift from John Cronan at the University of Illinois at Urbana-Champaign. It was modified from the pARA13 expression vector, and encodes E. coli BirA under control of an arabinose-inducible promoter in a chloramphenicol resistance plasmid containing a p15a origin of replication.
Plasmid pRK586 was modified to produce the E. coli biotin synthase (BioB). The bioB gene was amplified from K-12 E. coli with the primers BioB1, 5'-GGTGGTGGTACCATGCATATGGCTCACCGCCCACGCTG-3', and BioB2, 5'-GGTGGTGGATCCGCGGCCGCTCATAATGCTGCCGCGTTGTAATATTC-3'. The resulting amplicon, which contained 5' KpnI/NsiI sites and 3' NotI/BamHI sites (underlined), was digested with KpnI and BamHI and ligated into pRK586 digested with the same endonucleases. The resulting plasmid, pSPr058, was digested with BclI and SacI to excise the kanamycin resistance gene (encoding aminoglycoside 3'-phosphotransferase). Primers Amp1, 5'-GGTGGTTGATCAGCCTTTTTGCGTTTCTACAAACTC-3' and Amp2, 5'-GGTGGTGAGCTCTTACCAATGCTTAATCAGTGAGGC-3' were used to amplify the ampicillin resistance gene (encoding β-lactamase) and 118 upstream bases from plasmid pDB1282. The resulting amplicon was digested with BclI and SacI (underlined) and ligated into the pSPr058 fragment described above. The resulting plasmid, pSPr059, encodes BioB under control of the λPL/tetO promoter in an ampicillin resistant plasmid containing a pSC101 origin of replication.
In vivo biotinylation of PfBCCP-79 and western blotting
The methionine auxotroph E. coli strain B834(DE3) (Novagen) was transformed with plasmid pTDe010 (encoding PfBCCP-79), in combination with either pSPr059 (encoding BioB), pCY216 (encoding BirA) or pDB1282 (encoding the AvIsc proteins). Transformed cells were grown to an OD600 of 0.8 at 37°C with the appropriate antibiotics in minimal medium composed of sulfur-free minimal E medium (0.83 mM MgCl2, 9.5 mM citric acid, 58 mM K2HPO4, 29.7 mM NH4Cl, 16.7 mM NaH2PO4) supplemented with 0.4% (w/v) glucose, 100 μM FeCl3, and 2 mM methionine. Protein expression was induced with 0.4 mM IPTG and 0.025% arabinose, and the BioB substrate desthiobiotin (Sigma-Aldrich) was added at a concentration of 1 mM. Cells were grown for 10 hours at 20°C. Cultures were normalized based on their cell density, and equal amounts of cells were harvested by centrifugation at 16,000 g for 5 min in 1.5 ml Eppendorf tubes. Cell pellets were lysed in NuPAGE sample buffer (Invitrogen) and vortexed to shear genomic DNA. For western blot analysis, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4-12% bis-tris acrylamide gradient gels. The gels were blotted onto 0.2 μm nitrocellulose membranes (Invitrogen) for 120 min at 5 V using a Semi-Dry transfer cell (BioRad). Membranes were blocked in 5% non-fat dry milk (Carnation) in PBS, washed in PBS, and probed with 1:4,000 streptavidin-HRP, ultrasensitive (Sigma-Aldrich). After PBS washes, HRP was detected using the Supersignal® West Pico chemiluminescent kit (Pierce). Membranes were then stripped using 4% (w/v) trichloroacetic acid (TCA), blocked as before, washed in PBS, and probed with rabbit antiserum specific for PfBCCP-79 (1:4,000). After PBS washes, the blot was probed with 1:5,000 donkey anti-rabbit immunoglobulin antibody conjugated to horseradish peroxidase (GE Healthcare). Excess antibody was removed with PBS, and HRP was detected using the chemiluminescent kit.
