Cloning, Expression, and Purification of Insecticidal ProteinPr596 from Locust Pathogen Serratia marcescens HR-3

Ke Tao Æ Xiaoqi Yu Æ Yun Liu Æ Guanying Shi ÆShigui Liu Æ Taiping Hou

Received: 12 February 2007 / Accepted: 16 May 2007

� Springer Science+Business Media, LLC 2007

Abstract A novel insecticidal protein (Pr596) produced

by Serratia marcescens HR-3 was found be a metallopro-

tease and responsible for insecticidal activity toward lo-

custs. Two pairs of primers were designed to amplify Pr596,

a putative open reading frame (ORF) by similarity search

and the N-terminal amino-acid sequence of insecticidal

protein. The results revealed that the ORF consisted of 1464

nucleotides encoding a protein of 487 amino-acid residues.

Pr596 was cloned into expression vector pET32a(+) and

was expressed in Escherichia coli BL21 (DE3)/pLysS strain

with isopropyl-b-D-thiogalactopyranoside induction. The

Pr596 was found to be highly expressed as inclusion bodies

by sodium dodecyl sulfate–polyacrylamide gel electropho-

resis (SDS-PAGE). Pr596 inclusion bodies were isolated

and subjected to Ni-NTA His Bind Resins (Pharmacia,

Germany). Pr596 purified and refolded was revealed by

SDS-PAGE and had proteolytic activity and insecticidal

activity. Results suggested that there is a potential to de-

velop this protein to be used as an alternative locus control

agent.

Keywords Insecticidal protein � Cloning � Expression �Purification � Serratia marcescens

Introduction

Serratia marcescens has received some attention because

of its potential to control locusts [7, 8, 16, 17]. To develop

the potential of microorganisms as biocontrol agents as

supplements to chemical pesticides, we screened high

toxicity–producing strain S. marcescens HR-3 to control

China’s locusts [17].

S. marcescens secretes a number of extracellular pro-

teins into the medium, including nuclease, phospholipase,

hemolysin, siderophore, chitinase, protease, and lipase [2,

10]. Of these, a novel metalloprotease in S. marcescens

HR3 was shown to be involved in the pathogenesis in lo-

custs [17].

Extracellular metalloproteases are widely distributed in

the bacterial world. They are mostly associated with

pathogenic bacteria or bacteria that have industrial signif-

icance [9]. Analysis of a variety of different S. marcescens

strains revealed that ‡ 4 different proteases are produced

from these strains [6, 12]. Among these proteases, the gene

for the major protease has been cloned and sequenced from

S. marcescens E-15 [13]. The complete genome of S.

marcescens Db11 is also now complete (available at: http://

www.sanger.ac.uk/Projects/S_marcescens/). Metallopro-

tease from S. marcescens HR3 was found be the insecti-

cidal protein for controlling locusts; therefore, we will give

special attention to the molecular genetic approaches for

studying insecticidal protein (metalloprotease) as a part of

our plan for industrial consideration of locust control.

In our previous study, insecticidal protein was purified

and characterized and its first 18 amino-acid residues

determined. We describe here the results of cloning,

expression, and purification of insecticidal protein in

Escherichia coli.

K. Tao � Y. Liu � G. Shi � S. Liu � T. Hou (&)

Key Laboratory of Bio-resource and Eco-environment, Ministry

of Education, Sichuan University, Chengdu 610064, PRC

e-mail: guo596@hotmail.com

K. Tao � X. Yu (&)

Key Laboratory of Green Chemistry and Technology, Ministry

of Education, Sichuan University, Chengdu 610064, PRC

e-mail: xqyu@tfol.com

123

Curr Microbiol (2007) 55:228–233

DOI 10.1007/s00284-007-0096-z

Materials and Methods

Bacterial Strains and Plasmids

Pr596 was isolated from S. marcescens HR3. E. coli DH5a(Novagen, Madison, WI) was used as the host strain for

cloning, and E. coli BL-21(DE3) (Novagen) was used for

expression. pMD18-T (Takara, Dalian, China) was used as

cloning vector, and pET32a(+) (Novagen) was used as

expression vector.

Media and Growth Conditions

All bacterial strains were maintained on Luria-Bertani (LB)

medium [15] containing 1.0% tryptone, 0.5% yeast extract,

and 0.5% NaCl. When required, antibiotic ampicillin (50

mM) was added. The standard fermentation conditions

were defined as follows: 37�C for 24 hours and shaking

speed of 180 rpm.

