Microsoft Word - Latest one.doc Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  1  –  15                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   1 ORIGINAL RESEARCH ARTICLE Cloning,  Expression,  Purification,  and  Characterization  of  Clostridium  botulinum  Neurotoxin   Serotype  F  Domains   Santosh Dulal1*, Bhupal Ban2, Gi Hyoek Yang2, and Hyun Ho Jung1, 2 1Department of Applied Molecular Biology, Sun Moon University, Asan-si, Chung-nam, 336-708, The Republic of Korea 2Medy-Tox Inc., Cheongwon-gun, Chungbuk-do, 363-883, The Republic of Korea *Correspondence Author recent address: Microbiology Graduate Program, Department of Biology, New Mexico State University, Las Cruces, NM 88003, United States of America. E-mail: santoshd@nmsu.edu Abstract The use of recombinant BoNT domains has been proposed as a means to develop strategies to treat and prevent botulism. Here, details on the molecular cloning, protein expression, purification, and immunoreactivity of BoNT/F domains from Clostridium botulinum are presented. Initially, full- length synthetic genes encoding recombinant BoNT/F domains (catalytic, translocation, and receptor binding) were designed and cloned into Escherichia coli for expression. Recombinant proteins were then purified through GST affinity chromatography preceding elution of GST-free recombinant domains by thrombin protease. Soluble recombinant proteins encoding catalytic light chain and translocation N-terminal heavy chain were subsequently used to perform in vivo immunization. Polyclonal mouse antibodies specific to these domains were raised, confirmed by Western blot analysis and elevated immunoreactivity was identified through indirect ELISA. In conclusion, availability of the recombinant protein provides an effective system to study the immunological aspects of BoNT/F and corresponding applications in pathogen detection and vaccine candidacy. Keywords: Clostridium botulunium; Botulinum Neurotoxin Type F (BoNT/F) domains; cloning; recombinant protein expression; immunoreactivity Introduction Botulinum neurotoxins (BoNTs), the most potent of all biological substances known to date, are produced by several species of the genera Clostridia (C. botulinum, C. butyricum, and C. baratii) [1-3]. Seven immunologically distinct botulinum neurotoxin serotypes BoNTs/A-G are produced and implicated in botulism poisoning [4]. Botulism is a serious neuroparalytic disease [5], which generally occurs through ingestion of preformed toxin or rarely, through infection of wounds. The Center for Disease Control and Prevention (CDC) classifies BoNTs, among the six highest risk threat agents for bioterrorism “class A biological warfare agent” [6, 7]. Despite their potential to be used for deleterious purposes, BoNTs have increasing Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  1  –  15                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   2 applications in cosmetics [8] and therapeutics for the treatment of numerous dystonias, inflammation, and chronic pain [9-11]. Botulinum neurotoxin serotype F (BoNT/F), is a member of the botulinum neurotoxin family as a single ~150 kDa inactive polypeptide chain post-translationally nicked, forming a dichain consisting of a C-terminal ~100 kDa heavy chain (HC) and a N-terminal ~50 kDa light chain (LC) linked by a disulphide bond [12, 13]. BoNT/F cleaves its substrate vesicle associated membrane protein (VAMP) at position (Gln58-Lys59) [14, 15], one of three neuronal proteins associated with exocytosis; subsequently inhibiting acetylcholine release, resulting in death by flaccid paralysis [16, 17]. Each BoNT/F partial fragment; light chain catalytic domain (rF-LC), N-terminal half of the heavy chain translocation domain (rF- HN), the C-terminal half of the heavy chain receptor binding domain (rF-HC), and C- terminal quarter part of the heavy chain receptor binding domain (rF-HCc) plays a specific role in the toxicity mechanism [18, 19]. Botulinum neurotoxin LCs operate by zinc dependent proteolysis involved in neurotransmitter exocytosis from presynaptic termini [20]. Botulinum neurotoxin HNs possesses channel-forming capability in the acidic environment of the endosome, allowing internalization