Acta Biochim Biophys Sin 2010, 42: 388– 395 | ª The Author 2010. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmq033.
Original Article
Prokaryotic expression, purification, and polyclonal antibody production of a
hydrophobin from Grifola frondosa
Zefang Wang 1, Shuren Feng 1, Yujian Huang 1, Mingqiang Qiao 1 *, Baohua Zhang 2, and Haijin Xu 2 *
1
The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China
The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai University,
Tianjin 300071, China
*Correspondence address. Tel: þ86-22-23503340; Fax: þ86-22-23503692; E-mail: [email protected] (M.Q.).
Tel: þ86-13002212878; Fax: þ86-22-23503692; E-mail: [email protected] (H.X.)
2
Hydrophobins are small fungal proteins that self-assemble
spontaneously at hydrophilic – hydrophobic interfaces and
change the polar nature of the surfaces to which they
attach. A new hydrophobin gene hgfI was identified
recently from the edible mushroom Grifola frondosa. In
this paper, the cloning, expression, purification, and polyclonal antibody preparation of the HGFI were described.
The hgfI gene was cloned into pET-28a expression
plasmid at the EcoRI and NdeI restriction sites and then
transformed into Escherichia coli BL21 strain. SDSPAGE analysis showed that recombinant HGFI protein
was satisfactorily expressed by optimizing the concentration and induction time of IPTG. The expressed recombinant HGFI protein was purified by electroelution
because its inclusion body was insoluble in traditional
processing method. After a desalting procedure with
Sephadex G-25, the recombinant HGFI protein was used
to immunize adult rabbits following standard protocol.
ELISA and western blot analysis indicated that the produced antiserum could detect both HGFI protein
expressed in the prokaryotic (E. coli) and in the eukaryotic cells (G. frondosa). Furthermore, the antiserum was
used to determine the localization of HGFI protein in
G. frondosa cells using an immunofluorescence technique.
The results demonstrated that HGFI protein was localized
in the cell wall, especially at the budding position of
hypha. The polyclonal antibody against HGFI will facilitate further production and functional study of HGFI
protein.
Keywords
hydrophobin; HGFI; prokaryotic expression;
polyclonal antibody
Received: January 20, 2010
Accepted: March 21, 2010
Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 6 | Page 388
Introduction
Hydrophobins are small proteins that are uniquely produced by filamentous fungi [1,2]. All hydrophobins
contain eight cysteine residues at conserved positions [3].
These proteins containing both hydrophobic and hydrophilic parts are considered as one of the most surface-active
proteins. The main characteristic property of hydrophobins
is that they can self-assemble into amphipathic membranes
of 10-nm thick at the hydrophobic/hydrophilic interfaces,
reversing the hydrophobicity of the surfaces coated with
them [4,5].
Different hydrophobins seem to fulfill a variety of tasks
in fungal development and growth [6]. In fungi, the hydrophobic surface with a hydrophobin coating facilitates the
attachment of hyphae to the hydrophobic surfaces, aerial
growth of the hypha, dispersal of aerial spores, and proper
gas exchange in fungal air channels [7–10]. Moreover,
some hydrophobins implicated in the pathogenicity of
several fungi are involved in the interaction between the
pathogenic fungi and their host plants [11].
The properties of hydrophobins are so interesting that
they have gained many applications [12,13], including the
use of hydrophobins as personal care and emulsions [8,14],
separation technologies [15], biosensors and electrodes
[16], biomaterials and the gushing factor detection [17,18].
Although different applications of hydrophobins are found,
there is currently no mature product in the market that
exploits the features of these proteins. It has been known
that the production level is one of the biggest problems for
reducing the cost and opening up new applications of
hydrophobins [19].
In order to overcome the production limitation of hydrophobins, large-scale submerged fermentation of Grifola
Prokaryotic production of fungal hydrophobin HGFI for antibody preparation
frondosa could be achieved to produce adequate amount of
HGFI hydrophobin [20–23]. However, the fermentation
period of G. frondosa is normally over 1 week and contaminations happen frequently during the fermentation. If
we determine the exact time when the expression amount
of HGFI hydrophobin achieves the maximum level by
using the immunoassay, the possibility of contaminations
and fermentable cost can be reduced during the fermentation. In the immunoassays, such as enzyme-linked immunosorbent assay, western blot or immunofluorescence,
antibody against HGFI hydrophobin must be available. In
this study, we presented the prokaryotic expression, purification, and polyclonal antibody production of the HGFI
protein. According to our results, the antibody can successfully detect the expression of HGFI and elucidate the function of HGFI in the development of G. frondosa.
