I. INTRODUCTION
7. Circular dichroism
Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise because of structural asymmetry. CD determines whether a protein is folded and characterizes protein’s secondary structure, tertiary structure. Secondary structure can be measured by CD in the far-UV spectral region (190 - 250 nm). At these wavelengths, the chromophore is the peptide bond. Alpha-helix, beta-sheet, and random coil structures each give rise to a characteristic shape and magnitude of CD spectrum (Fig. 5). The CD signal
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reflects an average of the entire molecular population. The DC spectrum of a protein in the near-UV (250 – 350 nm) can be characterized tertiary structure of protein. At these wavelengths, the chromophores are the aromatic amino acids and disulfide bonds. The CD signals they produce are susceptible to the overall tertiary structure of the protein.
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Fig. 1. Metabolic conversion of aflatoxin B1 and following DNA adduct formation mutating hepatocyte DNA to carcinogenesis
(Smela et al., 2001 and Iain et al., 2006)
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Fig. 2. Subunit composition and domain distribution of immunoglobulin and single-chain variable fragment (scFv) antibody
(Ahamad et al., 2012)
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Table 1. Antibody classes
Charateristic/Fuction IgG IgA IgM IgD IgE Molecular Weight (KDa) 150 400 900 180 190
Heavy chain Γ α Μ Δ ε
Number of subunits 1 2 5 1 1
Serum level (㎍/㎖) 13.5 3.5 1.5 0.03 0.003 Allotypes G1,2,3,4 A1,2
Additional components J chain J chain
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Table 2. Rarely used codons in E. coli (Dominic et al., 2006)
Amino acid Rare codon(s)
Arginine AGG, AGA, CGG, CGA
Leucine CUA, CUC
Isoleucine AUA
Serine UCG, UCA, AGU, UCC
Glycine GGA, GGG
Proline CCC, CCU, CCA
Threonine ACA
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Fig. 3. Protein expression using solubility tags (Dominic et al., 2006)
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Table 3. Generally used solubility-enhancing fusion partners
Tag Source organism
MBP (Maltose-binding protein) Escherichia coli GST (Glutathione-S-trasferase) Schistosoma japonicum
Trx (Thioredoxin) Escherichia coli
NusA (N-Utilization substance) Escherichia coli SUMO (Small Ubiquitin-modifier) Homo sapiens
SET (Solubility-enhancing tag) Synthetic DsbC (Disulfide bond C) Escherichia coli Skp (Seventeen kilodalton protein) Escherichia coli T7 PK (Phage T7 protein kinase) Bacteriophage T7
GB1 (Protein G B1 domain) Streptococcus sp.
ZZ (Protein A IgG ZZ repeat domain) Staphylococcus aureus
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Fig. 4. Toxic protein such as green fluorescent protein (top) or red fluorescence inducing protein (bottom) expressed in C41 and BL21
(Lucigen, United States)
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Fig. 5. Standard curve of secondary structure; alpha-helix, beta sheet, and random coil by circular dichroism
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7. O
BJECTIVES OF THE THESISThis study is focused on the production of single chain variable fragment (scFv) in active form in engineered Escherichia coli. The specific objectives of this research were as follows:
1) Evaluation of factors such as codon optimization, maltose binding protein affinity tags, or E. coli strains affecting soluble expression of aflatoxin B1 scFv.
2) Applications of the expression strategies to other types of scFv.
3) Analysis of the physico-chemical and immunological properties of the purified aflatoxin B1 scFv.
