Media and solutions
All reagents used in this work were purchased from Sigma-Aldrich (St Louis, MO, USA) or VWR International Eurolab S.L. (Mollet del Vallés, Barcelona, Spain). The compositions of the media used in this study were as follows:
Minimal methanol medium (MM): 13.4 g·L-1 yeast nitrogen base with ammonium sulphate and without amino acids (YNB); 0.0004 g·L-1biotin; 15 g·L-1 agar and 0.5% methanol.
Minimal methanol medium + Histidine (MMH): 13.4 g·L-1 YNB; 0.0004 g·L-1 biotin; 15 g·L-1 agar; 0.04 g·L-1 histidine and 0.5% methanol.
Minimal dextrose medium (MD): 13.4 g·L-1 YNB; 0.0004 g·L-1 biotin; 15 g·L-1 agar and 20 g·L-1 dextrose.
Minimal dextrose medium + Histidine (MDH): 13.4 g·L-1 YNB; 0.0004 g·L-1 biotin; 15 g·L-1 agar; 20 g·L-1 dextrose and 20 g·L-1 dextrose.
Yeast Extract Peptone medium (YP): 10 g·L-1 yeast extract and 20 g·L-1 peptone.
Yeast Extract Peptone Dextrose medium (YPD): 10 g·L-1 yeast extract; 20 g·L-1 peptone and 20 g·L-1 glucose.
Yeast Extract Peptone Dextrose Sorbitol medium (YPDS): 10 g·L-1yeast extract; 20 g·L-1 peptone; 20 g·L-1 glucose; 182 g·L-1 sorbitol and 20 g·L-1 agar.
Trace element solution (TES): 2.0 g·L-1 ZnSO4 × 7H2O; 0.02 g·L-1 CuSO4 × 5H2O; 0.08 g·L-1 KI; 0.3 g·L-1 MnSO4 × H2O; 0.19 g·L-1Na2MoO4 × H2O; 0.02 g·L-1 H3BO3 and 2.9 g·L-1 FeCl3.
Vitamin solution (VT): 0.4 g·L-1 calcium pantothenate; 0.4 g·L-1 tyamine; 4 g·L-1 myo-inositol; 0.1 g·L-1 nicotinic acid; 0.4 g·L-1 pyridoxine and 0.4 g·L-1 biotin.
Production medium (PM): 13 g·L-1 KH2PO4; 8.75 g·L-1 (NH4)2SO4; 4.5 g·L-1 MgSO4; 0.5 g·L-1 CaCl2 × 2H2O; 2.5 g·L-1 yeast extract; 5 ml·L-1 TES and 5 ml·L-1 VT.
Cloning of R. microplus, R. annulatus and R. decoloratus Bm86 orthologs and sequence analysis
Tick strains were obtained from laboratory colonies maintained at the Utrecht Centre for Tick-borne Diseases, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Originally, tick strains were collected from infested cattle in Mozambique (R. microplus), Israel (R. annulatus) and South Africa (R. decoloratus).