of the toxin, while HCs are involved in specific binding to the presynaptic membrane via gangliosides and a protein co- receptor [21]. Lastly, the HCc region of BoNTs are known to harbor the receptor binding neutralizing epitopes which are targets for antibodies that can specifically bind to the receptor, and show neutralizing activity against BoNT toxicity [22]. Thus, it is imperative to construct each domain for the analysis of their molecular and biochemical activities as well as for the development of potential neutralizing antibodies specific to each. To achieve this goal, BoNT/F domains from C. botulinum were cloned and expressed using a high expression vector and compatible host E. coli strains to obtain high quality of recombinant proteins suitable for administration into mice. Methods Chemicals, Buffers, and Reagents Components related to DNA manipulation, including Ex-Taq polymerase, dNTP, and restriction enzymes were purchased from Takara Bio. Inc., (Shiga, Japan). Luria Bertani (LB) media and cell culture media were purchased from Becton Dickinson and Company (MD, USA) and Hyclone (UT, USA) respectively. Additional chemicals including ampicillin, IPTG, Freund’s complete and incomplete adjuvant, and buffers were purchased from Sigma Aldrich (MO, USA). Bacterial strains, plasmids, and purification Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  1  –  15                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   3 systems C. botulinum type F strain Langeland [23] and BoNT/F (Wako Pure Chemicals Ind., Osaka, Japan) was kindly supplied by Medy-Tox Inc. Plasmids pGEX-4T-1 (Amersham Pharmacia Biotech acquired by GE Healthcare, Uppsala, Sweden) and pET-32a(+) (Novagen, EMD Chemicals Inc., affiliate of Merck KGaA, Darmstadt, Germany) were used for the construction of expression vectors. E. coli strains DH5α (Takara Bio Inc.), BL21- CodonPlus-RIL and BL21-CodonPlus(DE3)- RIL (Stratagene, CA, USA) were used as host strains for propagation and expression of recombinant proteins, respectively. Glutathione-S-Transferase (GST) purification modules and thrombin protease were purchased from Amersham Pharmacia Biotech. Construction of BoNT/F domains Chromosomal DNA was isolated from C. botulinum F str. Langeland, which was used as the template for polymerase chain reaction (PCR) for all domains (Fig. 1). Forward and reverse primers for each domain were designed (Table 1) based on the published NCBI sequence of the Langeland genome (NC_009699). Synthetic genes encoding BoNT/F LC (light chain), catalytic domain (1308 bp); HN (N-terminal 1/2 of heavy chain), translocation domain (1266 bp); HC (C-terminal 1/2 of heavy chain), receptor binding domain (1263 bp); and HCc (far C- terminal quarter part of heavy chain), (501 bp) were designed based on the published BoNT/F sequence (ORF: 883635-887471) Langeland str. (NCBI GenBank Accession: NC_009699) for the plasmid construction of each domain. bp indicates base pair of DNA. 1kb marker is provided for scale. TABLE 1. Primers used for amplification of BoNT/F domains. aPrimer direction: F, forward; R, reverse. bDirection of each sequence is in 5' to 3' orientation (underlined) including restriction endonuclease sequence (bold) and inserted stop codon sequences (blue) for LC and HN reverse primers. cNucleotide positions based on published C. botulinum F str. Langeland genome sequence (NCBI Genbank accession: NC_009699). dRestriction endonucleases utilized to digest PCR amplified domain and vector for plasmid construction. Isolation of genomic DNA was done utilizing protocols and chemicals mentioned by Wizard Genomic DNA Purification Kit (Promega, WI, USA). PCR was performed using a Bio-Rad iCycler (CA, USA). Reaction mixtures were preheated for 5 min at 94 °C for initial denaturation and then 30 cycles of PCR (denaturation, annealing, and elongation) were performed for amplification of domains as follows: 1 min at 94 °C, 1 min at 57 °C, and 1 min 30 sec at 72 °C for F-LC; 1 min at 94 °C, 1 min at 50 °C, and 1 min 30 sec at 72 °C for F-HN and F-HC; 1 min at 94 °C, 1 min at 54.5 °C, and 40 sec at 72 °C for F-HCc. Following completion of cycles, a final extension was LC 1 kb Scale S S HN HC 1308 bp 1266 bp 1263 bp HCc 501 bp Native BoNT/F (3837 bp) 883635 887471 _____________________________________________________________________________________________________________________________ Primera Sequence 5' ! 