Materials

The restriction enzymes EcoRI and HindIII, the Taq DNA

polymerase, the GeneJETTM Plasmid Miniprep Kit, and

the Agarose Gel DNA Extraction Kit were purchased from

MBI Fermentas (Vilnius, Lithuania). The Rnase A, the

proteinase K, and the low molecular–weight protein marker

were from Sino-American Biotechnology Company

(Shanghai, China).

The polymerase chain reaction (PCR) primers were

synthesized by Shanghai Invitrogen Biotechnology Co.,

Ltd. The primers for Pr596 were as follows: A1: 5¢-GAG

GAA TTC ATG CAA TCT ACT AAA AAG GCA A-3¢(EcoRI) and A2: 5¢-TAT AAG CTT TTA CAC GAT AAA

GTC CGT GGC-3¢ (HindIII).

Cloning Insecticidal Protein

All molecular techniques were performed essentially as

outlined by Sambrook et al. [15]. Pr596 was amplified by

PCR. Mixtures consisted of 2 mM MgCl2, 0.2 mM dNTPs,

0.2 lM each primer, 40 ng DNA template, 5 ll 10 · Taq

buffer, and 2.5 U Taq polymerase. Cycling conditions for

the PCR reactions were: 94�C for 4 minutes, 30 cycles of

denaturation at 94�C, annealing at 58�C, and extension at

72�C for 1 minute each, followed by a 10-minute extension

at 72�C. The PCR product was purified and inserted into

the pMD18-T vector to construct the recombinant plasmid

pMD18-T- Pr596 (Fig. 1). The recombinant plasmid was

further identified by its restriction enzyme digestion pat-

tern, and the DNA sequence determination was performed

by DaLian Taka Biotechnology with an ABI PRISM 377

DNA Sequencer.

Expression of the Recombinant Insecticidal Protein

The recombinant plasmid pMD18-T- Pr596 was digested

with EcoRI and HindIII. The retrieved Pr596 was subcl-

oned into the EcoRI and HindIII sites vector of pET-32a(+)

to create the recombinant expression vector, pET-32a(+)-

Pr596 (Fig. 1) and was transformed into E. coli DH5a. The

recombinant plasmid was further identified by restriction

enzyme digestion. The expression plasmid was trans-

formed into E. coli BL21 (DE3) to express the pr596 fusion

protein. A single transformed colony was inoculated to LB

broth containing 50 mM ampicillin and grown at 37�C on a

shaking incubator until optical density at 600 nm (OD600)

reached 0.5 to 0.6. Isopropyl thiogalactose (IPTG) was then

added for final concentrations of 0.5, 1, 2.5, and 5 mM,

respectively. The induced cells were incubated 37�C for 4

hours and harvested by centrifugation at 6000 g for 15

minutes. The localization and expression level of Pr596

were analyzed by SDS-PAGE and gel-analysis software.

Purification of Recombinant Protein

The harvested cells as described previously were resus-

pended in 50 mM Tris-HCl (pH 8.0) lysed by sonication and

centrifuged at 10,000 g for 15 minutes to isolate the inclu-

sion bodies. Pr596 inclusion bodies were washed two times

with 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 mM

ethylenediaminetetraacetic acid, 0.5 M NaCl, 0.5% Triton

X-100, 5% glycerin, and 1 M urea, and then they were

Fig. 1 Strategy of cloning Pr596 into pET32a(+). The insecticidal

protein ORF Pr596 was cloned into pMD18-T. The resulting plasmid

pMD18-T- Pr596 was digested with EcoRI and HindIII, cloned into

pET-32a(+), and digested with the same set of restriction enzymes.

The resulting plasmid pET-32a(+)-Pr596 was transformed into E. coliBL21 (DE3)/pLysS strain

K. Tao et al.: Cloning, Expression, and Purification of Insecticidal Protein Pr596 229

123

washed with 50 mM Tris-HCl buffer, pH 8.0. The purified

inclusion bodies were then suspended in denaturation buffer

(50 mM Tris buffer [pH 8.0], 6 M guanidine hydrochloride,

and 0.1 M b-mercaptoethanol) at room temperature. After 1

hour of incubation, the solution was centrifuged at 10,000

rpm for 20 minutes, and the supernatant collected was

subsequently renatured overnight in 50 mM Tris-HCI (pH

8.0) and 0.6 M arginine. The solution was then centrifuged

at 12,000 rpm for 30 minutes, and the supernatant was

collected again and applied to Ni-NTA His Bind Resins

(Pharmacia, Germany). The column was washed with

binding buffer (50 mM Tris-HCl [pH 8.0], 5 mM imidazole,

500 mM NaCl, and 8 M urea), then eluted with wash buffer

(50 mM Tris–HCl buffer [pH 8.0], 30 mM imidazole, and

500 mM NaCl), and finally eluted with elution buffer (50

mM Tris–HCl [pH 8.0], 0.5 M imidazole, and 0.5 M NaCl).

