Chapter II Materials and Methods
2.9 Western blotting
To conduct the experiments of Western blotting of Der p 2, the supernatant was
mixed with 5x loading dye and was boiling at 100°C for 8 min, first the samples and the
rDerp 2 produced by yeasts as a standard were run on SDS-polyacrylamide gels,
respectively (running gel: 15%, stacking gel: 5%) and accompanied with LandMarker
Mid range Prestained Protein Marker (Mbiotech, Seoul, Korea). The electrophoresis
was performed under 150 V for 40 min. The gels were blotted onto Hybond-C Extra
membrane (Amersham, London, UK) at 85 V for 50 min followed by blocking with 5%
skim milk (Becton, Dickinson and Company, MD, USA) overnight. The membranes
were incubated in blocking buffer with the anti-Der p 2 polyclonal antibody as the 1st
antibody. Then the membranes were incubated with Goat anti-Rabbit IgG (H+L) HRP
conjugated affinity purified antibody as the 2nd antibody. All the incubation process was
performed at 50 rpm rotary shaker (Firstek, Middlesex, UK) at room temperature for 1 h.
Antibody binding was detected after incubation with chemifluorescence reagents
(PerkinElmer Life Science, Inc., MA, USA) and detected by UVP BioImaging System
(AutoChemTM System, CA, USA).
35
Chapter III
Results
36
3.1 Construction of the fusion allergen gene
Because the linker sequence was only 78 base pairs (Table 1), it is difficult to see
the PCR product with DNA gel electrophoresis during gene cloning, so we directly
ligated the PCR products of linker sequence and Der p 2 gene with SacII site, and then
the Der p 1 gene was added to the front with XhoI site for a full-length of our design.
The possibility of mutation during PCR is high, but with a strategy “constructed the
full-length first, and then replaced the wrong ones second”, we successfully constructed
the vector containing Der p 1 linked with Der p 2 without any incorrect nucleotide.
Compared with the data in NCBI website, the full length of Der p 1 gene is 907
bp and Der p 2 is 591 bp. In figure 2, the Der p 1 we cloned was 684 bp and Der p 2
was 395 bp. This is that Der p 1 belongs to a cysteine protease with a pre-sequence and
a pro-sequence in the front of its N-termini. The pre-sequence was thought to be used
to bring the protease to the right position within house dust mite. Mature Der p 1 with
cysteine protease activity is forming after the pro-sequence was deposited. Der p 2 also
contains a pro-sequence although the function remains unknown. Based on the above
information, we directly cloned the mature domain of Der p 1 and Der p 2, respectively,
for efficient production of the recombinant allergens. Fig 3 showed the sequencing
result of constructed gene in pPICZαA.
37
3.2 Selection of a higher productivity strain
Figure 4 shows the results of Zeocin selection. After 4 days of culture, these strains expressed different tolerance abilities at different concentration of Zeocin (500μg/mL
and 1000μg/mL). Strains showed higher tolerance to Zeocin were kept on YPD plate
and then transferred to 2 mL of YPD medium for further methanol induction.
After Zeocin selection, colony PCR were taken to confirm the fusion gene were
insert into the right position. Figure 5-1 showed the result of colony PCR using 5’AOX
and 3’AOX as primer, the insert DNA (1146 bp) plus AOX gene (588 bp) were 1734 bp.
Strain 31 were deleted for no result in colony PCR. Two more colony PCR used
pEcoRI-Der p 1 combined with plinker-SacII or pXhoI-linker combined with pDer p
2-XbaI were taken to confirm the gene were correctly fused (Fig 5-2 and Fig 5-3).
Figure 7 shows the ELISA results (for fusion allergen) in small scale methanol
inductions (Figure 6: Standard curve of Der p 2 ELISA). We found strain 6 and strain
33 both were higher expression strains. Therefore we chose strain 6, 33 and a randomly
selected (lower productivity) strain 10 for further comparisons. The supernatants were
concentrated 10-fold and were analyzed by Western blotting and ELISA.
