Chapter I Introduction
1.6 Aim of this study
The Pichia pastoris which can produce rDer p 2 was kindly provided by prof. B.L.
Chiang. The rDer p 2 can be secreted during high cell density culture in the total
synthetic medium. The secretion of rDer p 2 eased the recovery process from cultured
medium. We also constructed the ELISA and Western blotting systems that specific to
Der p 2 for quantification and qualitative analysis (LinWan-Yi, 2007).
In this study, we want to construct a recombinant fusion allergen gene encoding
Der p 1 and Der p 2 by an α-helix linker in pPICZαA vector, and then transformed into
18
Pichia pastoris. The linker was designed to be adequate not long for efficient expression
and not short to lose the function. We hope the α-helix linker could separate the two
allergens with a proper distance. If the α-helix linker was useful and fused Der p 2 and
Der p 1 could fold independently, the Der p 2 ELISA and Western blotting system will
show the signal during conduction. We can also observe the glycosylation of Der p 1
during Western blotting. The α-factor signal was included in the plasmid construction
for protein secretion. Histaq was designed to be located after the sequence of the fusion
allergen. This vector was transformed into Pichia pastoris X33 for recombinant protein
expression. A high productivity strain was chosen and expressed in Hinton’s flask and
then scale up cultured in a fermentor. This is the first report for fusion major house dust
mite production. We hope that this recombinant fusion allergen could be a good material
for oral tolerance research and a pretest for development of the edible vaccine.
19
Chapter II
Materials and Methods
20
2.1 Microorganisms and vectors
Escherichia coli JM109 (Yeastern Biotech, Taipei, Taiwan) was chosen for vector
preservation. Pichia pastoris X33 (Invitrogen, Sandiego, USA) were chosen as the
yeast expression hosts. yT&A vector (Yeastern Biotech, Taipei, Taiwan) was used as a
gene preservation vector for constructing the fusion allergen expression. The
expression vector for P. pastoris was pPICZαA (Invitrogen, Sandiego, USA). Der p 1
and Der p 2 gene respectively inserted in pGEX vector (named pGEX-Der p 1 and
pGEX-Der p 2) was kindly provided by Professor Chiang (Graduate Institute of
Clinical Medicine, Medicinal College, NTU) as a gift. The synthesized linker
nucleotide sequence and primers were purchased from Blossom biotechnologies,
Taipei, Taiwan.
2.2 Construction of plasmids and the P. pastoris expression host
pPICZαA vector contained an α-factor signal sequence which allowed the
recombinant fusion allergen protein downstream of the signal sequence to be secreted
in P. pastoris system. The complete sequence of pPICZαA is available for
downloading from Invitrogen company World Wide Web site (www.invitrogen.com). A
map of pPICZαA vector and the multiple cloning sites are shown in Fig 1. At the
21
C-terminus of the α-factor secretion signal peptide, there is a Kex2 signal cleavage site
right before the last four amino acids (Glu-Ala-Glu-Ala), which could be cleavaged by
KEX2 gene product for separating the α-factor signal peptide and the fusion allergen.
Fusion allergen gene was attached immediately behind Kex2 signal sequence cleavage
site by the polymerase chain reaction (PCR) methods between EcoRI and XbaI
restriction sites. Table 3 shows the linker sequence and the primers used. Figure 1 is
the schematic representation of Der p 1 - Der p 2 fusion protein. We constructed fusion
allergen gene in yT&A vector, and then send it into pPICZαA for transformation.
2.2.1 Construction of the fusion allergen gene in yT＆A vector and then pPICZαA vector
PCR which used pEcoRI-Der p 1 combined with pDer p 1-XhoI as the primer,
and pGEX-Der p 1 as the template constructed a full-length mature Der p 1 gene. PCR
which used pSacII-Der p 2 combined with pDer p 2-XbaI as the primer, and
pGEX-Der p 2 as the template constructed a full-length of Der p 2 gene. PCR which
used pXhoI-linker combined with plinker-SacII as the primer, and the synthesized
single strand linker sequence as the template constructing a 78 base pair of double
strand linker sequence. All PCR introduced restriction site as the primer name they
22
used followed by a 6 base pair sequence to the 5’-end and the 3’-end as the underlined
letters in Table 3.
The PCR mixture and the thermalcycling conditions were as following:
PCR mixture:
Superthem DNA polymerase (0.2 U)
5 μL
The PCR product was confirmed by electrophoresis using 1.2% agarose gel under
100 voltage in 1XTAE buffer. Product of the appropriate size was recovered by
NucleoSpin kit (Qiagen, Germany) with the protocol suggested in the user manual
(Vogelstein and Gillespie, 1979). After cutting out the region of the appropriate size
with a razer, this portion of agarose gel was mixed with 600 μL of NT1 buffer and
23
incubated in 50oC for 10 min. During this step, the agarose gel completely dissolved
and the solution was transferred into an extraction column adapting to a collection tube.