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II. MATERIALS AND METHODS
1. Plasmids and strains 1.1 Enzymes and reagents
AFB1 conjugated to bovine serum albumin (AFB1-BSA conjugate) was bought from Sigma Aldrich Co. (St. Louis, MO, USA). Restriction endonucleases, T4 DNA ligase, Taq polymerase, Klenow fragment, and calf intestinal alkaline phosphatase (CIP) were purchased from New England Biolabs (Beverly, MA). Pwo DNA polymerase and dNTPs were from Hoffmann-La Roche (Basel, Switzerland). Phosphate buffered saline (PBS: 0.01 M phosphate buffer with 0.138 M NaCl, 0.0027 M KCl), phosphate buffered saline tween 20 (PBST: 0.01 M phosphate buffer with 0.138 M NaCl, 0.0027 M KCl, 0.05 % Tween 20), carbonate-bicarbonate buffer capsules (0.05M carbonate-bicarbonate buffer, pH 9.6), bovine serum albumin (BSA), phosphate-citrate buffer tablets (0.05M phosphate-citrate buffer, pH 5.0, 1 tablet/100 ml), 5'-tetramethyl benzidine dihydrochloride (TMB), hydrogen peroxide were obtained from Sigma Chemical Co. (St. Louis, USA.). A molecular weight standard of DNA was obtained from New England Biolabs (Beverly, MA) and a protein standard for electrophoresis from Sigma Chemical Co. (St. Louis, MO). Agarose, ampicillin, ethidium bromide, Trizma base, imidazole were purchased from
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Sigma Chemical Co. (St. Louis, MO). Bacto-peptone, tryptone, yeast extract, and bacto-agar was purchased from Difco Laboratories (USA). All chemicals were of reagent grade. HiTrapFFfor purification of proteins fused with the His 6 residues and PD-10 desalting column were purchased from GE healthcare (Sweden). Quick Start Bradford Protein Assay Kit 2 was purchased from Bio-rad (USA).
1.2 Oligonucleotides
Oligonucleotides and codon optimized scFv gene were synthesized by Bioneer (Korea) (Fig. 9 and Fig. 10). They were synthesized with different restriction enzyme sites on the ends as primers according to the purpose of experiments, which are PCR amplication, construction of vector and recombination of gene.
1.3 Strains and plasmids
Escherichia coli Top10 [F- mcrA ∆(mrr-hsdRMS-mcrBC) φ80lacZ∆M15
∆lacX74 recA1 araD139 ∆(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG] (Invitrogen, CA, USA) was used for plasmid preparation for DNA manipulation. E. coli BL21(DE3) [F- ompT gal dcm lon hsdSB (rB-, mB-) λ(DE3 [lacI lacUB6-T7 gene 1 lnd1 sam7 nin5])] (Novagen, Germany) and E. coli C41(DE3) [F- ompT hsdSB (rB-, mB-) gal dcm (DE3)] (Lucigen, United
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States) were used as host strains for the expression of target proteins. E. coli Origami (DE3) [ F-ompT hsdSB(rB- mB-) gal dcm lacY1 ahpC (DE3) gor522::
Tn10 trxB (KanR, TetR)] was used as host strains for expression of target protein without signal sequence to periplasm.
Plasmids, pET19b were used as mother vectors which has the T7 promoter for expression of AFB1 scFv and maltose binding protein (MBP) fusion proteins. Aflatoxin B1 scFv genes was from plasmids of Won-Ki Min and Jae-Chan Park (Fig. 6). Fumonisin B1 and deoxynivalenol scFv genes were respectively from Jun-Bock Park (Fig. 7) and Gyu-Ho Choi (Fig. 8). MBP genes were from pMAL-p2X vector (New England Biolabs, USA) by polymerase chain reaction (PCR).
1.4 Recombinant DNA techniques
All of cloning steps were carried out according to the procedure of Sambrook et al (Sambrook et al., 1989). Mini-scale plasmid preparation was carried out with the High Pure Plasmid Isolation kit (Roche, Switzerland) and large-scale plasmid preparation was achieved using Plasmid Midi Kit (Qiagen, USA). Digestion of DNAs with restriction endonuclease, and dephosphorylation were achieved under the conditions recommended by the supplier, and the results of digestion were analyzed by agarose gel electrophoresis. This system was provided by Bio-Rad (USA). The isolation
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of DNA fragments from agarose gel, solubilization of the gels and recovery of the DNA fragments were conducted by “High Pure PCR Product Purification Kit” from Roche (Switzerland) or “QIAquick Gel Extraction Kit”
from Qiagen (USA). Ligation of the DNA fragments was carried out by
“DNA Ligation Kit” from Takara (Japan).