Total RNA was extracted from the viscera of partially fed R. annulatus and R. microplus females and from eggs of R. annulatus and R. decoloratus using TriReagent (Sigma-Aldrich, St Louis, MO, USA) and following manufacturer's recommendations. Bm86 (R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) coding regions (nucleotides 58–1884 of the coding region of Bm86 reference sequence; GenBank accession number M29321) lacking the signal peptide and GPI anchor sequences were amplified by RT-PCR. The RT-PCR was done using 10 pmol of each primer (CZABM5: 5'-A CTC GAG AAA AGA GAG TCA TCC ATT TGC TCT GAC TTC GG and CZABM3: 5'-A TCT AGA TTA AGC ACT TGA CTT TCC AGG ATC TG; Bm86 homologous regions are underlined) in a 50-μl volume (1.5 mM MgSO4, 1 × avian myeloblastosis virus (AMV) RT/Thermus flavus (Tfl) reaction buffer, 0.2 mM each deoxynucleoside triphosphate (dNTP), 5 u AMV RT, 5 u Tfl DNA polymerase) employing the Access RT-PCR system (Promega, Madison, WI, USA). Reactions were performed in an automated DNA thermal cycler (Techne model TC-512, Cambridge, England, UK). RNA was reverse transcribed for 45 min at 45°C prior to PCR consisting of an initial step of 2 min at 94°C followed by 35 cycles of a denaturing step of 30 sec at 94°C and an annealing-extension step of 2 min at 68°C. Control reactions were done using the same procedures, but without RNA added to control contamination of the PCR. PCR products were electrophoresed on 1% agarose gels to check the size of amplified fragments by comparison to a DNA molecular weight marker (1 Kb Plus DNA Ladder, Promega). The amplicon was resin purified (Wizard, Promega) and cloned into pGEM-T vector (Promega). Partial sequences of cloned Bm86 orthologs were obtained by double-stranded dye-termination cycle sequencing (Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University and Secugen S.L, Madrid, Spain). At least three clones from independent PCR reactions were sequenced for each gene. Multiple sequence alignment was performed using the program AlignX (Vector NTI Suite V 8.0, InforMax, Invitrogen, Carlsbad, CA, USA) with an engine based on the Clustal W algorithm . Searches for sequence similarity were performed at the ncbi with the BLASTN program against the nonredundant sequence database nr.
The GenBank accession numbers for Bm86 (R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) are EU191620–EU191622.
Construction of expression plasmids
Bm86, Ba86 and Bd86 coding regions were excised from pGEM-T by Xho I and Xba I digestion (restriction sites introduced during PCR by CZABM5 and CZABM3 primers, respectively) and cloned into P. pastoris expression vector pPICZαA (Invitrogen) digested with Xba I and Xho I. In this way, Bm86 orthologs were cloned under the control of the alcohol oxidase (AOX1) promoter, in frame with the yeast alfa-factor secretion signal but without the C-terminal c-myc/His tag due to a translation termination site introduced by CZABM3 primer during PCR. The expression constructs were sequenced at both ends and selected constructs with correct sequences were named pPAMoz9 (Bm86), pPADec8 (Bd86) and pBaI (Ba86) and used for transformation of P. pastoris.
Pichia pastoris transformation and screening for recombinant protein expression
Expression plasmids were linearized by restriction with Sac I and transformed into P. pastoris strains GS115, KM71H and X33 (Invitrogen) by electroporation as described . Transformants were selected on YPDS plates containing 100 μg·ml-1 Zeocin and incubated at 30°C. A functional assay to directly screen for high expression recombinant clones was made by culturing the transformants in an orbital shaker at 250 rpm and 30°C. Single colonies were inoculated in 1 ml YPDS containing 100 μg·ml-1 Zeocin and grown overnight. Cultures were divided into two parts of 500 μl each. Five hundred μl were transferred to 5 ml fresh YP medium with 20 g·L-1 glycerol, grown for 24 hrs and inoculated into 250 ml fresh YP medium supplemented with 20 g·L-1 glycerol. Growth in glycerol was resumed after 24 hrs and then methanol was added daily to 1% (v/v) during the course of induction. After 5 days growing on methanol, supernatants were collected by centrifugation for 15 min at 15,000 × g in a Beckman Allegra™ X-22R centrifuge, rotor F2402H (Beckman-Coulter, Palo Alto, CA, USA) and dot blots were made to screen for expression of recombinant proteins. The other 500 μl were also transferred to 5 ml fresh YP medium with 20 g·L-1 glycerol, grown for 24 hrs and mixed with glycerol to 250 g·L-1. Long term stocks were prepared as 100 μl aliquots and stored frozen at -80°C.