Spin down the column under 8,000xg for 1 min and then discard the flow through. Add
600 μL of NT3 buffer into the extraction column and spin down under 12,000xg for 1
min. Add 200 μL of NT3 buffer and spin down under 12,000xg for 1 min. Transfer the
extraction column to a sterilized microcentrifuge tube. Drip 10 μL of NE buffer on the
membrane of the extraction column and let it stay for 1 min, spin down under
12,000xg for 1 min, then repeat this step again. The PCR product of the appropriate
size was collected in the microcentrifuge tube.
Der p 1, Der p 2 and the linker gene coding sequence with tailored end
sequence possessing restriction enzyme sites obtained from PCR is then immediately
transferred into a yT&A cloning vector and preserved in E. coli strain JM109. The
ligation reaction of tailored Der p 1, Der p 2 and linker gene into yT&A cloning vector
is as following:
Ligation reaction of TA cloning:
yT&A cloning vector 2 μL
buffer A 1 μL
buffer B 1 μL
tailored Der p 1, Der p 2 and the linker gene or other genes should be preserved during the manipulation of genes
5 μL
yT&A T4 DNA ligase 1 μL
24
Total volume: 10 μL
The ligation mixture was kept at 4oC overnight and then added into 100 μL
half-thawed E. coli JM109 competent cells. Heat shock at 42oC for 45 sec, the
transformants were grown on LBA plates and selected by blue/white screening (Sambrook and Russell, 2001). Prior to spreading the transformants, 100 μL 100mM
IPTG and 20 μl 50mg/mL X-gal was added on the surface of the plates. Colonies
grown were first checked by colony PCR to see whether the appropriate base pair size
was presented and the sequences were confirmed by sending transformed colonies to
Tri-I Biotech (Taipei, Taiwan) for sequencing service.
With the correct sequence confirmed, we started to construct the fusion allergen
gene. Because of the restriction of linker size that can’t be seen within electrophoresis,
we directly ligate the Der p 2 gene and linker sequence after processed with SacII
endonucleases right after PCR of Der p 2 gene and the linker sequences. The reaction
mixture for restriction enzyme digestion and ligation were as follows and the
remaining reactions for restriction enzyme reactions were as follows with modification
of enzymes and buffers:
Restriction enzyme reaction:
10X reaction buffer 2 μL
Substrate DNA 2 μL
25
SacII 2 μL
H2O 14 μL
Total volume: 20 μL
After ligation, linker-Der p 2 gene was further ligated into a yT&A vector. For the
full-length of Der p 1-linker-Der p 2 gene construction, Der p 1 and linker–Der p 2
genes preserved in different yT&A vectors were digested with XhoI and XbaI
endonucleases and then electrophoresis to separate the products. We cut the yT&A-Der
p 1 and linker-Der p 2 from the gel and recovered them with NucleoSpin kit.
yT&A-Der p 1 and linker-Der p 2 genes were ligated into yT&A -Der p 1-linker-Der p
2. The full-length of Der p 1-linker-Der p 2 gene with proper restriction enzyme sites
were constructed in yT&A.
Because of the ligation of Der p 2 and the linker was conducted directly after
digestion of the PCR products, unconfirmed Der p 2 and linker genes were further
switched for the previously confirmed genes preserved in yT&A vector. With the
correct sequences, yT&A vector containing tailored Der p 1-linker-Der p 2 gene was then processed with XhoI and XbaI endonucleases. Empty pPICZαA vector was
processed with the corresponding restriction enzymes as well. Then, the Der p 1-linker-Der p 2 was ligated into a pPICZαA within XhoI and XbaI restriction enzyme sites. The shuttle vector pPICZαA-Der p 1-linker-Der p 2 was then transformed into E.
26
coli JM109 and sending for sequencing. With the correct sequence confirmed, the
shuttle vector containing the right sequence with proper reading frame was preserved
in E. coli JM109 and selected by LSLBZ plate (Low salt LB plate with 25 μg/mL of
Zeocin.)