Expression and purification of [35S]-biotin-PfBCCP-79
For radiolabeling studies, the methionine auxotroph E. coli strain B834(DE3) (Novagen) was transformed with plasmids pTDe010, pSPr059 and pCY216. Cells were cultured at 37°C in 10 mL sulfur-free minimal medium (described above). When the culture reached an OD600 of 0.8, expression of PfBCCP-79 and BirA were induced with 0.4 mM IPTG and 0.025% arabinose, respectively. The culture was transferred to 20°C, and after one hour, 1 mCi Na35SO4 (American Radiolabeled Chemicals, 10 mCi/ml) and 1 mM desthiobiotin were added. The culture was maintained at 20°C for an additional 10 hours. Cells were then harvested by centrifugation for 20 min at 3,000 g and frozen at -20°C for later purification.
Cell pellets were lysed at room temperature for 10 min in 1 mL BugBuster (Novagen) supplemented with 1 mg/mL lysozyme and 2.5 μg/mL DNAseI. The cell lysate was cleared by centrifugation for 5 min at 16,000 g and applied to a 1 ml HiTrap Metal Chelate HP Column (GE Healthcare) equilibrated with Buffer A (20 mM Na/K phosphate pH 7.5). The column was washed with 7 mL Buffer A, and the combined flow through was collected and labeled FT1. The column was washed with 5 mL 30 mM imidazole in Buffer A (W1), followed by 5 mL 1 M NaCl in Buffer A (W2), and 5 mL 1% Triton X-100 in Buffer A (W3). The metal chelate column was then connected to a 4.6 mm × 100 mm PEEK™column (Applied Biosystems) packed with 1.7 mL SoftLink™ Soft Release Avidin Resin (Promega), and bound proteins were eluted from the metal chelate column with 5 mL 400 mM imidazole in Buffer A (FT2). Both columns in tandem were washed with 8 mL Buffer A (W4), and then the metal chelate column was removed. Bound protein was eluted from the avidin column in 4 mL 50 mM glycine pH 2.9 (E1). 400 μl 1 M Tris was added to the collection tube prior to eluting to prevent damage over time from the low pH of the elution buffer.
The purified biotinylated PfBCCP-79 protein was precipitated by adding 100% (w/v) TCA to a final concentration of 10%, and centrifuging for 15 min at 16,000 g. The resulting protein pellet was resuspended in 200 μL 5 M HCl and incubated at 95°C for 8 hours. One μL 1 g/L phenol red was added as a pH indicator, and the resulting hydrolysate was buffered by adding 50 μL 1 M K2HPO4, and neutralized by adding 10 M NaOH until the pH indicator turned pink.
Verification and quantification of biologically active [35S]-biotin in E. coli
A biotin bioassay was developed using a biotin auxotroph E. coli strain in which the biotin synthase (bioB) gene had been replaced with a kanamycin resistance cassette (National BioResource Project (NIG, Japan):Keio JW0758). These cells were grown to full density in LB with 50 μg/mL kanamycin. To deplete biotin levels prior to the assay, 1 μL of the LB culture was added to 4 mL biotin-free minimal medium composed of minimal E medium (0.81 mM MgSO4, 9.5 mM citric acid, 58 mM K2HPO4, 7.4 mM (NH4)2SO4, 16.7 mM NaH2PO4) supplemented with 0.4% (w/v) glucose, 30 μM FeSO4, and 50 μg/mL kanamycin. This culture grew to full density over a one day period at 37°C, and 1 μL of this culture was used to start a second 4 mL culture in biotin-free minimal medium. This culture achieved a lower density, presumably due to the 16,000,000 fold dilution of biotin from the original LB culture. For the bioassay, 2 μL of the biotin depleted culture was added to 2 mL biotin-free minimal medium supplemented with known concentrations of biotin. Assay cultures were grown at 37°C for 16 hours, after which the optical density was measured at 600 nm. These cultures were used to construct a standard growth curve. Parallel cultures were supplemented with serial dilutions of PfBCCP-79 hydrolysate. Cell densities of cultures supplemented with the hydrolysate were compared with the standard curve in order to quantify the biologically active biotin in the hydrolysate. In order to detect [35S]-biotin incorporation into the E. coli BCCP protein, cells were harvested and proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane and exposed to autoradiography film.