Materials and Methods
Materials
The Escherichia coli strains DH5a and BL21(DE3), and
the pET-28a vector were preserved at the Nankai University
(Tianjin, China). The G. frondosa strain used in this study
was purchased from the Committee on Type Culture
Collection of Chinese Academy of Sciences (Beijing,
China). The pMD18-T, T4 DNA ligase, Taq DNA polymerase, all restriction enzymes and DNA molecular mass
markers and PCR product purification kit were purchased
from TaKaRa (Dalian, China). Tryptone, yeast extract and
granulated agar were purchased from Difco (Detroit, USA).
Cloning of pET-28-hgfI and construction of expression
vector
The full-length cDNA (genbank accession no. EF486307)
of hgfI contains 324 nucleotides, which encodes a precursor protein of 108 amino acid residues. The 57 nucleotides
of N-terminal encode a predicted signal peptide. The hgfI
coding sequence (from nucleotide 58 to the last nucleotide
of its cDNA) was amplified by PCR from the template
vector pBSK-hgfI constructed by our laboratory. Oligos
correspond to the N-terminus (50 -AAACATATGAC
CCCTGTCCGCCGC-30 ) and C-terminus (50 -CCCGAATTC
TCAGACGTTAACCGGAACACAT-30 ) of the mature
peptide, containing the NdeI and EcoRI restriction sites,
respectively (underlined). The reaction was carried out
using the following reaction cycles in a Peltier Thermal
Cycler (MJ Research, Watertown, USA): initial denaturation at 948C for 3 min followed by 30 consecutive cycles
of denaturation at 948C for 30 s, annealing for 30 s at
608C, extension at 728C for 30 s, then final extension at
728C for 10 min. The amplified hgfI gene was gel-purified
by highly pure PCR product purification kit. After digestion
with NdeI and EcoRI, the purified product was inserted into
corresponding region of pET-28a expression vector and confirmed by restriction enzyme digestion and sequencing. The
correct recombinant prokaryotic expression vector was
named as pET-28a-hgfI.
Expression of recombinant protein in E. coli
Expression of recombinant protein in E. coli BL21(DE3)
cells followed with the transformation of the recombinant
plasmid, pET-28a-hgfI. We optimized the conditions for
induction to obtain as much recombinant protein as possible, different concentrations of the isopropyl-beta-Dthiogalactopyranoside (IPTG) (0.2, 0.3, 0.5, 0.7 and
0.9 mM) and different induction time points (1, 2, 3, 4, 5,
6 and 7 h) were used. The recombinant protein was
expressed on a satisfactory scale as follows. The transformants were cultured in 5 ml of LB medium containing
15 mg/ml kanamycin and grew overnight at 378C and
200 rpm. In the next step, 0.5 ml of cultures were taken out
and transferred to 50 ml of fresh medium, and incubated
for about 4 h until the optical density (OD600) of the cultured cells reached 0.7. Expression of the recombinant
protein was induced with 0.7 mM IPTG at 258C for 5 h.
Extraction of recombinant HGFI protein
The 50 ml of induced culture pellet was harvested by centrifugation at 11,000 g for 15 min, and the supernatant was
saved. The cell pellet was washed three times with ddH2O
and resuspended in 5 ml of lysis buffer (50 mM NaH2PO4,
0.5 M NaCl, pH 8.0). Then the cell suspension was sonicated 10 times for 60 s and the lysate was centrifuged at
11,000 g for 30 min. The clear supernatant (soluble fraction) was collected and the remaining pellet (insoluble fraction) containing inclusion bodies were resuspended in an
equal volume of lysis buffer. All the fractions of the extraction procedure were analyzed by 16% Tricine-SDS-PAGE
and western blot analysis to detect the presence of recombinant HGFI protein.