Analysis of the Mut phenotype in P. pastoris transformed strains
The high expression transformants of X33 and GS115 strains were analyzed for Mut+ or MutS phenotype using the functional assay described in the Invitrogen user's manuals K1710-01 and K1750-01 . The KM71H strain always produces a MutS phenotype . Briefly, 50 μl from the long term stocks of the high expression X33 and GS115 transformants were streaked in YPDS plates containing 100 μg·ml-1 Zeocin and incubated at 30°C for 24 hrs. One colony of each transformant was streaked in both MMH and MDH plates for the GS115 and X33 strains. To differentiate Mut+ from MutS, control GS115/Albumin (MutS) and GS115/pPicz/lacZ (Mut+) strains (Invitrogen) were streaked in the MMH and MDH plates. Plates were incubated at 30°C for 3 days and cell growth was observed and compared to controls.
Pre-inoculums and inoculums for bioreactor cultures were grown in a shaker at 30°C and 250 rpm. Two 100 μl long term stock vials were seeded in 1 ml YP medium, grown for 12 hrs and transferred into 4 × 50 ml tubes containing 5 ml of YP medium with 20 g·L-1 glycerol. After 24 hrs, cultures were mixed again and 5 ml were used to inoculate 2 L Erlenmeyer flasks containing 250 ml of YP medium with 20 g·L-1glycerol. Cells were grown to an O.D.600 nm between 15 and 20 and then cultures were inoculated into a 5-L working volume Biostat B bioreactor (B. Braun Biotech, Melsungen, Germany) containing 3.5 L of PM with 40 g·L-1 glycerol.
During the fermentation process, temperature was kept at 30°C and dissolved oxygen was maintained at 30% saturation by regulating agitation and aeration rates. A three-phase cultivation protocol was used in the fermentation. The glycerol growth phase included a 12 to 14 hrs batch stage from the starting point followed by a 10 to 12 hrs glycerol fed-batch stage. A glycerol solution of 50% (v/v) was added to the fermentor for 4 hrs to reach an equivalent total quantity of 60 g·L-1 in the culture medium. Upon exhaustion of glycerol, indicated by a sharp increase in dissolved oxygen, methanol induction was made by adding 1% (v/v) methanol to the culture medium and 3 hrs later the fed-batch phase was started by feeding methanol according to the P. pastoris Fermentation Process Guideline . The pH was allowed to drop to 3.5 during the whole glycerol phase and it was maintained in this value by the addition of NH4OH. Prior to methanol induction, pH was adjusted and maintained at 5.5 by adding NH4OH or H3PO3. Throughout the fermentation processes, supplements of 20 ml TES and VT solutions were added to the culture medium every 24 hrs. Additionally, GS115 strain cultures were supplemented with 0.04 g·L-1 L-Histidine every 24 hrs.
Biomass analysis during fermentation
Time-course samples were withdrawn from the fermentor at regular intervals to check growth rate and protein concentration in the supernatant. Cell density was expressed as O.D.600 nm, either measured as grams of wet weight per litter broth (O.D.600 nm = 1.39 × wet weight (g·L-1) - 27.26), which was obtained by centrifugation of the samples at 15,000 × g for 15 min or measured directly in the culture medium. Total protein concentration in the culture medium was quantified using the Bradford method with BSA as standard .
Cells harvesting, recovery and purification of recombinant proteins
Cultures from the 5-L fermentor were centrifuged at 3,900 × g for 15 min in a Beckman Allegra™ X-22R centrifuge, rotor SX4250 (Beckman-Coulter) to separate cells. Supernatants were then collected and filtered through a tandem filtration system with a 20 μm cartridge (Sartorius AG, Goettingen, Germany), 5 μm and 0.45-0.22 μm cartridges (Millipore, Billerica, MA, USA) and checked for total and recombinant protein content using the Bradford method with BSA as standard  and the Experion semiautomated electrophoresis system (Bio-Rad, Hercules, CA, USA). For the Experion, 4 μl of denatured proteins were loaded into a Pro 260 Chip and protein concentration was determined following manufacturer's recommendations. Recombinant proteins were separated by size exclusion using a Sartocon® Slice 200 ultrafiltration system having a Hydrosart membrane with a molecular weight cut-off of 50 kDa (Sartorius). Finally, protein solutions were concentrated and diafiltrated against four volumes of phosphate buffer pH 8.3 using a centrifugal concentrator VIVACELL 100 (50 kDa cut-off; Sartorius) in a Beckman Allegra™ X-22R centrifuge, rotor SX4250 (Beckman-Coulter) at 3,900 × g.