3'b Nucleotide positionsc Restriction enzymesd _____________________________________________________________________________________________________________________________ F-LC-F CGTGTCGACATGCCAGTTGTAATAAATAGTTTT 883635-883658 SalI F-LC-R CCGGCGGCCGCTTATTTTCTAGGAATAACGCTCTT 884942-884922 NotI F-HN-F CGTGGATCCGGTACAAAGGCGCCACCGCGACTA 884943-884966 BamHI F-HN-R CCGCTCGAGTTAATATAAAATTAGAATTTTATC 886209-886188 XhoI F-HC-F CGTGGATCCTTTAATAAATTATATAAAAAAATT 886209-886232 BamHI F-HC-R CCGCTCGAGTTAGTTTTCTTGCCATCCATGCTC 887471-887448 XhoI F-HCc-F CGTGGATCCGATAAGTCTATTACTCAGAATTCA 886971-886994 BamHI F-HCc-R CCGCTCGAGTTAGTTTTCTTGCCATCCATGCTC 887471-887448 XhoI _____________________________________________________________________________________________________________________________ Fig.1: Schematic diagram demonstrating BoNT/F domains for cloning. Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  1  –  15                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   4 carried out for an additional 7 min at 72 °C. Amplified PCR products were purified by agarose gel elution kit (Intron Biotechnology, The Republic of Korea), and resultant PCR products were digested with restriction enzymes (Table 1) and subcloned into pGEX- 4T-1 vector using T4 ligase kit (Promega) for overnight at 16 °C, so that the correct reading frame was incorporated along the thrombin cleavage site under the GST gene (representative constructs made from MacVector 10.0.2 (NC, USA) software containing F-LC and F-HN domains are shown in Fig. 2). Ligated samples were transformed into DH5α by heat-shock (42 °C for 50 sec) method and positive clones with appropriate insert were screened out by use of LB agar plates containing 100 µg/ml ampicillin. Positive colonies containing the ligated constructs were transformed into BL21-RIL for the expression of type F recombinant domains. Expression and purification of recombinant proteins To monitor the induction of recombinant domains in BL21-RIL, 2 ml of transformant cultures were inoculated and grown in LB broth and induced at various time intervals using different concentrations of isopropyl β-D-1-thiogalactopyranoside (IPTG). In order to purify recombinant proteins, mass production was performed by inoculating 2 ml induced culture in 1 liter followed by centrifugation (12,000 xg) at 4 °C for 20 min. Pellet was then resuspended in 10 ml of 50 mM phosphate buffered saline (PBS) and cells were then lysed by sonication, with 10 short bursts of 30 sec followed by intervals of placing samples on ice for 1 min cooling. 2 (a) 2 (b) Fig.2: Schematic diagram of constructed plasmids generated by MacVector 10.0.2 software (Symantec Corporation). Plasmids pGEX-4T-1-F-LC (a) and pGEX-4T-1-F-HN (b) encoding catalytic and translocation domains. Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  1  –  15                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   5 After centrifugation at 12,000 xg for 30 min, the pellet was resuspended in 10 ml PBS overnight at 4 °C and supernatant was placed in a GST affinity chromatography column after column was initially washed multiple times with deionized water and PBS, followed by normalization with GST resin. The large scale of GST-fusion proteins was eluted by single step affinity chromatography (containing sepharose 4B beads). Fractions containing desired proteins were pooled and dialyzed for 2 h at 4 °C against PBS. For thrombin protease treatment, resin bound GST-fusion proteins were cleaved with 20 units thrombin incubating for 30 min at 37 °C followed by 15 min at room temp (20 - 25 °C) and final elution with 500 µl PBS. The eluted proteins were further dialyzed to maintain salt concentrations and pH. Protein concentrations were measured according to the Bradford method using a Bio-Rad model 550 microtiter plate reader. All proteins were labeled, aliquoted and stored at – 70 °C prior to use. Lastly, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on a 12% gel under reducing conditions in order to determine solubility of the