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J. Microbiol. Biotechnol.
J. Microbiol. Biotechnol. (2017), 27(1), 84–91https://doi.org/10.4014/jmb.1608.08042 Research Article jmbReview
One-Step Purification of Melittin Derived from Apis mellifera BeeVenomAngela Ching Ling Teoh1, Kyoung-Hwa Ryu2, and Eun Gyo Lee1,2*
1Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea2Biotechnology Process Engineering Centre, KRIBB, Cheongju 28116, Republic of Korea
Introduction
The venom of the European honey bee Apis mellifera is an
intricate mixture of chemical compositions, including
proteins, peptides, enzymes, and other small molecules.
Lately, there has been growing interest in the use of
melittin, due to its wide range of biological and potential
therapeutic applications. Melittin, which is considered to
be an antimicrobial, antitumor, and anti-inflammatory
peptide, is the main component (≥50% (w/w)) of honey
bee venom and is widely used in oriental medicine [1] and
studied as an alternative for treating drug-resistant
infections [2-4]. In parallel to antimicrobial peptides for
therapeutic use in humans, melittin can be used to fight
economically important plant pathogens that limit crop
production globally [5].
Phospholipase A2 (PLA2) and hyaluronidase (HYA) are
the two major enzymatic proteins present in the bee venom
[6, 7]. Both of these enzymes are classified as major allergens
according to the International Union of Immunological
Societies, as they are capable of inducing the IgE response
in susceptible individuals [6-8]. Thus, both of these enzymes
must be efficiently removed in order to fully employ
melittin and its wide range of therapeutic applications.
Previous studies have stated that the purification process
of bee venom is considered to be a challenging task as it
involves a chain of separation and purification steps, whereby
different separation techniques such as gel filtration, affinity,
and ion-exchange chromatography methods are used [2, 9]
(Zhang W. 2009. Melittin purification method. Patent
CN20071158989). These purification processes aim to obtain
the highest possible product recovery yield and purity of
melittin. Yet, the yield and purity were usually low even
though it went through a series of complicated purification
processes [2]. Researchers have also tried to increase the
yield and purity of melittin by incorporating intermediate
steps between purification to enhance its yield and purity
[2] (Zhang W. 2009. Melittin purification method. Patent
CN20071158989). Intermediate steps such as adding invasive
additives and incubating melittin fractions for hours [2] are
Received: August 22, 2016
Revised: September 20, 2016
Accepted: September 20, 2016
First published online
September 23, 2016
*Corresponding author
Phone: +82-43-240-6633;
Fax: +82-43-240-6609;
E-mail: [email protected]
pISSN 1017-7825, eISSN 1738-8872
Copyright© 2017 by
The Korean Society for Microbiology
and Biotechnology
The concern over the use of melittin in honey bee venom due to its adverse reaction caused by
allergens such as phospholipase A2 (PLA2) and hyaluronidase (HYA) has been an obstacle
towards its usage. We developed a novel single-step method for melittin purification and the
removal of PLA2 and HYA. This study explores the influence of pH, buffer compositions, salt
concentration, and types of cation-exchange chromatography resins on the recovery of
melittin and the removal of both HYA and PLA2. Melittin was readily purified with a strong
cation-exchange resin at pH 6.0 with sodium phosphate buffer. It resulted in a recovery yield
of melittin up to 93% (5.87 mg from a total of 6.32 mg of initial melittin in crude bee venom),
which is higher than any previously reported studies on melittin purification. PLA2 (99%) and
HYA (96%) were also successfully removed. Our study generates a single-step purification
method for melittin with a high removal rate of PLA2 and HYA, enabling melittin to be fully
utilized for its therapeutic purposes.
Keywords: Melittin, purification, cation-exchange chromatography, hyaluronidase, phospholipase
A2
Melittin Purification 85
January 2017⎪Vol. 27⎪No. 1
very uneconomical and time consuming. Therefore, an
efficient one-step purification of melittin is definitely
required to obtain a high-yield and pure melittin.