Elution peak fraction (3 ml) was pooled. Purified Pr596 was

analyzed by SDS-PAGE and gel-analysis software.

Proteolytic Activity Assay and Insecticidal Activity Test

Purified Pr596 was assayed for proteolytic and insecticidal

activities in accordance with previously published proce-

dures [17].

Results

Cloning and Sequence Analysis of the Insecticidal

Protein Gene

Based on the N-terminal amino-acid analysis of insecti-

cidal protein form S. marcescens HR-3 [17] and the

homologous metalloprotease sequences reported [4, 13],

the suitable primer pair was synthesized. A mature

insecticidal protein region approximately 1.46 kb in

length was amplified by nested PCR from HR3 genomic

DNA. The fragment was sequenced and deposited to the

GenBank database (accession number EF070725). The

nucleotide sequence and the predicted amino-acid se-

quence for the Pr596 are shown in Fig. 2. Sequence

analysis indicated that the fragment consisted of 1464

nucleotides encoding a protein of 487 amino-acid resi-

dues. A comparison of the amino-acid sequence of the

Pr596 with that in the National Center for Biotechnology

Information’s conserved domain databases (available at:

http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi)

clearly showed that the Pr596 consisted of a conserved

domain, a zinc-dependent metalloprotease domain (amino

acids from 113 to 280). The conserved domain is

responsible for the zinc binding and contains the motif

H192E193I194G195H196 (Fig. 2), the best conserved zinc-

binding active site. No signal peptide was found using

online signal peptide (SignalP 3.0 Server; available at:

http://www.cbs.dtu.dk/services/signalp/). Although the

deduced amino-acid sequence of the Pr596 from strain

HR3 exhibited a high sequence similarity with metallo-

protease from S. marcescens SM6 (98%) [4] and S.

marcescens Db11 (95%) (available at: http://www.san-

ger.ac.uk/projects/s_marcescens/) and relatively lower

similarity (10% to 22%) to other metalloproteases—i.e.,

Bacillus subtilis neutral protease [18] and Pseudomonas

aeruginosa alkaline proteinase [14]—it all showed high

similarity to these proteases in the regions corresponding

to the zinc ligands and active sites. ‘‘B. thermoproteolyt-

icus’’ thermolysin was the first zinc metalloprotease for

Fig. 2 The nucleotide sequence

and the deduced amino-acid

sequence of Pr596 (accession

number EF070725) from S.marcescens HR-3. The highly

conserved region of Pr596 is

underlined. The active site is

boxed. Three zinc ligands are

shown by an asterisk

230 K. Tao et al.: Cloning, Expression, and Purification of Insecticidal Protein Pr596

123

which the three-dimensional structure was determined

[5]; therefore, the location for three zinc ligands and the

active site for Pr596 were predicted on the basis of

amino-acid sequence similarity (Fig. 2).

Expression of the Recombinant Insecticidal Protein

Pr596 was subcloned into pET-32a(+) to construct the

recombinant expression vector pET-32a(+)- Pr596 and

was then identified by its restriction-enzyme digestion

pattern (data not shown) and DNA sequencing. Plasmid

pET-32a(+)-Pr596, containing Pr596, T7 promoter for

high levels of expression, Trx-Tag for fusion expression,

and His-Tag for purification, was expressed by transfor-

mation into E. coli BL-21. After IPTG induction at 37�Cfor 4 hours, SDS-PAGE image analysis showed the

E. coli transformants produced a large amount of a new

fusion protein (molecular weight approximately 78 kDa).

Furthermore, after sonicating the induced E. coli trans-

formants, it was shown that the inclusion bodies in the

precipitate were formed largely by induction with IPTG

(Fig. 3). The expression of the fusion protein was ob-

served at 1 hour after induction, and its maximal

expression was achieved at 3 hours after induction. This

result also indicated that expression reached a plateau

after induction for 3 hours. Finally, the expression of

fusion protein with different IPTG concentration titration

was tested. No significant difference was observed when

0.5 to 5 M IPTG was added to induce expression (data

not shown).

