One-Step Purification of Melittin Derived from Apis mellifera Bee … · 2017. 1. 24. · 86 Teoh...

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.


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.



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.


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]


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]


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. 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.


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.



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.

References

1. Moon DO, Park SY, Lee KJ, Heo MS, Kim KC, Kim MO, et

al. 2007. Bee venom and melittin reduce proinflammatory

mediators in lipopolysaccharide-stimulated BV2 microglia.

Int. Immunopharmacol. 7: 1092-1101.

2. Maulet Y, Brodbeck U, Fulpius BW. 1982. Purification from

bee venom of melittin devoid of phospholipase A2

contamination. Anal. Biochem. 127: 61-67.

3. Vila-Farres X, Giralt E, Vila J. 2012. Update of peptides with

antibacterial activity. Curr. Med. Chem. 19: 6188-6198.

4. Wu R, Wang Q, Zheng Z, Zhao L, Shang Y, Wei X, et al.

2014. Design, characterization and expression of a novel

hybrid peptides melittin (1-13)-LL37 (17-30). Mol. Biol. Rep.

41: 4163-4169.

5. Stockwell VO, Duffy B. 2012. Use of antibiotics in plant

agriculture. Rev. Sci. Tech. 31: 199-210.

6. Cichocka-Jarosz E. 2012. Hymenoptera venom allergy in

humans. Folia Med. Cracov. 52: 43-60.

7. Moreno M, Giralt E. 2015. Three valuable peptides from bee

and wasp venoms for therapeutic and biotechnological use:

melittin, apamin and mastropan. Toxins (Basel) 7: 1126-1150.

8. WHO/IUIS. 2016. Allergen Nomenclature. Available from

http://www.allergen.org. Accessed August 12, 2016.

9. Zhu W, Wang B, Zhu X. 2002. Isolation and purification of

BV I-2H from bee venom and analysis of its biological

action. Chin. Sci. Bull. 47: 910-914.

10. Stenholm A, Lindgren H, Shaffie J. 2006. Comparison of

amine-selective properties of weak and strong cation-

exchangers. J. Chromatogr. A 1128: 73-78.

11. Schmidt M, Hafner M, Frech C. 2014. Modeling of salt and

pH gradient elution in ion-exchange chromatography. J. Sep.

Sci. 37: 5-13.

12. GE Healthcare. Ion Exchange Chromatography and Chromatofocusing:

Principles and Methods. GE Healthcare Handbooks.

13. Corporation WB. 1993. Worthington Enzyme manual. Available

from http://www.worthington-biochem.com/PLA/assay.html.

Accessed January 05, 2016.

14. Sigma-Aldrich. Enzymatic Assay of Hyaluronidase. Available

from http://www.sigmaaldrich.com/technical-documents/

protocols/biology/enzymatic-assay-of-hyaluronidase.html.

Accessed January 20, 2016.

15. Hall T, Wilson JJ, Brownlee TJ, Swartling JR, Langan SE,

Lambooy PK. 2015. Alkaline cation-exchange chromatography

for the reduction of aggregate and a mis-formed disulfide

variant in a bispecific antibody purification process. J.

Chromatogr. B Analyt. Technol. Biomed. Life Sci. 975: 1-8.

16. McDonald P, Tran B, Williams CR, Wong M, Zhao T, Kelley

BD, Lester P. 2016. The rapid identification of elution

conditions for therapeutic antibodies from cation-exchange

chromatography resin using high-throughput screening. J.

Chromatogr. A 1433: 66-74.

17. Staby A, Jacobsen JH, Hansen RG, Bruus UK, Jensen IH.

2006. Comparison of chromatographic ion-exchange resins

V. Strong and weak cation-exchange resins. J. Chromatogr. A

1118: 168-179.

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.

One-Step Purification of Melittin Derived from Apis mellifera Bee … · 2017. 1. 24. · 86 Teoh...

Documents

Transcript of One-Step Purification of Melittin Derived from Apis mellifera Bee … · 2017. 1. 24. · 86 Teoh...