1.4.1 Polymerase chain reaction (PCR)
All PCRs for amplification were performed with the GeneAmp 2400 (Applied Bioscience, USA). AccuPower PCR PreMix (Bioneer Co., Daejon, Korea), ready-to-use PCR reagent fully optimized for more accurate PCR amplification, was used in the PCR reaction. PCRs for cloning of genes were performed containing 10 pM each of forward and reverse primers, and plasmid DNA as a template. Reaction time and temperature were optimized according to each reaction condition. The amplified gene was confirmed by gel electrophoresis. Primers in this study are listed in Table 4.
1.4.2 Construction of expression plasmids
Expression plasmids, pET19b s.s malE Xa scFv H6, pET19b H6 scFv Xa malE, pET19b s.s malE Xa scFV op H6, pET19b pelB scFv op Xa malE H6, pET19b malE Xa scFV op H6, and pET19b H6 scFv op Xa
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malE were constructed from pET19b, which purchased from Novagen (USA). The factors to be analyzed are codon optimization gene, direction of MBP fusion protein, existence of signal sequence, and plasmids expression hosts. The 6 histidine residues were fused to the N and C-terminal for efficient purification of proteins. The ligation of the expression vectors was performed using the DNA ligation kit according to manufacturer’s protocols.
1.5 DNA sequencing
DNA sequencing was performed by Mbiotech (Hanam, Korea). The results of DNA sequencing for selected clone candidates were compared each other, and then the selected DNAs were used in the subsequent experiments.
2. Expression of proteins
2.1 Transformation and expression of fusion proteins
Transformation of E. coli was carried out as described by Sambrook et al.
(Sambrook et al., 1989). An E. coli strain Top10 was incoculated in 5 mL LB medium, and precultured at 37°C overnight. A 1% aliquot of the cultured cells was transferred into 50 mL of fresh LB medium and incubated with
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shaking until OD at 600 nm reached 0.5. The pellet which had been harvested by centrifugation at 6,000 rpm for 5 min at 4°C was resuspended cautiously in 5 mL of ice-cold 100 mM CaCl2 solution and stored on ice for 30 min. The cell suspension was centrifuged (6,000 rpm for 5 min at 4°C) and the pellet was resuspended in 5 mL of 100 mM CaCl2 solution.
Resuspended cells were aliquoted to 100 ㎕, mixed with ligated DNA, and
kept on ice for 30 min. They were subjected to heat-shock at 42°C for 45
E. coli BL21(DE3) and C41(DE3) colonies were picked from LB agar plates with ampicillin containing fresh transformed cells and cultured in 5 mL LB medium in the presence of ampicillin for about 12 hr at 37°C. Flask cultures were carried out with LB medium in 500 mL baffled flasks with a 100 mL working volume. Each flask was inoculated with 1.0 mL of the transformed
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cell, pre-cultured in the log phase and was grown at 37°C in LB medium containing selective markers. Expression of scFv and MBP fusion proteins were induced by the addition of isopropyl-2-D-thio-galactopyranoside (IPTG) to final concentration of 0.2 mM.