Figure 8 shows the ELISA results of strain 6, 10 and 33 cultured for different time
of methanol induction. The fusion allergen yield increased with a longer induction time,
38
and strain 33 was apparently a higher yield strain. Our results showed strain 33 grew
better than strain 10, but similar to strain 6. It was consistent to the results of Zeocin
selections. It demonstrated that Zeocin selection was efficient for selection of a high
yield strain. The ELISA system specific to Der p 2 was used for quantification of the
fusion protein, and the yield was further calibrated by a ratio of 45k/15k (molecular
weight of fusion allergen protein / molecular of rDer p 2 standard).
3.3 Western blotting of products of strain 33
Figure 9 shows the results of Western blotting. rDer p 2 standard showed a sharp
band at the lower site in the page however, the target samples showed a smeare
phenomenon at the higher site. Compared with the primary membrane of this Western
blotting which loaded with a prestained marker, rDer p 2 standard was at the right
position around 15 kDa (date not shown). Recombinant fusion allergen was predicted
with a 45kDa molecular, however, it showed smear and shifted to the position during
65 to 90 kDa. It may be caused by the N-glycosylation of P. pastoris, because of a
N-glycosylation could be happened at the matured amino acid of Der p 1 (N52). It had
been known that one site of N-glycosylation for a protein could result in smear at the
sites of higher molecular weight (Yasuhara et al., 2001; Takai et al., 2002; van Oort et
39
al., 2002). The appearance of smear could be detected in our Western blotting system
also suggests that Der p 1 had been expressed and fused with Der p 2.
3.4 Cultures of strain 33 in Hinton’s flask and in a fermentor
Before cultured in a fermentor for high cell density cultures, strain 33 was
cultured and then treated 72 h of methanol induction for fusion allergen expression in
Hinton’s flasks (Fig.10). The fusion allergen in medium achieved 35.5 μg/mL (n=3),
and the wet biomass dropped after 48 h of induction. It suggests that the carbon source
were depleted at the stage. Comparison to the flask cultures contained BMG (M)
medium, which only produced 50 g/L in wet biomass.
Figure 11 shows the production results of strain 33 cultured in a fermentor.
During the initial 24 h, the wet biomass accumulated slowly and reached to 99.6 g/L.
During 24 h to 30 h, 250 mL of 50% glycerol was fed and the wet biomass rapidly
increased to 227.9 g/L. Methanol induction started at the 30th h and the wet biomass
raised slowly before 60th h because P. pastoris needed time to adapt to the new carbon
source (methanol feeding rate was increased from 9 ml/h to 18 mL/h at this stage).
During the stage of P. pastoris adapted to methanol (60~96th h), the wet biomass
increased with a higher growth rate and achieved 400 g/L in wet biomass (methanol
40
feeding rate was kept at 18 mL/h at this stage). In the last stage of methanol feeding,
the wet biomass accumulated slowly and finally achieved 427 g/L in wet biomass at
132th h (methanol feeding rate was gradually adjusted from 18 mL/h to 12 mL/h at this
stage for not to accumulate that would be toxic to cells). The OD600 also showed a
similar trend compared to the accumulation of wet biomass. In the stage of initial log
phase (18~30th h) and the stage of methanol adaption (30~60th h) the fluctuation of
dissolve oxygen was observed, and we gradually increased the air flow rate from 1
vvm to 3 vvm, to ensure the affordance of enough oxygen, and the DO was sustained
at 20~15% during 60~144th h. After 144th h, air flow rate was adjusted back to 1 v.v.m.
because of an increment of oxygen tension was observed.
After conducing ELISA for fusion protein assay and Bradford assay for total
soluble protein quantification, the results showed that the fusion allergen appeared from
30th h. From 36th to 60th h, fusion allergen in medium increased rapidly and achieved a
higher efficiency at 60th h. During 60th to 96th h, the fusion protein in medium dropped
quickly and turned to slowly increase after 96th h. The concentration of fusion achieved
another peak at 171st h, but the cultivation time was long and Pichia pastoris obviously
began to die. The Western blotting also showed a similar trend to the ELISA results (Fig.