Purification of recombinant protein by electroelution
After electrophoresis, the gel was briefly stained with
Coomassie Brilliant Blue R250 for 20 min followed by
destaining with 250 mM KCl till the background of gel was
clear, and then target protein bands were cut off from the
whole gel with a clean scalpel. The excised gel pieces were
placed in a dialysis bag (Sangon, Shanghai, China) containing the electroelution buffer (25 mM Tris–HCl, 192 mM
glycine, 0.5% SDS, pH 8.3) with 6 K molecular weight
cut-off. Electroelution was carried out for about 3 h at
100 mA in the same buffer. The process of electroelution
was stopped when the Coomassie Brilliant Blue R250 run
out from the gel pieces. The supernatant was added with 10
volumes of acetone and kept still at 2208C for 1 h. Then
the centrifugation was performed at 12,000 g for 20 min,
Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 6 | Page 389
Prokaryotic production of fungal hydrophobin HGFI for antibody preparation
the pellet was collected and re-dissolved in ddH2O, and the
protein solution was desalted by Sephadex G-25.
At the same time, another method of staining and
destaining was performed to compare the result with that
of the above method [24].
Water contact angle measurements
Adsorptions of purified HGFI (or native HGFI) to the
hydrophobic surface gold surface was studied by water
contact angle (WCA) measurements. Taking recombinant
HGFI for example, the gold surface was coated with 20 ml
of recombinant HGFI solution (0.1 mg/ml) and incubated
at room temperature for 30 min. After removing the protein
solution gently, the gold surface was dried in a nitrogen
stream and kept in room temperature overnight. The
surface was rinsed with water and WCA measurements
were used to analyze the surface. WCA were measured
with a 5-ml of water droplet on the modified surface at
room temperature. At least three water droplet readings
were analyzed on different areas of the sample surfaces.
Production and purification of polyclonal antibodies
against the recombinant protein
Antibodies against recombinant HGFI were raised in a
male New Zealand white rabbit. The rabbit was injected
subcutaneously with 1 mg of highly purified recombinant
HGFI protein dissolved in 0.2 M NaCl and emulsified in
1 ml of Freund’s complete adjuvant to enhance the
response to the immunogen. Two booster injections were
given with 0.5 mg recombinant protein each in incomplete
Freund’s adjuvant at 2-week interval to obtain a prolonged
persistence of the immunogen in tissues and a continuous
stimulation to the immune system. Ten days after the final
injection, 60 ml of blood was collected and kept overnight
at room temperature to allow clotting of blood. The crude
antiserum was collected by centrifugation (4200 g for
5 min) and the globulin fraction was isolated by three
rounds of selective precipitation with ammonium sulfate
(40% saturation). After the final precipitation, the proteins
were dissolved in 25 ml of 0.2 M NaCl. At this stage, the
titer and the specificity of the antibody were checked by
ELISA and western blot analysis, respectively.
Flask culture of G. frondosa and preparation of the
crude HGFI protein
The stock culture was maintained on potato dextrose agar
(PDA) slants and subcultured every 2 months. The seed
cultures medium contained 40 g/L glucose, 5 g/L peptone,
2.5 g/L KH2PO4, and 3 g/L MgSO4.7H2O. For the preparation of the inoculum, five pieces (about 1 cm 0.5 cm)
of the mycelia of G. frondosa were each transferred from a
slant into a Erlenmeyer flask containing 50 ml of seed
Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 6 | Page 390
medium with the sterilized self-designed cutter. The flasks
were then placed in a rotary shaker incubator at 160 rpm
and 258C for 5 days. The next formal fermentation cultures
were performed in 250 ml flasks containing 100 ml of fermentation medium after inoculating with 10% (v/v) of the
seed culture. The fermentation medium composition was
20 g/L glucose, 4 g/L peptone, 1 g/L KH2PO4, 1 g/L
MgSO4.7H2O, and 15 g/L corn steep liquor. The initial pH
value of the medium was adjusted to 5.5. The fermentations were carried out in a rotary shaker incubator at
160 rpm, 258C for 7 days, and then the mycelium was harvested. The crude HGFI protein was prepared as previously
described [25]. Before using, a portion of lyophilized crude
HGFI potion was treated with trifluoroacetic acid and dried
under a flow of nitrogen gas. The dried material was dissolved in a desired buffer.