2.2 SDS-PAGE
After induction, the induced cells were grown for about additional 4 h, were centrifuged at 10,000 rpm, resuspended in 100 mM sodium phosphate buffer (pH 7.4) and then were treated by sonication for cellular protein assay. The crushed cells were centrifuged at 12,000 rpm at 4°C for 15 min and the supernatant, taken as the soluble fraction, was recovered carefully. The pellet, taken as the insoluble fraction, was resuspended in an equivalent volume of the same phosphate buffer. Separation of proteins by molecular weight in SDS-PAGE, soluble and insoluble proteins were resuspended in sodium dodecyl sulfate (SDS) sample buffer and boiled at 100°C for 5 min. Heat-denatured fractions were analyzed by 10 ~ 15% SDS-polyacrylamide gel electrophoresis. Protein samples were electrophoresed on an SDS-containing discontinuous polyacrylamide gel electrophoresis unit using the Mini-Protein II system (Bio-Rad, USA). 12.5% (w/v) separating gel was prepared usually from 33.5% (w/v) acrylamide/ 0.3% (w/v) N, N’-methylenebisacrylamide stock solution in 0.38 M Tris-HCl (pH 9.1) and 0.1% (w/v) SDS. The 4%
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(w/v) stacking gel was prepared from 30% (w/v) acrylamide/0.44% (w/v) N, N’-methylenebisacryl-amide stock solution in 0.125 M Tris-HCl (pH 6.8) and 0.1% (w/v) SDS. Both gels were polymerized with ammonium persulfate and TEMED. The running buffer was composed of 25 mM Tris base, 192 mM glycine, and 0.1% (w/v) SDS. Samples were mixed with equal volumes of 2X loading buffer [0.125 M Tris-Cl buffer (pH 6.8); 10% (v/v) β-mercaptoethanol; 4% (w/v) SDS; 20% (v/v) glycerol; a pinch of bromophenol blue] and boiled for 3 min before loading on the gel.
Electrophoresis was carried out at 90 V for stacking and at 120 V for separating. When electrophoresis was finished, the gel was stained with commassie blue R-250 solution [0.2 % (w/v) Coomassie blue R-250; 50 % (w/v) methanol; 10 % (v/v) acetic acid] for 30 min with gentle shaking and destained with destaining solution [20 % (v/v) methanol; 10 % (v/v) acetic acid].
2.3 Fed-batch fermentation
Fed-batch culture was carried out in a 2.5 L jar fermentor (Kobiotech, Seoul, Korea) with a 1 L start working volume of a Riesenberg medium. The 100 ml seed culture was prepared in a 500 ml flask and grown in a shaking incubator at 37°C and 250 rpm for 12 hr, and main culture was carried out.
To maintain the dissolved oxygen (DO) level, agitation speed and aeration
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rate were in between 1200 rpm and 1 vvm of air supply, respectively. The pH was automatically controlled at 6.8 by the pH-stat strategy. To keep the cell growth and basal level of sugar after depletion of 20 g/L sugar initially added, 28% ammonia water and high concentration of carbon source feeding solutions (800 g/L glucose and magnesium sulfate heptahydrate 20 g/L) that were used. Feeding solutions were conversed to organic acids by the metabolic processes of cells. Expression of scFv and MBP fusion proteins was induced by the addition of IPTG when O.D.at 600 nm reached 95.
3. Purification and quantitative analysis of scFv 3.1 Purification
3.1.1 Affinity chromatography
The purification of the expressed protein was carried out by the Äcta prime system (Amersham Bioscience, Sweden) using the HisTrap FF column (GE healthcare, Sweden) used in His-tagged protein purification. This column was washed with distilled water. After equilibrating the column with binding buffer [20 mM sodium phosphate (pH 8.0), 500 mM NaCl], the sample resuspended in binding buffer [20 mM sodium phosphate (pH 8.0), 500 mM NaCl] was loaded. The column loaded with the sample was washed with binding buffer, and bound proteins were eluted with elution buffer [20 mM
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sodium phosphate (pH 8.0), 500 mM NaCl, 500 mM immidazole] under constant level of immidazole concentration. Purified proteins were analyzed by SDS-PAGE (Fig. 11).
3.1.2 Desalting column
Desalting of purified protein was carried out using PD-10 desalting columns (GE healthcare, Sweden) by gravity force. The columns were washed with PBST buffer. After equilibrating the columns with PBST buffer, 2.5 mL of the protein samples was loaded to the columns. Bound proteins were eluted with 3.5 mL of PBST buffer (Fig. 11).
3.2 Quantitative analysis 3.2.1 Bradford assay
BSA (Bovine Serum Albumin) standard protein was prepared to determine standard curve for quantitative analysis of scFv and MBP fusion proteins.
BSA with concentrations of 0, 2, 4, 5 µg/mL for the standard assay was used.
scFv and MBP fusion protein samples were diluted with distilled water.