Purification of Recombinant Protein

Insecticidal protein was successfully denatured, renatured,

and purified in E. coli as fusion protein. Ni-NTA His Bind

Resins resulted in a protein peak (Fig. 4). Calculations

based on the amount of protein at approximately 16-

ml fractions from Ni-NTA His Bind Resins indicated that

the bacteria secreted approximately 4.5 mg insecticidal

protein/l 24-hour culture broth. Protein purity was calcu-

lated to be 90% by SDS-PAGE (Fig. 5).

Proteolytic and Insecticidal Activities of Purified

Fusion Protein

Insecticidal activity of the purified fusion protein was

therefore tested against grassland locusts. Results showed

that the purified fusion protein was still highly toxic to

grassland locusts. Comparable with insecticidal protein

from S. marcescens HR-3 [17], the LD50 value (with 95%

confidence interval) of the insecticidal protein to grassland

locusts was 11.25 (range 4.97 to 25.21). Significant pro-

teolytic activity (132 U/ml) was detected compared with

the insecticidal protein from S. marcescens HR-3.

Discussion

Low yield is one of the major problems in the study and

application of insecticidal protein from S. marcescens

HR-3. Considering the unique properties of the insecticidal

protein (metalloprotease) from locust pathogen S. mar-

cescens HR-3 and its high yield requirement for biotech-

nologic application, the high-level expression of

insecticidal protein in E. coli is a good solution.

Fig. 3 SDS-PAGE analysis of the fusion protein induced by IPTG.

Proteins were separated by SDS-PAGE (8%) and visualized by

Coomassie brilliant blue G-250 staining. Lane 1 = uninduced BL-21/

pET-32a(+)/Pr596. Lane 2 = supernatant after sonication of induced

BL-21/ pET-32a(+)/Pr596 for 4 hours. Lane 3 = precipitate after

sonication of induced BL-21/pET-32a(+)/Pr596 for 4 hours. Lanes 4

through 7 = pET-32a(+)/Pr596 in BL21 (DE3) induced for 4 hours, 3

hours, 2 hours, and 1 hour, respectively. Lane 8 = protein molecular-

weight marker

Fig. 4 Purification curve of insecticidal protein. Purification curve of

insecticidal protein was determined after application of Ni-NTA His

Bind Resins. Closed circle, protein concentration; arrow, the

insecticidal activity peak

K. Tao et al.: Cloning, Expression, and Purification of Insecticidal Protein Pr596 231

123

In the previous study, locust pathogen HR-3 was iso-

lated from naturally dead grassland locusts and tested

against grassland locusts in China. The molecular identi-

fication of strain HR-3 confirmed that it corresponded to

S. marcescens (Jin et al. 2005 [11]; 16S rRNA genes partial

sequence, GenBank accession number AY538657). An

extracellular insecticidal protein secreted by HR-3 was

purified and characterized. The results of testing the puri-

fied insecticidal protein for enzymatic activities and

N-terminal amino-acid analysis both demonstrated that the

insecticidal protein was a metalloprotease [17]. In this

study, Pr596 encoding an extracellular insecticidal protein

from S. marcescens HR-3 was cloned and the fusion pro-

tein successfully expressed in E. coli and then purified. The

capability to produce the insecticidal protein in E. coli will

facilitate large-scale protein production and structural and

functional studies on the insecticidal protein.

According to the analysis of the insecticidal protein

gene, the mature insecticidal protein does not have the

characteristics of a signal peptide. Therefore, the expressed

protease was an enzymatically inactive form in E. coli and

therefore not excreted into the medium. However, insecti-

cidal protein in Serratia could be secreted to the culture

filtrates. Further studies of the secretion mechanism of

insecticidal protein in Serratia for this phenomenon will be

interesting.

In an important biocontrol agent, Bacillus thuringiensis,

the site-directed mutagenesis of Cry1Ab could improve

the toxicity to Manduca sexta (linnaeus) [1], and the site-

directed mutagenesis of the ORF of Cry1Ca also had

higher toxic comparable with that of the control [3].

Because of the importance of the insecticidal protein pro-

duction, one approach to obtain more stable and highly

toxic insecticidal protein stains are to modify some resi-

dues in the protease cleavage sites using site-directed

mutagenesis. This modification is now in progress.

Acknowledgments The authors acknowledge financial support

from the Program for New Century Excellent Talents in University of

China (NCET-04-0868), National Natural Science Foundation of

China (NSFC 20572076), and National Key Technology R&D Pro-

gram of China (2006BAE01A01-14).

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