Antiserum titer determination by ELISA
Antibody titer was measured using an indirect ELISA.
Purified antigens were diluted to 5 mg/ml in 50 mM carbonate –bicarbonate buffer ( pH 9.6) and then coated on
plates at 100 ml aliquot per well in 96-well immunoplates
(Sangon), at 48C overnight. The plates were washed three
times for 5 min with PBS ( pH 7.6) containing 0.5%
Tween 20. For the assay, 200 ml of 3% BSA were added to
each well, and the plates were incubated for 40 min at
room temperature, then incubated with 150 ml polyclonal
antibodies against recombinant HGFI with different
dilutions (from 1:50 to 1:781,250). After incubation for 2 h
at 378C, the wells were incubated with 150 ml of horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma,
St. Louis, USA) (dilution 1:5000) for 1 h at 378C after
thorough washing. Peroxidase activity on the immunoplate
was detected using 3,30 ,5,50 -tetramethylbenzidine (TMB)
and H2O2 as enzyme substrates. Color development was
stopped with 2 M of H2SO4 and the absorbance was
measured at 490 nm using Microplate Reader (Bio-Rad,
Hercules, USA).
Western blot analysis of the recombinant proteins
Protein samples were analyzed on 16% SDS-PAGE, and
then electroblotted onto a 0.22-mm pore size nitrocellulose
membrane at a constant current of 80 mA at 48C for 1 h.
After being blocked in TBS (150 mM NaCl, 20 mM
Tris-base, pH 7.4) with 5% (w/v) skimmed milk, the membrane was incubated with the polyclonal antibodies against
HGFI (1:2000 diluted in TBS) at 378C for 2 h. After
washing three times, goat-anti-rabbit IgG conjugated with
HRP was added and incubated for 1 h at room temperature.
The membrane was visualized with TMB membrane substrate (Amresco, Solon, USA). As a control, the formal
serum control was treated with the same protocol.
Prokaryotic production of fungal hydrophobin HGFI for antibody preparation
Immunofluorescence assay
Indirect immunofluorescence analysis was carried out as
reported by Haido et al. [26]. Briefly, fermentable mycelia
were incubated in blocking buffer (3% BSA in PBS) for
1 h at 378C, then treated with rabbit anti-HGFI serum at
a dilution of 1:500 and incubated for 1 h at 378C in a
moist chamber. After washing, fluorescein-isothiocyanateconjugated goat anti-rabbit IgG diluted (1:100) in PBS
(10 mM, pH 7.2) was added. The mycelia were incubated
for 1 h, washed and examined in a fluorescence microscope. A control experiment was performed on the hyphae
of Trichoderma reesei by using the same procedure.
Results
Construction of the recombinant plasmid pET-28a-hgfI
The nucleotides sequence encoding HGFI protein was
amplified from the template vector DH10B (pBSK-hgfI)
using the gene-specific primers, which contained the NdeI
and EcoRI sites to facilitate cloning in the pET-28a vector.
The location of the resulting DNA fragment is about
260 bp on an agarose gel (Fig. 1). Subsequently, the PCR
product was ligated into the pET-28a vector and transformed into the competent E. coli DH5a cells. The clone
was identified by PCR and restriction analysis, and then a
positive clone was sequenced and the result confirmed the
hgfI gene in frame with C-terminal His6 tag in the pET-28a
multiple cloning sites.
Expression of the recombinant protein
The confirmed recombinant vector was transformed into
E. coli BL21(DE3) cells. In order to make the recombinant
protein expressed maximally, we carefully optimized the
expression conditions as described in ‘Materials and
Methods’. Small-scale cultures were first subjected to
IPTG induction to identify the capacity of expression.
Correct recombinant protein with molecular weight of
Figure 1 PCR amplification of hydrophobin gene hgfI from the
plasmid DH10B ( pBSK-hgfI) Lane 1, molecular mass marker; lane 2,
the amplified product of 260 bp.