200 µl of Quick Start Bradford protein 1x dye reagent from Quick Start Bradford Assay Kit and 800 µl of protein were combined and vortexed.
Combined samples were incubated at room temperature for 5 minutes, and
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measured absorbance at 595 nm.
4. Immunological and physico-chemical analysis 4.1 Immunological analysis
4.1.1 Indirect ELISA
Antigen-binding affinity of scFv and MBP fusion proteins was examined by indirect ELISA (Enzyme linked immunosorbent assay) (Fig. 12).
Antigen-binding activity was determined by increasing signal of scFv and MBP fusion proteins according to increasing concentration of antigen.
Immune 96 Microwell Plate was coated with 0, 1, 5, 10, 50, 100, 1000, 10000 ppb of AFB1-BSA conjugate at room temperature overnight. The plate was washed by filling the wells with 200 µl of PBST buffer. After washing, plate was coated with 200 µl of 5% skim milk in 2 hours. The plate was washed with PBST buffer and coated with 100 µl of the prepared each of soluble scFv (0.01 g/L) and MBP fusion proteins and incubated at room temperature 2 hour. After washing, 100 µl of HisProbe-HRP was respectively added to each well and incubated 15 minutes. After washing, 100 µl of 5'-tetramethyl benzidine dihydrochloride (TMB) substrate solution was added and incubated in 30 minutes. 2 M sulfuric acid solution was added
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to each well to stop color development. Absorbance of samples was measured with a microplate reader at 450 nm.
4.2 Physico-chemical analysis
4.2.1 Circular Dichroism Detector
Secondary structure of scFv and MBP fusion proteins in PBST buffer was analyzed by circular dichroism detector (Chirascan plus, Applied Photophysics, UK). Detector was High performance UV-Vis-IR avalanche photo-diode fluorescence monochro detector. Analyzing temperature was 25°C. Cell path length was 0.5 mm. Wavelength of CD was 190 ~ 250 nm to analyze secondary structure of proteins. PBST buffer was analyzed to determined baseline. Factor Xa protease treatment was conducted to obtain scFv without MBP to analyze secondary structure. Though, pure scFv itself was extremely unstable and remained as insoluble aggregate (Fig. 30). Thus, MBP and scFv-MBP fusion protein were analyzed. Absorbance difference of MBP and MBO-scFv was analyzed.
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5. Application of expression system to other types of scFv 5.1 Fumonisin B
1scFv
5.1.1 Expression of fumonisin B
1scFv and MBP fusion protein in E. coli C41(DE3) strain
E. coli BL21(DE3) and C41(DE3) carrying fumonisin B1 scFv and MBP fusion protein plamids were inoculated in LB medium baffled flask and grown at 37°C, 250 rpm. Expression of proteins was induced by the addition of isopropyl-2-D-thio-galactopyranoside (IPTG) to final concentration of 0.2 mM. Expression was analyzed by SDS-PAGE.
5.2 Deoxynivalenol scFv
5.2.1 Expression of deoxynivalenol scFv and MBP fusion protein in E. coli C41(DE3) strain
E. coli BL21(DE3) and C41(DE3) carrying deoxynivalenol scFv and MBP fusion protein plamids were inoculated in LB medium baffled flask and grown at 37°C, 250 rpm. Expression of proteins was induced by the addition of isopropyl-2-D-thio-galactopyranoside (IPTG) to final concentration of 0.2 mM. Expression was analyzed by SDS-PAGE.