2.2.2 Construction of P. pastoris expression host
The pPICZαA vector did not contain yeast replication origin, thus, transformants
could be isolated only recombination did occur between the plasmid and the host
genome. P. pastoris X33 was chosen as the expression hosts for expressing fusion
allergen. X33 represents the wild-type of P. pastoris. Expression vectors were
transformed into the host by electroporation according to the protocol provided by
Invitrogen with a few improvements (Becker and Guarente, 1991; Chang, 1992). P.
pastoris strain was incubated in 5 mL YPD medium at 30oC, 150 rpm shaking overnight, and then transferred into 100 mL YPD medium, incubated overnight again
at 30oC, 150 rpm shaking until OD600 reached 1.3~1.5. The overnight cultured cells
were collected by centrifuging at 2,000xg for 5 min at 4oC. Then the cells were washed
2 times with ice-cold H2O, once with 50 mL and the second time with 25 mL, cells
were collected by centrifuging at 2000xg for 5 min at 4oC. The cells were then washed
27
with ice-cold 1M sorbitol, once with 2 mL and the second time resuspended in 100 μL.
These cells were kept on ice and used that day.
The expression vector pPICZαA-Der p 1-linker-Der p 2 was linearized for
electroporation. For linearization, SacI endonuclease was used under the following
reaction mixtures:
Reaction mixtures were kept at 37oC for 90 min. The linearized expression
vectors were obtained and checked by electrophoresis using 1.2% agarose gel and
purified by Nucleospin kit (Qiagen, Germany) with the protocol described above.
The electroporation mixture was setup by mixing 80 μL of cells prepared above
and 2 μL of linearized expression vector previously mentioned. Transferred the
mixture to an ice-cold 0.2 cm electroporation cuvette and stayed on ice for 5 min. Then
the mixture was pulse under 1.5 KV, 25 μF, 200 ohm for 5 msec, and then quickly
added 1 mL ice-cold 1M sorbitol. Gently resuspended the cells, and the mixture was
transferred into a sterilized glass tube and the tube was incubated at 30oC without
shaking for 90 min. Spread every 300 μL of incubation mixture on a YPDSZ plate,
28
incubated the plates at 30oC until colony forms (5~6 days). Colonies were streaked
again on YPDZ plates, and incubated at 30oC until colony formed. Colony PCR were
taken during further selection to confirm the fusion gene. Primer used were 5’ AOX
combined with 3’AOX to confirm fusion gene were insert into the right position;
pEcoRI-Der p 1 combined with plinker-SacII or pXhoI-linker combined with pDer p
2-XbaI to confirm the fused gene.
2.3 Selection of a proper expression strain
2.3.1 Selection of a Mut
+expression strain
Recombination of plasmid and Pichia pastoris genome may result in the
destruction of AOX1 gene because of the substitution of AOX1 gene by the
heterologous gene. Destruction of AOX1 gene leads to that metabolites alcohol poorly
is called a Muts strain, and the preservation of AOX1 gene called a Mut+ strain that
sustains a good ability in alcohol metabolite.
We used MD (Dextrose) and MM (Methonal) plates which with different carbon
source to select the Mut+ strains. Bigger colonies from the YPDSZ plates were selected
to streak on the MM and MD plate, respectively. Colonies showed high growth rate on
MM plate were picked for further selection.
29
2.3.2 Selection of a strain with high copy-number
During gene recombination, multiple gene-insertion events at a single locus in a
cell do occur spontaneously but with a low, detectable frequency--between 1 and 10%
of all selected Zeocin resistant transformants (Daly and Hearn, 2005). With higher
copy number, colonies could grow on higher concentration of Zeocin plate. We used a
series of concentration of Zeocin on YPDZ plates (from 50 μg/mL to 1000 μg/mL) to
select strains with higher copy number.
After Mut+ selection, selected colonies were streaked on YPD plate and cultivated
at 30oC until colonies formed. Colonies were picked and suspensed in 10 μL of YPD medium respectively. One μL suspension of each colony was dropped on YPDZ
plates with different concentrate of Zeocin. Colonies grown on 1000 μg/mL Zeocin
were picked and streaked on YPD plates for the next selection.
2.3.3 ELISA selection for higher productivity in small scale cultures
After Zeocin selection, strains were cultured in tubes for further selection for
strains with high expression efficiency. Colonies with high copy numbers were picked
from the YPD plate and cultured in 2 mL of YPD medium for 24 hours. After 24
30
hours, we added 1 μL of 100% methanol to achieve 0.5% of methanol induction
within the YPD medium for further 24 h cultures. Finally, supernatant was collected
by centrifuging at 14,000xg for 10 min at 4oC. Supernatant was conducted the
sandwich-ELISA (as described below) for selection of high productivity strains.
2.4 Cultures in the Hinton’s flasks
After the above selections, the two strains (strain 6 and strain 33) expected with
high productivity and a random chosen strain (strain 10) for comparison were
cultured in Hinton’s flasks to investigate the strategy we took was right. First, three
strains were respectively cultured in 25 mL of BMG medium which contained
glucose as the sole carbon source at 30oC, 150 rpm shaking overnight. The overnight
cultured cells were collected by centrifugation at 2,000xg for 10 min at 4oC and
further resuspensed in 100 mL of BMM medium which contained methanol as the
sole carbon source for expression induction. Induction was carried out for 24 hours
and added 0.5 mL of methanol per 24 h for 72 hours totally continuous induction.