eluted proteins. Production and analysis of polyclonal antibodies against rF-LC and rF-HN For immunization, three female Balb/c mice (four weeks old having around 18-22 gram body weight) were immunized intraperitoneally with 10 µg of antigen mixtures containing recombinant proteins (25 µg GST free F-LC or 25 µg GST free F-HN) in PBS. Additionally, an equal volume of Freund’s complete adjuvant was separately injected into each mouse. After two weeks lapse, the mice were immunized with the mixture containing 20 µg of recombinant protein and equal volume of Freund’s incomplete adjuvant. At the start of the fourth week following the initial immunization, the mice were finally boosted by intravenous injection of the recombinant proteins (30 µg) without adjuvant. Three days following final boost, mice were then bled from the tail and tested by indirect ELISA. Preparation of monoclonal antibodies (hybridoma technology) Cell fusion and culture of hybridomas was carried out according to the protocol by Kohler et al. [24]. Briefly, cultures were incubated at 37 °C in an incubator with 5% CO2-in-air and 98% relative humidity. Four days following final boost, the mouse showing the highest antibody titer by ELISA was sacrificed by cervical dislocation and its spleen was removed aseptically. Spleen cells (1x108 viable cells) were mixed with SP2/0- Ag 14 myeloma cells (1x107viable cells) and Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  1  –  15                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   6 grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone). The addition of 50% polyethylene glycol 4000 (PEG) (Sigma Aldrich) allowed for the fusion of mixed cells; resulting in hybrid cells. Hybrid cells were screened by incubation in 96-well plates (Nunc) containing hypoxanthine-aminopterin-thymidine (HAT) selection medium in combination with mouse feeder cells for one week. Antibody production from hybrid cells was monitored through indirect ELISA; positive cells were expanded into 24-well plates (Nunc) containing hypoxanthine-thymidine (HT) media supplemented with dilute aminopterin according to the standard protocol by Harlow and Lane [25]. ELISA-positive hybridomas were selected and cloned twice via limiting dilution; one cell per well into 96-well plates supplemented with HT media and feeder cells. Finally, cloned cell lines were grown in DMEM supplemented with 10% FBS and stored in liquid nitrogen. Indirect enzyme linked immunosorbent assay (ELISA) For the measurement of serum antibody titers, ELISA was performed as a standard protocol mentioned by Sigma Aldrich, with only minor modifications. Each well of 96 well plates (Nunc, Copenhagen, Denmark) was coated with antigen (100 µl containing 100 ng of either GST free recombinant protein or 100 ng BoNT/F) and incubated at 4 °C overnight. Each well was washed once with 200 µl of PBS containing 0.05% Tween 20 (PBST) and blocking solution (100 µl 1% skim milk) was added and incubated for 30 min at 37 °C. Following two more washes, the serum (1: 1000 dilution) in PBS was added as the primary antibody. The plates were incubated at 37 °C for 90 min, and then washed three times as described above. Goat anti-mouse IgG conjugated with alkaline phosphatase was added as the secondary antibody (diluted 1:2000 with PBS) and incubated for 2 h. After incubation with IgG, plates were washed four times prior to substrate treatment and visualization. Immobilized antigens were visualized with the p-Nitrophenyl phosphate (disodium) in substrate buffer containing 9.7% diethanolamine, and 1 mM MgCl2 and the resultant absorbance was measured at 415 nm with microplate reader (Model 550 Bio-Rad) for 5 - 10 min at 37 °C. Western blot analysis Western blot experimentation was adapted from previous methodology [26]. The protein samples were separated by SDS-PAGE and electroblotted for ~70 min by using 230 mA current onto a nitrocellulose membrane (Schleicher and Schuell Inc., NH, USA). Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  1  –  15                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   7 Subsequently, the membrane was blocked for nonspecific binding incubating with blocking solution for 1 h and then incubated with primary antibody overnight at room temp. The membrane was washed two times with PBST and incubated with alkaline phosphate conjugated goat anti-mouse IgG as a secondary antibody for 2 h. After washing three more times with PBST, the membrane was color developed by using 10 ml alkaline phosphate substrate solution, 50 µl nitro blue tetrazolium chloride, and 50 µl 5-bromo-4- chloro-3-indolyl phosphate. Results and Discussion The cloning of both light and heavy chain domains using synthetic genes has been reported for various BoNTs [27-33]. However, there exist few studies on the cloning and expression of BoNT/F domains [34, 35]. Construction of recombinant plasmids containing pGEX-4T-1-F-LC, -HN, -HC, - HCc was made for subsequent GST-tagged expression in E. coli. Initially, sequences of BoNT/F DNA domains were PCR amplified from genomic DNA of C. botulinum F str. Langeland and flanked by restriction sites (Table 1). Specifically, the amplified products were cloned into pGEX-4T-1 after digestion with respective restriction enzymes. Ligated constructs (for reference see schematic diagrams of constructed plasmids made by MacVector software 10.0.2, Fig. 2) were transformed into DH5α for propagation and positive colonies were identified after screening on plates containing ampicillin. Subsequently the recombinant plasmids were digested with restriction enzymes (Table 1) and appropriate band sizes were identified for both pGEX-4T-1 (4.9 kb) and all individual fragment sizes (Fig. 3). For confirmation of domain homology incorporated into our vector, sequencing was performed. After sequence analysis, plasmid DNA of positive clones had 100% identity with native sequences (data not shown). For protein expression, the four recombinant clones were transformed into E. coli BL21- CodonPlus-RIL (strain used for protein expression with vectors driven by non-T7 promoters). The BL21-CodonPlus-RIL was chosen because of its capability to express rare codons which allows for the high-level expression of recombinants [36]. Thus, BL21- CodonPlus-RIL was a compatible host strain for our pGEX-4T-1 plasmid systems (containing a tac promoter). All domains were expressed in the form of fusion proteins with GST localized at the N-terminal of the fusion protein, enabling the Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  1  –  15                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   8 (i) Visible bands of amplified F-LC product (3a), F-HN (3b), F-HC (3c), and F-HCc (3d); (ii) Endonuclease restricted bands of purified pGEX- 4T-1-F-LC with SalI/NotI (3a), pGEX-4T-1-F-HN (3b), pGEX-4T-1-F-HC (3c), and pGEX-4T-1-F-HCc (3d) with BamI/XhoI. M denotes DNA marker and Lane 1, DNA sample loaded. ease of purification using GST affinity chromatography. Expression of clostridial proteins at 37 °C has been shown to increase protein degradation [37], thus we chose an optimal temperature range of 20 - 25 °C for our expression studies. Conditions for expression of recombinant proteins were as follows: 7 h at 24 °C using 0.5 mM IPTG concentration for GST-F-LC, 8 h at 22 °C using 0.4 mM IPTG concentration for GST-F-HN, 8 h at 20 °C using 0.25 mM IPTG concentration for GST-F- HC, and 8 h at 20 °C using 0.10 mM IPTG concentration for GST-F-HCc. GST-F-LC and GST-F-HN induction was monitored by SDS- PAGE (Fig. 4) and were found to be highly over-expressed in soluble form and purified at high concentrations. Despite induction at low temperature and IPTG concentrations, GST-F-HC and GST-F-HCc were expressed in inclusion bodies. Change in expression vectors from pGEX-4T-1 to pET- 32a(+) was then carried out in attempt to obtain soluble forms of GST-F-HC and GST-F-HCc. pET-32a(+) was subsequently transformed into BL21-CodonPlus(DE3)-RIL host cells, that utilizes the T7 RNA polymerase promoter. M 1 M 1 Insert F-LC (1308 bp) Vector pGEX-4T-1 (4.9 kb) bp 300 400 850 650 1650 3000 2000 4000 5000 500 1000 100 400 500 3000 2000 5000 1000 300 200 650 850 1650 4000 Amplified F-LC PCR product (1308 bp) a (i) bp a (ii) Amplified F-HN PCR product (1266 bp) 3000 M 1 500 1000 850 650 1650 2000 bp b (i) M 1 Insert F-HN (1266 bp) 500 650 1000 850 1650 3000 2000 4000 5000 bp Vector pGEX-4T-1 (4.9 kb) b (ii) M 1 Amplified