Cation-exchange chromatography resins possessing ionized
–NH2 groups are frequently used for the recovery and
purification of protein [10]. To achieve an ideal experimental
setup for the purification of melittin by cation-exchange
chromatography, a series of parameters such as the influence
of pH, buffer composition, salt concentration, and type of
the resin used must be taken into consideration [10, 11].
The purpose of this study was to obtain a high recovery
yield and purity of melittin and removing HYA and PLA2
with a single-step purification method. This study explores
the influence of pH, buffer composition, and strength of the
cation-exchange resins on the recovery yield of melittin
and the removal of HYA and PLA2 from crude bee venom.
We report that a high recovery yield of pure and
concentrated melittin was recovered with a single-step
purification when a suitable buffer and pH were used. This
is the first report in which melittin has been purified using
a single-step purification method with HYA and PLA2 both
being removed.
Materials and Methods
Bee Venom
Lyophilized crude bee venom (300 mg) (Chungjin Biotech Co.,
Ltd, South Korea) was dissolved in 6.0 ml of HPLC grade water
(Honeywell, Burdick & Jackson, USA) to give a bee venom
solution. As bee venom is readily dissolved in water, nonsoluble
foreign substances remain undissolved and thus cleared by
centrifugation at 8,050 ×g for 20 min at 4ºC. Filtration using a
0.20 µm (Corning Incorporated, USA) filter was also carried out to
remove any tiny debris, bee glue, and pollen remaining. The bee
venom solution was prepared 1 h before use.
Buffer Preparation
Sodium phosphate, sodium acetate, and sodium citrate at a
50 mM concentration were used as the binding buffer. Each buffer
was added with 1 M of NaCl with the same concentration as
stated above to make the elution buffer. Salt (1 M NaCl) was
added to alter the ionic strength of the buffer and prevent
nonspecific bindings [12]. All the buffers were prepared with a
broad range of pH values ranging from pH 4 to 7 at intervals of
0.5 for buffer screening. The buffers were prepared by dissolving
the appropriate amount of material in water and adjusted to the
appropriate pH with 5 M HCl and 5 M NaOH. Buffers were
filtered through a 0.45 µm filter (Corning Incorporated) before
running in the AKTAfplc system (GE Healthcare Life Sciences,
Sweden).
Melittin Purification
Crude bee venom (200 µl) was mixed with 300 µl of binding
buffer to a total loading volume of 500 µl fixed at a 20 mg/ml
concentration and applied to a (7 × 25 mm) HiTrap SP FF column
(GE Healthcare, Life Sciences, UK) coupled to an AKTAfplc
system equilibrated with binding buffer. The sample was
chromatographed at room temperature at 1 ml/min flow rate.
Before sample loading, the column was equilibrated with 5
column volume (CV) of binding buffer. As soon as the sample was
loaded into the column, the column was washed with 15 CV of
binding buffer to remove the unbound samples. The elution was
done by elution buffer with a linear concentration gradient of
NaCl from 0 to 1 M to enable effective elution of melittin. The
elution profile was determined by monitoring the absorbance at
280 nm. Fractions (1 ml) were collected. HiTrap CM FF (GE
Healthcare, Life Sciences, UK) was also used with the parameters
stated above. For purification of melittin in a larger mass, HiTrap
SP FF (16 × 25 mm) (GE Healthcare, Life Sciences, UK) was used
with a step gradient of NaCl at 0.55, 0.90, and 1 M.
SDS-PAGE Analysis
Melittin, HYA, and PLA2 peak fraction pools were analyzed by
SDS-PAGE in a 15% polyacrylamide gel. Electrophoresis was
performed in the presence of SDS, and a molecular weight marker
ranging from 2 to 250 kDa was run in parallel. The gels were
stained with InstantBlue (Expedeon Ltd, UK).