Vaccine formulation and analysis
Prior to adjuvation of the vaccine, protein solutions were adjusted to a concentration of 120 μg·ml-1 and filtered through 0.45 and 0.22 μm cartridges (Sartorius AG) under sterile conditions in a laminar flow to obtain a sterile antigen solution. Adjuvation was made by mixing a solution of anhydromannitoletheroctodecenoate (Montanide ISA 50 V; Seppic, Paris, France) with the recombinant protein solution in batch-by-batch processes using a high-speed mixer Heidolph DIAX 900 (Heidolph Elektro, Kelheim, Germany) at 8,000 rpm and the vaccine was filled manually under sterile conditions in glass bottles of 20 ml (Wheaton, Millville, NJ, USA). Quality controls were made by testing mechanical and thermal stability of vaccine emulsions as described by Canales et al. .
Rabbit immunization with recombinant proteins
Two New Zealand White rabbits per group was each immunized with 3 doses (weeks 0, 4 and 8) containing 50 μg/dose of purified recombinant proteins formulated as described above or Gavac (Revetmex, Mexico City, Mexico) as control. Rabbits were injected subcutaneously with 1 ml/dose using a 1 ml tuberculin syringe and a 27 1/2G needle. Two weeks after the last immunization, blood samples were collected from each rabbit into sterile tubes and maintained at 4°C until arrival at the laboratory. Serum was then separated after centrifugation and stored at -20°C. Rabbits were cared for in accordance with standards specified in the Guide for Care and Use of Laboratory Animals.
SDS-PAGE, dot blot and Western blot analyses
Protein samples were analyzed by denaturing SDS-PAGE with a 12% PAGEgel-SDS cassette gel (PAGE-gel Inc, San Diego, CA, USA) under reducing conditions. Protein bands were visualized by either Coomassie Brilliant Blue R250 or silver staining. Samples were treated with dithiothreitol (DTT) reducer (PAGE-gel Inc.), heated in boiling water for 5 min before loading onto the gel and electrophoresed for 80 min at 90 mA constant current.
Electrophoretic transfer of proteins from gels to nitrocellulose membranes (PROTRAN BA85; Schleicher and Schuell, Dassel, Germany) for Western blot analysis was carried out in a Minie-Genie Electroblotter semi-dry transfer unit (Idea Scientific, Corvallis, OR, USA) according to manufacture's protocol. Protein samples of 2 μl were absorbed onto nitrocellulose membrane by gravity flow to perform the dot blot analysis. A standard curve was constructed with known amounts of recombinant Bm86 extracted from Gavac (Revetmex) and was used for semi-quantitative analysis in dot-blots. The supernatant of the GS115/Albumin strain (Invitrogen) grown under the same conditions was used as a negative control in both dot- and Western-blots. Membranes for dot or Western blots were blocked with 5% skim milk for 1 hr at room temperature, washed three times in TBS (25 mmol/L Tris·HCl, 150 mmol/L NaCl, pH 7.6) and probed with sera from rabbits immunized with Gavac (Revetmex) (1:1000 dilution) or recombinant proteins (1:5000 dilution) as described above. The antisera were diluted in 3% BSA in TBS. Membranes were then washed three times with TBS and incubated with an anti-rabbit IgG horseradish peroxidase (HRP) conjugate (Sigma-Aldrich) diluted 1:1000 in TBS. After washing the membranes again, color was developed using TMB stabilized substrate for HRP (Promega).