F-HC PCR product (1263 bp) bp 100 400 500 3000 2000 5000 1000 300 200 650 850 1650 4000 c (i) M 1 Insert F-HC (1263 bp) Vector pGEX-4T-1 (4.9 kb) 300 400 850 650 1650 3000 2000 4000 5000 500 1000 bp c (ii) 2000 1000 1650 4000 M 1 Amplified F-HCc PCR product (501 bp) bp 100 400 500 300 200 650 850 d (i) M 1 Insert F-HCc (501 bp) Vector pGEX-4T-1 (4.9 kb) d (ii) 2000 1000 1650 4000 bp 100 400 500 300 200 650 850 Fig.3: Agarose gel (1%) electrophoresis of PCR product and restriction digested plasmid stained with ethidium bromide and visualized by UV Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  1  –  15                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   9 IPTG induction was carried out in a time dependent manner and SDS–PAGE (12 %) analysis of total lysate of E. coli BL21- (pGEX-4T-1-F-LC (a) and pGEX-4T-1-F-HN (b) with or without IPTG induction stained with Coomassie blue. Lane M: protein marker (Bio-Rad); Lanes 1 - 4: (0, 2, 5, and 7 h for F-LC and 0, 2, 5, and 8 h for F-HN) after IPTG induction. Lanes 2 - 4 demonstrate expressed GST-F-LC a) and GST-F-HN (b) with treatment of IPTG. kDa, protein size expressed in kiloDalton. Although both vectors contain thrombin cleavage sites and are ideal for production of soluble proteins, neither one was able to alter the formation of inclusion bodies. Protein- refolding procedures were performed using reducing reagents including various molar concentrations of urea or guanidine – HCl to solubilize the inclusion bodies but attempts were unsuccessful. As a result, only LC and HN domains were used for protein analysis and collection of polyclonal serum from immunized mice. GST affinity chromatography was then carried out to collect GST tagged LC and HN. After GST affinity chromatography and cleavage with thrombin protease treatment, recombinant GST free F-LC and GST free F-HN were analyzed on SDS-PAGE to monitor expected protein bands. SDS-PAGE revealed the appropriate size of both recombinants; GST fused 75 kDa and GST free 50 kDa (Fig. 5). Final recombinant protein concentrations were determined to be 1.0 mg/l. In order to analyze specificity of mice polyclonal sera against F- LC and F-HN, mice were immunized intraperitoneally with antigens; GST free F- LC and F-HN. To analyze the specificity of the polyclonal serums against F-LC and F- HN, mouse polyclonal serum was collected from the tail vain and tested by Western blotting and indirect ELISA (both of these techniques being highly valuable in the detection of candidate biomarkers). The mouse anti-F-LC polyclonal antibodies were found to specifically recognize F-LC and BoNT/F (Figs. 5a and 6a). Similarly, anti-F- HN polyclonal antibodies demonstrated a M 1 2 3 4 kDa 25 75 50 37 100 150 250 15 10 M 1 2 3 4 kDa 25 75 50 37 100 150 250 15 GST-F-LC GST-F-HN SDS-PAGE (12%) 4 (a) SDS-PAGE (12%) 4 (b) Fig.4: Induction and expression analysis of recombinant proteins Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  1  –  15                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   10 strong affinity towards F-HN and native type F toxin (Figs. 5b and 6b). However, neither anti-F-LC nor anti-F-HN polyclonal antibodies were able to detect the HC domain (Fig. 5), validating the specificity of the polyclonal serums towards their respective region of BoNT/F. The designated recombinant domains have native structure as holotoxin and it was not unexpected for them to produce anti-sera against native toxin (BoNT/F). Mouse showing highest immunoreactivity was then sacrificed in attempt to isolate a monoclonal antibody specific to F-LC or F- HN domains. This could allow further research into the development of a highly effective neutralizing monoclonal antibody for a subunit vaccine and/or passive immunotherapy against BoNT/F. (i) SDS–PAGE of purified GST F-LC and GST free F-LC (a) Purified GST F-HN and GST free F-HN (b) by single step GST affinity chromatography separated on a 12% gel and visualized by Coomassie blue stain; (ii) Western blot analysis performed by raising anti-F-LC (a) and anti-F-HN (b) mouse polyclonal antibody. Lane M: protein marker (Bio-Rad); Lane 1, GST-F-LC or HN; Lane 2, GST free-F-LC or HN; and Lane 3, unpurified GST-F-HC. Fig.5: Purification