RP-HPLC Analysis
The Varian Prostar HPLC system (Varian, Inc., USA) coupled to
a C18 monomeric column (4.6 mm × 150 mm × 5 µm) (Grace Vydac,
USA) was used at 1 ml/min flow rate. Elution was performed
with a linear gradient of 0% to 65% acetonitrile (Honeywell, USA)
in 0.1% trifluoroacetic acid (Sigma Aldrich, USA) for 38 min. The
elution profile was monitored at 280 nm. The area of the peak
detected was used to calculate the recovery of melittin. RP-HPLC
separation profiles of the bee venom fractions were also used to
assess the identity of the fractions compared with the standard
prepared.
PLA2 Enzyme Assay
PLA2 activity was assayed with the titrimetric assay method
according to the Worthington Biochemical Corporation enzyme
manual [13]. The substrate was prepared with 4 g of reagent-
grade soybean lecithin (MP Biomedicals, USA) dissolved in 30 ml
of 1 M NaCl, 10 ml of 0.1 M CaCl2, and 100 ml of reagent-grade
water. The substrate was stirred for 30 min at 4ºC and then
sonicated for 10 min at maximum power. The mixture was then
finally diluted with reagent-grade water to a final volume of
200 ml. Titration was carried out in a reaction vessel maintained at
25ºC and the change in pH was measured with a Orion 3 star pH
meter (Thermo Fisher Scientific, USA). The blank rate was first
determined by pipetting 15 ml of lecithin emulsion into the
86 Teoh et al.
J. Microbiol. Biotechnol.
reaction vessel, and the pH was then adjusted to pH 8.9 with
0.02 N NaOH. After a constant rate was obtained, the volume of
the titrant needed to maintain the pH at 8.9 for 5 min was
recorded. Then, 15 µl of sample from the melittin fraction pool
was added into the above emulsion. The amount of titrant (0.02 N
NaOH) required to maintain the pH at 8.9 for 5 min was recorded
as the sample rate. One unit releases one micromole of titratable
fatty acid per minute from lecithin emulsion at pH 8.9 and 25ºC
under the specific condition. The percentage of the remaining
PLA2 enzyme was calculated by the following formula:
HYA Enzyme Assay
HYA activity was assayed with the turbidimetric method [14].
A sample volume of 250 µl from the melittin peak fraction pool
was mixed with 250 µl of enzyme diluent (20 mM sodium
phosphate with 77 mM NaCl and 0.01% (w/v) bovine serum
albumin, pH 7.0 at 37ºC). The mixture was then mixed by swirling
and equilibrated to 37ºC for 10 min. Hyaluronic acid solution
(500 µl) (Sigma Aldrich) was then added and mixed immediately
by swirling followed by incubation at 37ºC for exactly 45 min.
After 45 min, 34 µl of mixture was added to 170 µl of acidic
albumin solution and transferred into a 96-well plate. The solution
was left to stand for 10 min at room temperature before being
analyzed with a microplate reader at 600 nm wavelength. The
percentage of the remaining HYA enzyme was calculated by the
following formula:
Results and Discussion
Buffer and pH Selection for High Recovery of Melittin
and Removal of HYA and PLA2
The specific effects of different buffers and pH values on
the purification of melittin were studied. Generally, cation-
exchange resins function by exploiting the protein net
charge [15]; thus, all the pHs selected were below the
isoelectric point (pI) of the melittin (12.01) so that the net
charge is positive. The melittin peak was eluted at an
average of 0.8 M NaCl and showed low HYA and PLA2
activity depending on the type of buffer and pH used.