12).
41
Chapter IV
Discussion and Conclusion
42
4.1 Discussion
Mut+ strains show metabolite methanol more efficient than Muts strains. P.
pastoris X33 is a wild strain belongs to Mut+ genotype, therefore the advantages of using X33 is mainly possessing a good growth rate when facing a methanol induction.
In this study, the Mut+ selection was conducted before Zoecin selections (for higher
copy number strains) in order to avoid choosing a higher copy number but with a Muts
genotype. During Mut+ selections, 35 colonies were chosen from 50 colonies for
growing well in MM plates and these strains were further screening in
Zeocin-containing plates. Our results showed all strain grew well on plates with lower
Zeocin concentrations (50 μg/mL and 100 μg/mL), but only few could grow on plates
with higher Zeocin concentrations (500 μg/mL and 1000 μg/mL). It suggested that
multiple recombination sites were happened after electroporation of vectors.
During Zeocin selections, 9 strains tolerant to 1000 μg/mL Zeocin were
discovered (Fig. 3). After ELISA quantification of the fusion allergen, 3 strains showed
higher productivity than 10 μg/mL after 24 h of methanol induction, and 5 strains
showed the productivity lower than 10 μg/mL. Among these strains, No. 16 was found
without the ability of fusion allergen expression. It suggests that strain 16 might be a
Muts strain.
43
Our ELISA and Western blotting results indicated that the fusion allergen was
secreted to the medium and the α-helix linker did work. Via a high cell density
cultured in a fermentor (Fig. 9), we observed that before adaption to methanol, for P.
pastoris methanol was used for induction of fusion allergen production and P. pastoris
grew slowly. Once P. pastoris adapted to methanol, methanol would be used as the
energy source to expand the biomass and secreted the Pichia-derived protease to
decompose the expressed fusion allergen as the nutrients. When the biomass came into
a saturation stage, the methanol was used for fusion allergen production again. In order
to solve the disadvantage of co-expression of protease, P. pastoris strain such as
SMD1168 and KM71 might be good choices. These strains are protease-deficient and
therefore the recombinant proteins could be preserved during the methanol induction.
In addition, to elongate the stage of glycerol-fed to achieve a higher biomass yield
before methanol feeding is an alternative way.
The reports regarding productivity of Der p 1 or Der p 2 expressed in different
hosts were summarized in Table 4. Reports show that crucial commitment of proteolytic
activity of a pichia-derived rDer p1 to sensitization toward IgE and IgG responses
(Takai et al., 2002; van Oort et al., 2004; Kikuchi et al., 2006). Der p 1 was first
expressed in E. coli in 1988 however produced in the form of inclusion body that rDer p
44
1 was hardly to be determined. During 2000-2001, rDer p 1 was reported to be
successfully expressed in Drosophila cells (20 μg/mL) and mammalian cells (34 μg/mL)
with a low productivity. Glycan test showed that N-glycosylation was not happened in
natural Der p 1, but were observed in Drosophila cells and mammalian cells (Jacquet et
al., 2000; Massaer et al., 2001). Natural Der p 1 showed a sharp band on SDS-PAGE
instead of a smear bands (Jacquet et al., 2002). In 2002, an expression of rDer p 1 in P.
pastoris was reported and rDer p 1 in cultured medium was 70 μg/mL. However, the
expressed rDer p 1 was found to be hyperglycosylated. Compared with the natural Der p
1, hyperglycosylation may lead rDer p 1 to be difficultly analyzed in vivo for losing its
histamine release activity, proteolytic activity and the inhibition of IgE binding ability.
After treated with N-Glycosidase, rDer p 1 recovered the similar activities to natural
Der p 1 (Jacquet et al., 2002). N-Glycosidase therefore can be a way to remove
glycosylation on the fusion allergen when we produce the rDer p 1 with a glycosylation.
And another strategy was to take a site-direct mutation of proDer p 1 (N52Q) which
optimized the rDer p 1 into a more similar character as the natural Der p 1 (van Oort et
al., 2004).