and characterization of recombinant BoNT/F domain proteins SDS-PAGE 5b (i) M 1 2 3 M 1 2 3 kDa 25 75 50 37 100 150 250 SDS-PAGE 5a (i) kDa 25 75 50 37 100 150 250 Western blot 5a (ii) GST-F-LC GST free-F-LC 25 75 50 37 100 150 250 M 1 2 3 M 1 2 3 kDa kDa Western blot 5b (ii) 25 75 50 37 100 150 250 GST-F-HN GST free-F-HN Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  1  –  15                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   11 Bar graph values represent interaction of anti F-LC (a) and anti-F-HN (b) mouse polyclonal serum antibody with: PC = BoNT/F as a coating antigen; S. 1 - 3 = samples of GST free F-LC (a) and GST free F-HN (b) coating antigens respectively & interaction of non-immunized mouse serum with: NC= GST free F-LC (a) and GST free F-HN (b) used as coating antigens respectively. Mouse polyclonal serum was harvested from three different mice (from different batches) immunized with GST free F-LC or GST free F-HN. PC- positive control, NC- negative control. n = 3 wells, two-tailed students t-test, S. 1 - 3 compared to NC, p < 0.05. Attempts were unsuccessful in isolating a monoclonal antibody specific towards our constructed BoNT/F domains (data not shown). However, we have successfully isolated a mouse monoclonal antibody that demonstrates neutralizing activity against native BoNT/F toxin resulting from BoNT/F toxiod in vivo immunization and hybridoma technology [38]. Conclusion This paper describes molecular and immunological studies on C. botulinum neurotoxin type F domains. Synthetic genes encoding BoNT/F partial fragments: catalytic domain (rF-LC), translocation domain (rF- HN), receptor binding domain (rF-HC) and quarter part of HC (rF-HCc) were designed and cloned into E. coli. Additionally, expression, purification, and immunoreactivites were analyzed by Western blotting and indirect ELISA for LC and HN domains. The use of GST tagged recombinant protein technology was chosen in order to optimize detection and acquisition of high purity, stable, and soluble proteins through use of single step affinity chromatography. Although host strains, vector types, and induction conditions for expression were optimized to recover recombinant proteins in the soluble fraction, we were unable to purify HC and HCc. Immunoreactivity was accessed through GST free F-LC and F-HN through in vivo immunization and polyclonal serum antibody collection, proceeded by Western blotting and indirect ELISA. The anti-F-LC and anti-F-HN mouse 6 (a) 6 (b) Fig.6:Immunoreactivity analysis by Indirect ELISA Nepal  Journal  of  Biotechnology.    Jan.  2012,  Vol.  2,  No.  1:  1  –  15                                                                                                                    Biotechnology  Society  of  Nepal  (BSN),  All  rights  reserved   12 polyclonal antibodies exhibited a strong affinity towards GST free and GST tagged F- LC and F-HN respectively, while both recognized the native type F toxin. From this work, we conclude that purified BoNT/F LC and HN domains are capable of producing highly effective immunogens. Moreover, if future attempts prove fruitful in the isolation of monoclonal antibodies (that possess neutralizing activity), towards BoNT/F fragments, this may lead to more efficient protection against a high neurotoxin dose. Implications of this research could span across the development of therapeutic approaches, diagnostic detection systems, and vaccine candidacy for the protection and treatment of botulism as has been elucidated in previous work [34, 37, 39-42]. In summary, we have expressed both recombinant LC and HN domains of BoNT/F. The recombinant proteins are soluble; elicit an active immune response (polyclonal antibodies) in mice, making them ideal for investigators to process potential subunit vaccines towards BoNT/F. Acknowledgements All authors wish to acknowledge the financial support from Medy-Tox Inc. and for their help in the acquisition of C. botulinum F str. Langeland and BoNT/F (Wako Pure Chemicals Ind., Osaka, Japan). We would also like to express our gratitude to Jesus Andres Cuaron for his contributions on the drafting of this article. References 1. 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