Among the buffers screened, sodium phosphate buffer
was proven to be the best and most versatile buffer for a
high recovery yield of melittin and removal of HYA and
PLA2. Although sodium phosphate buffer is stable in a
broad range of pHs, the results showed diminution of the
average recovery yield of melittin as the pH value increased,
as shown in Fig. 1A. The optimum pH for a high recovery
of melittin and the removal of its contaminants lies at pH 6,
whereby an average of 5.5 mg (79%) of melittin was
recovered from the crude bee venom with a high removal
of HYA (97%) (Fig. 3A) and PLA2 (99%) (Fig. 2A). A total of
3.38 U/mg of HYA and 0.55 U/mg PLA2 remained in the
fraction pool. The purity of melittin purified with sodium
phosphate buffer at pH 6 (data not shown) was also one of
the highest (92%) among all the buffers. Lower pH value
buffers such as pH 4 and 4.5 were not selected despite
showing a high yield of melittin because the removal of
HYA contaminants was unsatisfactory. The theoretical pI
for HYA and PLA2 is 8.72 and 8.07, respectively. Thus,
when low pH buffers are used for the separation of melittin
PLA2 remained %( )Amount of PLA2 in sample
Amount of PLA2 in crude bee venom---------------------------------------------------------------------------------------------------- 100×=
HYA remained %( )Amount of HYA in sample
Amount of HYA in crude bee venom----------------------------------------------------------------------------------------------------- 100×=
Fig. 1. Effect of buffer and pH on the recovery yield of melittin
with the strong cation-exchange SP FF resin.
(A) Sodium phosphate, (B) sodium acetate, and (C) sodium citrate
buffers with different pH value ranging from pH 4 to 7 were screened.
The results were expressed as the percentage of yield of melittin with
standard error from three independent experiments.
Melittin Purification 87
January 2017⎪Vol. 27⎪No. 1
from bee venom, HYA and PLA2 become highly protonated
and more positively charged, and therefore, will bind
strongly to the cation-exchange resins together with melittin,
resulting in a lower purity of melittin.
The melittin yield obtained with sodium citrate buffer
(73%, 5.13 mg), as shown in Fig. 1C, was significantly
higher than that obtained with sodium acetate buffer (62%,
5.10 mg) at pH 6 in Fig. 1B. The highest recovery yield of
melittin with sodium acetate buffer was observed at pH 4
and 4.5 with an average recovery yield of 73% (5.10 mg)
and 80.5% purity. Although the yield of melittin was
considerably high at low pH, the efficiency of removing the
contaminants was relatively low. Low HYA removal rates
were detected at pH 4 (48%, 58.6 U/mg) and pH 4.5 (80%,
22.82 U/mg) with HYA enzymatic assay (Fig. 3B). PLA2
removal was also significantly low at pH 4 (97%, 1.04 U/mg)
(Fig. 2B) detected with PLA2 enzymatic assay. The melittin
fraction pool obtained with sodium citrate at pH 6 has
higher HYA removal (86%, 16.03 U/mg) and slightly lower
PLA2 removal (98%, 0.86 U/mg) than sodium acetate
buffer. Therefore, we can conclude that sodium acetate is
the most unsuitable buffer among the chosen because the
Fig. 3. Percentage removal of HYA from purified melittin
fraction pool.
HYA enzymatic assays were carried out using the fraction pool
collected from the purification of melittin with (A) sodium phosphate,
(B) sodium acetate, and (C) sodium citrate buffers. The results were
expressed as the percentage of removal of HYA with standard error
from three independent experiments.
Fig. 2. Percentage removal of PLA2 from the purified melittin
fraction pool.
PLA2 enzymatic assays were carried out using the fraction pool
collected from the purification of melittin with (A) sodium phosphate,
(B) sodium acetate, and (C) sodium citrate buffers. The results were
expressed as the percentage of removal of PLA2 with standard error
from three independent experiments.
88 Teoh et al.
J. Microbiol. Biotechnol.
average recovery yield and contaminant removal ability
were significantly lower compared with sodium phosphate
and sodium citrate buffers.