In addition to the effect of glycosylation on its performance of protease activity,
existence of the prosequence of natural Der p 1 is also thought to be an important issue.
45
Reports shows that rDer p 1 expressed with proDer p 1 domain design achieved a
similar activity to natural Der p 1(Jacquet et al., 2000; Massaer et al., 2001). We realize
that protease activity of Der p 1 was directly associated with the prodomain that would
function as an intramolecular chaperon to help Der p 1 correctly folded into an active
form, and it suggests that prosequence of Der p1 may be necessary for its immuno-
efficacy in clinic trial. After the construction of full-length Der p 1 fused with Der p 2,
this fusion allergen may have ability to facilitate itself to pass through epithelium cells
and enhance the immune response. Besides the above reports, rDer p 1 had also already
expressed in the rice as an edible vaccine and achieved a content of 50μg/grains.
Der p 2 was also initially expressed in E. coli as an inclusion body. Expressed in
eukaryotic system of S. cerevisiae (7 μg/mL) however was mostly to be degraded.
Expression of rDer p 2 in tobacco showed a good activity in oral delivery in murine
model of asthma (Ho, 2002) however with a low productivity. Co-expression of Der p 1
and 2 and in the form of a fusion allergen by P. pastoris is now first reported in this
study. The productivity of fusion allergen achieved 203 μg/mL in 60 hours of the
culture. This is also the highest yield among the related articles which had been
reported.
In addition to the previous issues, switching the linker sequence into other linkers
46
with different characters is also an interesting work. Overall, to establish an economic,
efficient, and to get an active form of fusion allergen, the choice of an adequate link is
absolutely important.
4.2 Conclusion
In brief, we successfully linked the Der p 1 and Der p 2 genes with an α-helix
forming nucleotide sequences. Via constructed in a pPICZαA vector, it was
electroporated into P. pastoris wild strain. A high productivity strain (strain No. 33) for
production of fusion allergen was chosen. The productivity of the fusion allergen in
Hinton’s flask was 35.5 μg/mL after 72 h of methanol induction. A high cell density
culture in the Bioflo110 fermentor was achieved which caused to 203 mg/L fusion
allergen productivity at 60 h and got a 427 g/L wet biomass at 132 h. This combination
of the major allergens in Dermatophagoides pteronyssinus is suspected to be
developed as an efficient vaccine for allergen disease treatment.
47
Tables and Figures
48
Table 3House dust mite allergens. (Thomas et al., 2002)
Group Biochemical function MW cDNA1
(SDS-PAGE) Species2 IgE binding3
8 Glutathione-S-transferase 26,000 Dp 40
9 Collagenolytic serine protease no cDNA, (30 000) Dp 90
10 Tropomyosin 37,000 Dp, Df 50–95
11 Paramyosin 96,000 (92,000,
98,000) Df, Bt 80
12 Unknown 14,000 Bt 50
13 Fatty acid-binding protein 15,000 Bt, Ld, As 10–23 14 Vitellogenin/apolipophorin-like 177,000 (variable) Df, Dp, Em 90 15 98,000 Chitinase 62,500 (98,000,
105,000) Df 70
16 Gelsolin 55 Df 35
17 Ca-binding EF protein 30 Df 35
18 Chitinase 60,000 Df 60
19 Anti-microbial peptide 7,000 Bt 10
1 MW calculated from cDNA (SDS-PAGE of natural allergen, if different).
2 Allergen described for the species designated by initials: Dermatophagoides pteronyssinus, Dermatophagoides farinae, Euroglyphus maynei,Dermatophagoides siboney, Dermatophagoides microceaus, Lepidoglyphus destructor, Blomia tropicalis, Tyrophagus putrescentiae, Glycophagusdomesticus, Ascaris siro.