14 kDa was selectively expressed in the transformed E. coli
BL21(DE3) cells and this protein was almost absent in
non-induced cells transformed with the same vector
[Fig. 2(A), lane 1]. It was shown that the content of the
recombinant protein increased with the increasing induction
time and the concentrations of IPTG. The expression level
of the recombinant protein achieved culmination at a fusion
protein of 5 h [Fig. 2(A), lane 6] and 0.7 mM IPTG
[Fig. 2(B), lane 5]. Finally, the best induction condition for
the recombinant protein is: 0.7 mM IPTG, induction for
5 h at 258C with an initial cell density at OD600 ¼ 0.7.
After sonication, the supernatant and pellet of cell lysate
were analyzed to examine the solubility of the expressed
recombinant protein. We found that a majority of recombinant protein was distributed in the inclusion body in an
insoluble form (Fig. 3, lane 2).
Purification and identification of the recombinant
protein
Because the inclusion body of the recombinant HGFI
protein could not be dissolved in 8 M urea or 6 M guanidine hydrochloride, immobilized metal-chelated affinity
chromatography could not be used for purification. Protein
purification was carried out through the passive elution of
the recombinant HGFI protein from polyacrylamide gel
pieces. Comparing the results of staining and destaining,
we found that the first method described in the Materials
and Methods was better. After staining by the Coomassie
Brilliant Blue R250 and destaining by 250 mM KCl solution, the background of the gel was clear and the color of
the protein bands were so stable that we can cut plenty of
target bands easily. However, when the gel was only
stained with the 250 mM KCl solution directly according
to the Hon’s method, the protein was stained to ivory-white
as the background of the gel. Therefore, it was hard to distinguish the protein bands from the gels and the color of
the protein bands disappears within 10 min. The later
method was not convenient for incising lots of protein
bands from the stained gels. Electroelution allowed the
perfect recovery of pure recombinant HGFI protein
[Fig. 4(A)]. The purified recombinant HGFI protein was
recognized by anti-His polyclonal antibody in western blot
assay [Fig. 4(B)]. Based on spectrophotometric measurement of protein concentration in the eluted fraction, it was
calculated that at least 100 mg of purified recombinant
HGFI protein could be obtained per 100 ml of bacterial
culture after desalting by Sephadex G-25 (Fig. 4).
WCA measurements
The self-assembly property of recombinant HGFI was
determined by WCA measurements. The WCA of bare and
modified gold surface were shown in Fig. 5. The WCA of
the hydrophobic bare gold surface was about 758
Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 6 | Page 391
Prokaryotic production of fungal hydrophobin HGFI for antibody preparation
Figure 2 Optimization of expression conditions by the 16% gel electrophoresis of total cellular protein (A) Expression of recombinant protein at
different induction time points. Lane 1, induction for 8 h without IPTG treatment; lanes 2 – 8, induction with IPTG for 1, 2, 3, 4, 5, 6, and 7 h,
respectively. (B) Expression of recombinant protein at different concentrations of IPTG. Lane 1, without IPTG induction; lanes 2– 6, induction of 0.1,
0.3, 0.5, 0.7, and 0.9 mM IPTG, respectively.
Figure 3 Solubility analysis of the recombinant HGFI protein Lane
1, molecular weight markers; lane 2, total cellular protein from induced
E. coli cell; lane 3, the pellet of lysate from induced E. coli cell; lane 4,
the supernatant of lysate from induced E. coli cell; lane 5, total cellular
protein from E. coli cell without induction; lane 6, the pellet of lysate
from E. coli cell without induction; lane 7, the supernatant of lysate from
E. coli cell without induction.
[Fig. 5(A)]. After adsorption of recombinant HGFI, the
WCA decreased to 568 [Fig. 5(B)], which was slightly
bigger than that (478) of native HGFI-coated gold surface
[Fig. 5(C)].
Flask culture and preparation of the crude HGFI
protein
When the fermentation of G. frondosa was finished, the
mycelium was harvested by centrifugation. About 1.2 g
(dry weight) of mycelia of G. frondosa was obtained from
100 ml of fermentation solution after 9-day fermentation.
We could see clearly the appearance of the mycelia by the
common light microscope and the result was shown that
the mycelia were intact and very strong (date not shown).