RP-HPLC was used to analyze the fraction pools after
crude bee venom was purified using SP FF with sodium
phosphate buffer at pH 6 (Fig. 4). As shown in Figs. 4A and
4B, the chromatograms proved successful removal of
contaminants from bee venom. A single melittin peak was
observed from the chromatogram after purification.
Therefore, sodium phosphate buffer was selected as the
buffer for all the following steps in the study.
The use of a suitable buffer and pH actually led to an
increase in the recovery yield of melittin, as shown in this
study. Major recovery yield differences and peak broadening
were also observed between buffers and pH values. These
prove that buffers play an important role in the purification
of melittin. In principle, the difference in buffer strength
and pH values alters the polarity of the protein, which then
affects the separation efficiency of the venom [16].
Previous studies on the purification of melittin [2]
(Zhang W. 2009. Melittin purification method. Patent
CN20071158989) had employed different types of buffers
such as ammonium acetate and urea acetate at a pH range
of 4−4.75 as their working buffer. Purification of melittin
using such buffers seemed to be less effective compared
with sodium phosphate buffer. The recovery yields of
melittin using ammonium acetate (60.3%) [2] and urea
acetate (47%) (Zhang W. 2009. Melittin purification method.
Patent CN20071158989) were far lower than sodium
phosphate (79%) used in this study. As such, the purification
of melittin was more complicated involving multiple
intermediate steps [2] such as soaking the venom in water
for hours, multiple desalting, and adding invasive additives
such as guanidine hydrochloride to purify the melittin and
to separate its contaminants. A series of purification steps
were also carried out with size exclusion and cation-
exchange chromatography. Size exclusion chromatography
separates the proteins based on their size whereas cation-
exchange chromatography separates them based on their
charge. Melittin can be more readily purified with charge
rather than its size because bee venom is composed of a
cocktail of proteins, peptides, and enzymes. Based on the
studies obtained using a series of purification steps [2]
(Zhang W. 2009. Melittin purification method. Patent
CN20071158989), only PLA2 has been reported to be removed
from melittin whereas the removal of HYA has not been
reported at all. Previous studies that carried out purification
of melittin using cation-exchange chromatography utilized
a weak cation-exchange resin instead of a strong resin.
Comparison of Strong and Weak Cation-Exchange Resins
for the Purification of Melittin
The performances of weak carboxymethyl Sepharose
Fast Flow (CM FF) and strong sulfopropyl Sepharose Fast
Flow (SP FF) cation-exchange resins for the purification of
melittin were compared based on their pH stability,
recovery yield of melittin, and efficiency in removing HYA
and PLA2 under similar conditions.
As shown in Figs. 5A and 5B, purification of melittin
using SP FF resin showed less nonspecific peaks than CM
FF resin using sodium phosphate buffer at pH 6. The
melittin peak labelled as P1 (Figs. 5A and 5B) in the
chromatograms also proved that melittin was eluted from
CM FF resin slightly earlier than from SP FF. Peaks labelled
as FT1, FT2, and FT3 in Figs. 5A and 5B are peaks that
contain HYA and PLA2 as proved in SDS-PAGE (Figs. 6A
and 6B). The SDS-PAGE also indicated that purification
using SP FF resin was more efficient than CM FF, as the
HYA and PLA2 bands were denser.
The recommendation of most suppliers is to run CM FF
above pH 6 to obtain full performance of the resin,
however, the highest binding was still obtained below pH
6. Although CM FF resins at pH 5 using sodium phosphate
buffer showed a higher melittin recovery profile than pH 6,
the removal rate of HYA using CM FF at pH 5 was lower,
whereby only 71% of HYA was successfully removed and
65.27 U/mg HYA was detected in the melittin fraction
pool. This indicates that CM FF is poor at purifying
Fig. 4. RP-HPLC chromatograms of crude bee venom before
and after being purified with strong cation-exchange resin
using sodium phosphate at pH 6.