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Table 4. Sequence of the primers used in this research
Primer name Primer sequences
(5’-3’)
MalE-BspHI-F AAG CTT TC ATG AAA ATA AAA ACA GGT GCA CGC MalE-NdeI-R AAT CGC CAT ATG CCT TCC CTC GAT CCC GAG GTT pelBscFv AFB-BspHI-F AAG CTT TC ATG AAA TAC CTG CTG CCG ACC
pelBscFv AFB-NdeI AAT CGC CAT ATG CCT TCC CTC GAT ACC TAG GAC GAG TTT GGT TCC TC
scFv AFB H6-BamH1-R AAT CGC GGA TCC TTA GTG GTG GTG GTG GTG GTG C His6 scFv AFB-Nde1-F AAT CGC CAT ATG CAC CAC CAC CAC CAC CAC ATG GAG GTG AAG
CTG CAG
His6 scFv AFB Xa-xho1-R AAT CGC CTC GAG CCT TCC CTC GAT ACC TAG GAC GAG TTT GGT TCC TC
malE-xhoI-F2 AAT CGC CTC GAG AAA ATC GAA GAA GGT AAA CTG GTA ATC malE-BamHI-R2 AAT CGC GGA TCC TTA CCC GAG GTT GTT GTT ATT GTT ATT G scFv AFB1 H6-Nde1-IF-F TCGAGGGAAGGCATATGGAGGTGAAGCTGCAGGAGTCTG scFv AFB1 H6-BamH1-IF-R GTTAGCAGCCGGATCCTTAGTGGTGGTGGTGGTGGTGC
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scFv op. H6-Nde1-IF-F TC GAG GGA AGG CAT ATG GAA GTG AAA CTG CAG GAA AGC G scFv op. H6-BamH1-IF-R G TTA GCA GCC GGA TCC TCA GTG GTG GTG GTG GTG GTG C
pelB scFv op. - Nde1 – F AAT CGC CAT ATG AAA TAC CTG CTG CCG ACC GC pelB scFv op. Xa - xho1 – R AAT CGC CTC GAG CCT TCC CTC GAT GCC CAG CAC CAG TTT GGT G
malE H6-BamHI-R2 AAT CGC GGA TCC TTA GTG GTG GTG GTG GTG GTG CCC GAG GTT GTT GTT ATT GTT ATT G
malE Xa – BspH1-F AAG CTT TC ATG AAA ATC GAA GAA GGT AAA CTG ATC H6 scFv op Xa – Nde1 – F AATCGCCATATGCACCACCACCACCACCAC ATG GAA GTG AAA CTG
CAG GAA AGC
H6 scFv op Xa – Xho1 – R AAT CGC CTC GAG CCT TCC CTC GAT GCC CAG CAC CAG TTT GGT G pelB scFv op. Xa – BspH1 – F AAG CTT TC ATG AAA TAC CTG CTG CCG ACC GC
pelB scFv op. Xa – Nde1 – R AAT CGC CAT ATG CCT TCC CTC GAT GCC CAG CAC CAG TTT GGT G malE H6-Nde1-F2 AAT CGC CAT ATG AAA ATC GAA GAA GGT AAA CTG GTA ATC malE H6-XhoI-R2 AAT CGC CAT ATG AAA ATC GAA GAA GGT AAA CTG GTA ATC FB1 scFv - Nde1 – F AAT CGC CAT ATG GAT GTA GTC ATG ACC CAG TCT CC FB1 scFv - BamH1 – R AAT CGC GGA TCC TCA GTG GTG GTG GTG GTG GTG
DON scFv - Nde1 – F AAT CGC CAT ATG CAG GTG AAG CTG CAG CAG TCT G DON scFv - BamH1 – R AAT CGC GGA TCC TCA GTG GTG GTG GTG GTG GTG
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MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLA EVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFG GYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSL IYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNA DTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKG QPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPL GAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRT AVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRHMEV KLQESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWV ATISSGGTYTYSPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYY CASHGLLWSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQE SALTTSPGETVTLTCRSSTGAVTTSNSANWVQEKPDHLFTGLIGGTN NRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHLVFG GGTKLVLGLEHHHHHH
Fig. 6. Amino acid sequence of AFB1 scFv and MBP fusion protein
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MDVVMTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPG QSPKRLIYLVSKLDSGFPDRFTGSGSGTDFTLKISRVEAEDLGVYYCW QGIHFPRTFGGGTKLEMGGGGSGGGGSGGGGSEVQLQQSGAELVKP GASVKLSCKTSGYTFTSYWIQWVKQRPGQGLGWIGEIFPGTGTTYY NEKFKGKATLTIDTSSSTVYMQLSSLTSEDSAVYFCASRRFAYWGQG TTVTVSSLEHHHHHH
Fig. 7. Amino acid sequence of fumonisin B1 scFv with 6 histidine tag
MQVKLQQSGTEVVKPGASVKLSCKASGYIFTSYDIDWVRQTPEQGL EWIGWIFPGEGSTEYNEKFKGRATLSVDKSSSTAYMELTRLTSEDSA VYFCARGDYYRRYFDLWGQGTTVTVSSGGGGSGGGGSGGGGSQAV VTQESALTTSPGGTVILTCRSSTGAVTTSNYANWVQEKPDHLFTGLI GGTSNRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYST HFVFGGGTKVTVLGLEHHHHHH