According to the standard procedure, the crude protein
extracts were prepared successfully. We calculated that
about 50 mg of the crude protein were extracted from 1 g
Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 6 | Page 392
Figure 4 Purification and identification of the recombinant
protein (A) Purification of the recombinant protein. Lane 1, induced
total cellular protein; lane 2, the purified recombinant HGFI protein; lane
3, molecular weight marker. (B) Western blotting assay using anti-His
polyclonal antibody.
of dry mycelia of G. frondosa using a BCA protein analyzer kit. After 16% Tricine-SDS-PAGE, we found three
main bands on the gel, the location of the bands were
around 10, 18 and 25 kDa, respectively.
Titer and specificity analysis by ELISA and western
blot analysis
Using the purified recombinant HGFI protein as the
antigen, its polyclonal antiserum was prepared successfully
through the procedure described in the Materials and
Methods. The polyclonal antiserum against recombinant
HGFI protein was purified from rabbit antiserum by
(NH4)2SO4 precipitation. ELISA was used to determine the
titers of the obtained antibody and we found that the
Prokaryotic production of fungal hydrophobin HGFI for antibody preparation
Figure 5 WCA measures of bare or modified gold surface
HGFI-coated gold surface.
(A) A bare gold surface. (B) A recombinant HGFI-coated gold surface. (C) A native
Sub-cellular localization analysis of HGFI
We used the prepared antiserum to detect the subcellular
localization of HGFI in mycelial cells of G. frondosa.
Immunofluorescence staining result suggested that HGFI
protein was localized predominantly in the cell wall of
mycelium [Fig. 7(A)]. Moreover, the intensity of the fluorescence at the budding position was higher than that at
other position on the hypha [Fig. 7(B)]. As shown in
Fig. 7(C), the fluorescence intensity on the hypha of
T. reesei was much lower than that on the hypha of
G. Frondosa.
Figure 6 Western blotting analysis Lane 1, the purified recombinant
HGFI protein from the E. coli BL21; lane 2, the native HGFI of crude
protein extract from G. frondosa.
antibody at different dilutions (1000 to 156,250 and 1000
to 31,250) was reacted with an equal amount of the recombinant protein and the crude protein from mycelium,
respectively. At the same time, the pre-immunized rabbit
serum was used as the negative control and we could not
detect the positive signal. The specificity of the antiserum
was determined by western blot analysis. The results were
shown in Fig. 6. It was shown that the polyclonal antiserum could detect not only recombinant HGFI protein
expressed in E. coli BL21(DE3) (Fig. 6, lane 1), but also
HGFI from crude protein extracts (Fig. 6, lane 2). It was
confirmed from the result that molecular weight of the
natural HGFI protein is 10 kDa.
Discussion
The self-assembly of hydrophobins is interesting for many
applications, the current and the potential applications
require large amounts of hydrophobin. However, there is
still a serious challenge on the production of these proteins
[4]. We intend to produce the HGFI hydrophobin in a satisfactory level by optimizing the fermentation condition of
G. frondosa. It is clear that antibody of HGFI is an important tool for the fermentation study of G. frondosa and
functional research of HGFI.
In this study, we chose pET-28a vector to facilitate the
following purification. This vector carried an N-terminal
His6 Tag, thus, the recombinant protein of HGFI should
carry a His6 fragment in its N-terminus. The comparatively
high level of expression could be achieved with 0.7 mM
IPTG and 5 h of induction at 258C. At the best expression
condition, cells of E. coli BL21 transformed with
Figure 7 Immunofluorescence labeling of fermentable hypha of G. frondosa with antibody of recombinant HGFI (A) A specific green labeling is
detected at the surface of the hyphae of G. frondosa. (B) More intense green fluorescence is present at the budding positions (arrows) than that of other
positions at the surface of the hyphae. (C) The control experiment performed on the hyphae of T. reesei.
Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 6 | Page 393
Prokaryotic production of fungal hydrophobin HGFI for antibody preparation
recombinant vector could produce a recombinant protein of
14 kDa that was absent in non-induced cells. The molecular
weight of the resulting protein was inconsistent with the
theoretical value (10.9 kDa) calculated from sequence of
the recombinant protein. This phenomenon was similar
with those found in other reports. For example, the apparent
molecular weight (17 kDa) of the recombinant HYDPt-1
hydrophobin produced in E. coli is slightly higher than the
expected size (14.5 kDa) [27]. The intrinsic characteristics
of hydrophobin are supposed to be contributed to this
result. It is well known that most of hydrophobins contain
eight cysteine residues and form four pairs of internal disulfide bonds, which are so stable that neither the DTT nor the
mercaptoethanol can permanently open the four disulfide
bonds. Therefore, the SDS monomer could not completely
cover on the protein peptide chain of the monomer of the
hydrophobin resulting in changes in gel mobility in the
process of the gel electrophoresis.
Most of the recombinant HGFI protein was in the form of
inclusion body. Moreover, it was very surprising that the
inclusion body of the recombinant HGFI could not be dissolved in the solution buffer containing 8 M urea or 6 M guanidine hydrochloride at common temperature. When the
inclusion bodies were boiled in the same buffer, it could dissolve very well but could not dissolve again when the temperature of the solution buffer was below 808C. The reason for
this phenomenon was unclear. Because all hydrophobin contained eight cysteine residues and formed four disulfide
bridges, we thought that numbers of disulfide bridges in this
recombinant protein played a important role for this problem.
Adsorption of hydrophobin molecules onto the different
surfaces has been studied by WCA analysis previously
[28,29]. Therefore, WCA analysis was used to prove the
biophysical activity of recombinant HGFI after purification.
In this study, a surface is considered to be hydrophobic if
its WCA is more than 608; while hydrophilic if it is lower.
The hydrophobic gold surface was reversed from hydrophobic to hydrophilic, suggesting that the biophysical
activity of recombinant HGFI was preserved. However, the
slightly bigger WCA of recombinant HGFI illuminated that
there were some difference between the structure of recombinant HGFI and that of native HGFI. In recent years, a
novel protein purification method and drug targeting
system was developed by using polyhydroxyalkanoate
(PHA) granule-binding protein named phasin [30–32]. In
these reports, phasin was specially used to attach the
hydrophobic PHA granule surfaces. It was supposed that
hydrophobins could be used as protein phasin to develop a
similar protein purification and specific drug targeting
systems, because of the intrinsic self-assembly properties
of hydrophobins on the hydrophobic surfaces.
The purity of the purified recombinant HGFI protein
was nearly 100%, so we obtained high titer of the
Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 6 | Page 394
antibody. Because we did not incise 20 extra residues
including a His6 tag from the recombinant protein, the
polyclonal antibody could be recognized as a kind of
recombinant antibody. Therefore, the antibody titer for the
recombinant HGFI was little higher than that of the crude
protein extracts containing the nature HGFI.
The aim of the indirect immunofluorescence analysis
was to localize the HGFI on the hypha of G. frondosa. The
results were shown that HGFI antibodies were located at
the surface of cell walls of G. frondosa. This observation
was in agreement with the presence of a putative signal
peptide in the nucleotide sequence of HGFI gene.
Moreover, it was interesting that the fluorescence intensity
at the budding position was higher than other position on
the hypha. Thus, we could believe that HGFI hydrophobin
not only fulfilled a function in the development of the
hypha, but also in the budding process of the new hypha.
The latter function of HGFI hydrophobin was first discovered in the hydrophobin family. The control experiment
performed on the hypha of T. reesei, which can produce
class II hydrophobin HFBI was to prove the specificity of
HGFI antibody. We believe the faint fluorescence was
resulted from the nonspecific adsorption of HGFI antibody
on the hypha of T. reesei. The result revealed that HGFI
antibody has no cross reaction with HFBI protein on the
cell wall of T. reesei due to the good specificity.
Acknowledgement
We thank Professor Yaoting Yu for revising this manuscript.
Funding
This work was supported by the grants from the Program
for New Century Excellent Talents in University
(NCET-06-0212), and Sino-Finnish Scientific and
Technological Cooperation Project from the Ministry of
Science and Technology of China (2006DFA32360).
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mutation of Schizophyllum commune, which suppresses formation of aerial
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Acta Biochim Biophys Sin (2010) | Volume 42 | Issue 6 | Page 395