(A) Crude bee venom before purification. (B) Crude bee venom after
purification.
Melittin Purification 89
January 2017⎪Vol. 27⎪No. 1
melittin. Fig. 7 also shows that SP FF has a higher recovery
yield of melittin and a better removal of HYA and PLA2
contaminants than CM FF, regardless of the pH values
selected.
pH values lower than pH 5 were not tested for CM FF
because the resins were unstable at lower pH. These resins
will gradually loses their charge as the pH decreases below
4 or 5 due to its carboxymethyl group. Although the CM FF
column was stable from pH 6 to 10, a pH higher than pH 6
was not considered because as buffer pH approaches
higher pH values such as pH 7, contaminants and melittin
will have weaker binding to the resins due to its pI value.
CM FF resins also showed drastic change in the melittin
recovery yield and its purity when a change in pH was
implemented. A previous study had proved that if a resin
displays too much variation in protein retention in the
operating or selected pH range, replacement with a less pH
sensitive resin may be necessary [17]. In this case, SP FF is a
much more suitable resin to be used in this study. Thus, SP
Fig. 5. Chromatograms of melittin purification and removal of
HYA and PLA2 from the crude bee venom with strong and
weak cation-exchange resins using sodium phosphate buffer
at pH 6.
(A) Chromatogram of melittin purification with SP FF resin. (B)
Chromatogram of melittin purification with CM FF resin.
Fig. 6. SDS-PAGE analysis of fraction pools of bee venom
collected from AKTAfplc after purification of melittin with
strong and weak cation-exchange resins.
The SDS-PAGE gels were labelled according to the chromatograms in
Figs. 5A and 5B. (A) SDS-PAGE for fraction pools collected from SP
FF resin. M: protein marker; C: crude bee venom; PLA2: phospholipase
A2 standard; Mel: melittin standard; FT1: peak 1 flow through; FT2:
peak 2 flow through; P1-1 to P1-3: chromatographic elution pools
containing melittin. (B) SDS-PAGE for fraction pools collected from
CM FF resin. M: protein marker; C: crude bee venom; PLA2:
phospholipase A2 standard; Mel: melittin standard; FT1: peak 1 flow
through; FT2: peak 2 flow through; FT3: peak 3 flow through; E1:
elution peak; P1-1 to P1-2: chromatographic elution pools containing
melittin.
Fig. 7. Comparison of percentage yield of melittin obtained
from SP FF and CM FF resins using sodium phosphate buffer
at pH 5 and 6.
90 Teoh et al.
J. Microbiol. Biotechnol.
FF proved to be a better resin for melittin purification than
CM FF.
Optimization of the Gradient Condition in Cation-
Exchange Chromatography
Optimization of the gradient condition for chromatography
can actually increase the concentration of the melittin
recovered. Linear gradient elution was initially carried out
to obtain the data on elution patterns of melittin and its
contaminants HYA and PLA2, and step-wise gradient
elution was then optimized and performed to elute high
concentrated melittin from crude bee venom.
As shown in Fig. 8, a three-step gradient elution was
designed to enhance the purification of melittin by increasing
the concentration and purity of melittin. The NaCl
concentration was increased to 0.55 M to remove any
contaminants remained in the column. Then, the NaCl
concentration was further increased to 0.90 M to efficiently
elute the melittin from the column. Finally, 1 M of NaCl
concentration was applied to confirm that melittin was
successfully eluted at 0.90 M salt concentration. Compared
with the linear gradient elution method, the separation
time was shortened remarkably, and the main fraction
containing melittin was concentrated. Successful recovery
of highly concentrated melittin from 0.42 mg/ml to
0.59 mg/ml was also achieved with a smaller fractionation
volume of melittin peak during the purification of melittin
by the step-wise gradient elution method from 0.55 to
0.90 M NaCl. Although the removal of HYA had observed
to be lower (96%) in step-wise gradient elution compared
with the linear gradient elution method (97%), the removal
of PLA2 had shown to remain unchanged (99%).