Samples were collected per 24 h and supernatant were collected by centrifugation at
14,000xg for 10 min at 4oC. Supernatant were assayed by ELISA and the high
productivity strain was chosen.
31
2.5 Cultures in fermentors
After the highest productivity strain was selected, we cultured it to produce the
fusion allergen in Bioflo 110 fermentor via high cell density cultures. Culture
conditions within the fermentor were set at 30oC, 1000 rpm and pH-stat at 5. DO probe
was used to monitor the oxygen tension and the air-flow set at 1vvm and gradually increased
to 3 vvm. when the cultures achieved a high cell density. The colony picked from YPD
plate was cultured in 100 mL BMG medium at 30oC, 150 rpm overnight as a seed
culture. One hundred mL of broth was inoculated into 2.5 L FBSM medium and grew
for 24 h to deplete the glycerol as the 1st stage, and the wet biomass reached to 100
g/L. During the continued 6 hours, 250 mL of 50% glycerol was fed at a rate of 18.15
ml/h/liter for the initial fermentation volume, and achieved 200 g/L wet biomass at
this stage. At the 30th h, methanol induction was started at a feeding rate of 3.6
ml/h/liter (to the initial fermentation volume). The methanol feeding rate rose to 7.2
ml/h/liter when the dissolved oxygen stopped fluctuation and rose to 20%. Finally,
the methanol induction continued until the wet biomass stop rising. Samples were
collected every 3 h during the first 36 h and every 12 h for the remaining culture.
Supernatant was collected by centrifugation at 12,000xg for 10 min at 4oC and the
wet biomass was measured at the same time. The fusion allergen in supernatant was
32
measured by sandwich-ELISA and the total soluble protein was determined by the
Bradford method (Bradford, 1976). Western blotting was also conducted to detect the
fusion allergen.
2.6 Quantification of total soluble protein
Total soluble protein was determined by the Bradford method according to the
Bio-Rad-Microassay Procedure (Bio-Rad, CA, USA). A standard curve was produced
with known concentrations of BSA (Sigma-Aldrich, MO, USA).
2.7 Preparation of recombinant Der p 2 protein as the standard in ELISA and the Western blotting
P. pastoris transformed with pPICZαΑ-Der p 2, kindly provided from Prof. B.L.
Chuang as a gift, was used to produce the rDer p 2 as the standard. After cultured in a
Bioflo 110 fermentor including 4 days of methanol induction, 3 litters of supernatant
were recovered. The supernatant contained mainly the 15 kDa molecular weight of rDer
p 2 and some minor 70K molecular weight of protease. After filtered with a 30 kDa
Amicon Ultra membrane (Millipore, USA), the protease was depleted. We further used
5 kDa Amicon Ultra membrane for desalting and to concentrate the rDer p 2. To make
33
sure the rDer p 2 had been correctly folded, the concentrate was treated with refolding
reagent (8 M urea) (Takai et al., 2005a).
2.8 Sandwich-ELISA
We used sandwich-ELISA specific for quantification of rDer p 2. First, 96-well
flat-bottomed polystyrene microplates (TPP® Techno Plastic Products AG, Trasadingen,
Switzerland) were coated with 100 μL of coating buffer (Carbonate-bicarbonate, pH 9.6)
which contained 1/100 (v/v) diluted anti-Der p 2 monoclonal antibody. After plates
blocked with gelating-NET (0.25% gelatin, 0.15 M NaCl, 5 mM EDTA, 0.05% Tween
in 50 mM Tris-HCl, pH 8.0), samples (100 μL) were respectively added to the wells and
incubated at 37°C for 1 hour. And then, anti-Der p 2 polyclonal antibody from rabbit at
a 1:1000 dilution was added to each well and incubated at 37°C for 1 h. After the wells
washed by phosphate buffer saline with 0.05% Tween-20 (PBST), 100 μL of Goat
anti-Rabbit IgG (H+L) HRP conjugated affinity purified antibody (Chemicon, CA, USA)
at a 1:3000 dilution was added to each well and incubated at 37°C for 1 h. After the
wells were washed, the 1-StepTM Ultra TMB-ELISA substrate (Pierce, IL, USA) was
added and incubated for 20 min to 30 min until the blue color appeared. The Der p 2
concentrations were determined by comparison to a standard curve of rDer p 2 from
34 yeast.
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.
and 3’AOX as primer, the insert DNA (1146 bp) plus AOX gene (588 bp) were 1734 bp.