Fig. 8. Amino acid sequence of deoxynivalenol scFv with 6 histidine tag
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Fig. 9. Codon usage table of non-codon optimized scFv gene in E. coli
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Fig. 10. Codon usage table of codon optimized scFv gene in E. coli
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Fig. 11. Purification process
Cell lysis by sonication
Collection of soluble fraction
Affinity chromatography (Histag purification)
Desalting
(Buffer change to PBST buffer
for ELISA)
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Fig. 12. Diagram of indirect ELISA
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III. RESULTS AND DISCUSSIONS
1. Plasmids and strains
1.1 Construction of expression plasmids and strains
To obtain soluble and functional scFv in vivo, maltose binding protein which is expressed in high solubility in E. coli was fused with AFB1 scFv.
Direction of MBP fusion to scFv such as N or C-terminal influences expression of proteins. Both direction fusion protein expression plasmids were constructed with scFv and codon optimized scFv gene. Factor Xa protease site was inserted between scFv and MBP gene. For purification, 6 hisitidine tag was combined into the expression plasmids. pET19b s.s. malE Xa scFv H6 and pET19b H6 scFv Xa malE plasmids were constructed for non-codon optimized gene. pET19b s.s malE Xa scFv op H6, pET19b pelB scFv op Xa malE H6, pET19 malE Xa scFv op H6, and pET19 H6 scFv op Xa malE were constructed for codon optimized scFv gene. All plasmids usded and constructec in this research are summarized from fig. 13 to 16 and Table 5.
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2. Expression of proteins
2.1 scFv and MBP fusion protein expression
In order to express soluble and active scFv and elevate the expression level of scFv, maltose binding protein was fused. pET19 s.s malE Xa scFv H6 and pET19 H6 scFv Xa malE were expressed in E. coli BL21(DE3).
Control strains are E. coli BL21(DE3) carrying scFv gene without MBP fusion protein.
Maltose binding protein fusion slightly elevated the expression level in E.
coli BL21(DE3) strains (Fig. 17 and Fig. 18). However, majority of expressed scFv and MBP fusion protein was insoluble. Experimental results gave no positive effect on sloluble expression.
2.2 Codon optimized and non-codon optimized scFv gene expression in diverse E. coli host strains.
Codon optimized scFv gene to optimized translation in E. coli was fused with maltose binding protein in N and C-terminal. Periplasmic targeting signal sequence of pelB or maltose binding protein itself was inserted in express vectors.
Some of MBP fusion optimized scFv expressed in BL21(DE3), though expression pattern was insoluble (Fig. 19 to 20). N-terminal MBP fusion
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scFv with signal sequence in C41(DE3) host expressed in soluble form. (Fig.
22) Fusion protein without signal sequence was expressed in Origami (DE3) strains (Fig. 23). scFv and MBP protein expression in Origami (DE3) strains was no positive effect (Fig 24).
To verify the direction of fusion protein and compare with these result of experiments with non-codon optimized scFv gene, non-codon optimized gene was fused with maltose binding protein in N and C-terminal of scFv.
Like the preceding, N-terminal MBP fusion scFv with signal sequence in
Like the preceding, N-terminal MBP fusion scFv with signal sequence in