3 Binding frequency (% patients, variation due to patient selection).
49
Table 2 Recombinant allergens expressed in various systems. (Schmidt and Hoffman, 2002)
Expressed in Allergen Comments
E. coli Api m 1 Renatured to full enzyme and IgE-binding activities Api m 2 Only 20–30% enzyme activity after renaturation Cyp c 1 Produced in fully active form when calcium added Ara h 1 Codon usage requires specially engineered strain Bet v 1 Isoforms with varying IgE-binding activity Cor a 1 Bet v 1 cross-reactive, isoforms
Alt a 1 IgE-reactive, glycosylated Mus m 1 Native conformation Bla g 4 High yield
Fel d 1 Glycosylated
Der p 1 Hyperglycosylated precursor Ves v 5 High yield, native conformation Saccharomyces Der p 1 Insoluble
Der p 2 IgE-binding activity
Procalin Immunoreactive
Baculovirus Api m 2 Full enzymatic and IgE-binding activities
Sol i 2 IgE-reactive, native conformation, natural cleavage of initiation sequence
Fel d 1 Fully immunoreactive Lep d 2 Isoforms produced
Mal f 1 Similar to that expressed in E. coli
50 Table 3 Primer design and the linker sequence.
Fragment nucleotides sequence
pEcoRI-Der p 1 5'-AAG-GAATTC-ACT AAC GCC TGC AGT ATC AA-3' pDer p 1-XhoI 5'-CTG-CTCGAG-GAG AAT GAC AAC ATA TGG AT-3'
pXhoI-linker 5'-ACA-CTCGAG-GGT TCT ACC TCT-3 plinker-SacII 5'-TTA-CCGCGG-GAT ACC AGA ACC-3'
pSacII-Der p 2 5'-AAT-CCGCGG-GAT CAA GTC GAT GTC AAA GA-3' pDer p 2-XbaI 5'-ACA-TCTAGA-CC-ATC GCG GAT TTT AGC ATG AGT-3' Linker sequence 5'-ACA-CTCGAG-GGT TCT ACC TCT GGT GGT TCT ACC TCT GGT
GGT TCT ACC TCT GGT TCT GGT TCT GGT ATC-CCGCGG-TAA-3'
51
Table 4 Comparisons of rDer p 1 and rDer p 2 expressed in different hosts
allergen system concentration year
Der p 1 E. coli low 1988
Der p 1 Drosophila cells 20 μg/mL 2000
Der p 1 mammalian cells 34 μg/mL 2001
Der p 1 Pichia pastoris 70 μg/mL 2002
Der p 1 rice 50μg/grains 2008
Der p 2 E. coli 50 μg/mL 1997
Der p 2 Saccharomyces cerevisiae 7 μg/mL 1998
Der p 2 Tobacco 8 μg/mL 2002
Der p 1+Der p 2 Pichia pastoris up to 203 μg/mL 2008
52
Figure 1 pPICZαA vector. (A) is the multiple cloning site of pPICZαA and (B) is the vector map. The figure and the complete sequence of pPICZαA is available for downloading from Invitrogen company’s World Wide Web site (www.invitrogen.com).
Figure 2 Schematic representation of Der p 1 and 2 fusion allergen.
53 Figure 3 Sequencing result of fusion gene.
54
Figure 4 Results of the Zeocin selection. Thirty five strains cultured on YPDZ plate for 4 days and the Zeocin concentrate was from 50 μg/mL to 1000 μg/mL.
55
Figure 5 Colony PCR result. 5-1, AOX primer. 5-2, Der p 1+linker primer. 5-3, linker+Der p 2 primer
5-1
5-2
5-3
56 Figure 6 Standard curve of Der p 2 ELISA.
57
Figure 7 Selection results of 9 strains cultured in a small scale methanol induction.
58
Figure 8 Fusion allergen expression efficiency of strain 6, 10 and 33.
59
Figure 9 Western blotting results of strain 33 cultured in Hinton’s flasks.
60 Figure 10 Strain 33 cultured in Hinton’s flasks.
.
61 Figure 11 Strain 33 cultured in a fermentor.
62
Figure 12 ELISA and the Western results of strain 33 cultured in a fermentor.
63
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