The overall purity of the melittin fractions had also
increased from 92% to 98% when the step-wise gradient
was applied instead of the linear gradient elution method.
Thus, these results indicate that the optimized step-wise
gradient elution method enhanced the purification of
melittin by shortening the purification time, increasing the
concentration, and obtaining higher purity melittin. Further
fine-tuning of the step elution in this study allowed successful
purification of melittin.
Evaluation of the Purification Efficiency of Melittin for a
Larger Quantity of Crude Bee Venom
Screening and optimization for melittin purification were
first performed at analytical scale, and then further
purification with an increase in mass of crude bee venom
was carried out. The initial concentration of crude bee
venom was set constant at 20 mg/ml. It is important that
criteria such as the recovery yield, purity, and concentration
of melittin are maintained after increasing the mass of the
crude bee venom to be purified. Five milliliters of 100 mg
of crude bee venom was loaded into two (16 × 25 mm)
HiTrap SP FF columns coupled to an AKTAfplc system and
was eluted using the step-wise gradient elution method.
Further increase in mass of crude bee venom was carried
out by loading 10 ml of 200 mg crude bee venom into the
same column and purified using the same condition as
stated above. The buffer used was sodium phosphate
buffer at pH 6.
Based on the results in Table 1, it was proven that an
increase in mass of crude bee venom for purification of
melittin is possible. The melittin recovered from 10 and
100 mg of crude bee venom had a purity as high as 98%.
The purity of melittin obtained from the purification of 200
mg crude bee venom was slightly lower (96%). Removal of
PLA2 was maintained at 99% even after purification of an
increased mass of crude bee venom had been carried out.
Although the percentage of removal of HYA was seen to
drop as the mass of crude bee venom increased, the rate of
removal of HYA was still high with a minimum of 88%. All
the experiments showed comparable elution patterns and
impurity profiles even though the loading mass of crude
bee venom had increased. Thus, a further increase in mass
of crude bee venom for purification of melittin has proved
to be possible.
In conclusion, the results of this study confirm that a
high purity and recovery yield of melittin can be obtained
with a one-step purification method with strong cation-
exchange chromatography resins using sodium phosphate
buffer at pH 6. The purity and recovery yield of melittin
Fig. 8. Chromatogram of melittin purified by the step-wise
gradient elution method using sodium phosphate buffer at
pH 6.
The elution profile was determined by monitoring the absorbance at
280 nm.
Melittin Purification 91
January 2017⎪Vol. 27⎪No. 1
were highly dependent on the types of buffer and pH used.
The optimal step-wise elution condition was designed to
increase the yield and concentration of the melittin collected.
An increased mass of crude bee venom for purification of
melittin was also made possible with the optimal condition
used in this study. Our study has proved to solve the
problem whereby melittin can be highly recovered with its
allergenic contaminants such as PLA2 and HYA removed in
a single-step purification.
Acknowledgments
This research was supported by a grant from the KRIBB
Research Initiative Program. BEESEN Co., Ltd. had also
fundamentally supported this study by donating crude bee
venom and provided information on this research.
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Table 1. Comparison of the purification of melittin from analytical scale (10 mg) with that of an increase of mass of crude bee
venom (100 and 200 mg).
Bee venoma
(mg)
Melittin
amountb (mg)
Purified melittin
concentration (mg/ml)
Purified
melittin (mg)
Melittin recovery
yield (%)
Melittin
purity (%)
HYA
removalc (%)
PLA2
removald (%)
10 6.32 0.59 5.87 93 98 96 99
100 64.4 1.16 57.91 90 98 88 99
200 128.8 2.46 122.93 95 96 90 99
aInitial mass of crude bee venom.bInitial amount of melittin in crude bee venom.cPercentage removal of hyaluronidase.dPercentage removal of phospholipase A2.