Protein Expression and Purification 69 (2010) 198–203

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Protein Expression and Purification

journal homepage: www.elsevier.com/locate/yprep

Expression, purification, and characterization of recombinant lumbrokinase PI239

in Escherichia coli
Zhe-rong Xu a, Yun-mei Yang a,*, Qi-feng Gui a, Li-na Zhang a, Lin Hu b
a
Department of Geriatrics, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China
b
Pharmaceutical Department, 302 Military Hospital of China, Beijing 100039, China

a r t i c l e i n f o a b s t r a c t

Article history: Lumbrokinase (LK) is an important fibrinolytic enzyme derived from earthworms. It has been found that
Received 17 July 2009 LK is composed of a group of isoenzymes. To construct and express the mature peptide of LK PI239 in
and in revised form 20 August 2009 Escherichia coli, we amplified and optimized the gene of LK which was then cloned into the prokaryotic
Available online 28 August 2009
expression vector pET-22b(). The recombinant LK (rLK) protein was expressed as inclusion bodies and
we have developed a purification process of rLK from these inclusion bodies. A step-down urea concen-
Keywords: tration strategy was applied to the rLK renaturation process. The purified and renatured rLK apparently
Lumbrokinase
ameliorated the conditions of the model thrombosis rats used, and may be developed into a therapeutic
Thrombosis model
Escherichia coli
agent for thrombotic-associated diseases.
Inclusion body Ó 2009 Elsevier Inc. All rights reserved.

Atherothrombotic diseases lead to about 26 million deaths every fied and refolded, and the anti-thrombotic and fibrinolytic effects
year around the world, and therefore more and more attention is were investigated using a thrombosis rat model. This work may serve
being given to anti-thrombotic drugs. Earthworms, which have long as a significant foundation for the bioengineering of lumbrokinases.
been used as a component in traditional Chinese medicine due to
their anti-pyretic and diuretic properties, have been found to have
Materials and methods
anti-thrombosis effects [1–6]. Further studies showed that lumbro-
kinases, which are present in the body cavity and digestive organs of
Materials and reagents
earthworms, are a group of fibrinolytic enzymes. Some lumbrokin-
ases have been isolated from earthworms or expressed by recombi-
Earthworms (Lumbricus bimastus), purchased from Beijing Baiao
nant DNA techniques and are being extensively investigated
Pharmaceuticals, were the LK source. Restriction enzymes, rTaq
[5,7–10]. It was found that many wild type lumbrokinases are highly
polymerase and T4 DNA ligase were purchased from TaKaRa (Dalian,
stable in common organic solutions in vitro over a long period of
China). Isopropyl-b-D-thiogalactoside (IPTG)1 was purchased from
time, and most importantly, tend to be absorbed through the intes-
Sigma (USA). Urea, guanidine hydrochloride, Triton X-100 and Tris
tine [2]. Therefore, they have the potential to be developed into ther-
were purchased from Serva (Germany). Sephacryl S-300 high reso-
apeutic drugs for thrombotic-associated diseases such as cerebral
lution resin was purchased from Amersham Pharmacia Biotech
thrombosis and post-apoplectic sequelae [1,3].
(USA). The expression vector pET-22b() and its host strain
Unfortunately, the life cycle of earthworm is long, and extraction
BL21(DE3) were purchased from Novagen. The t-PA (Actilyse) was
of lumbrokinases is generally a labor intensive and time-consuming
purchased from Boehringer Ingelheim Pharma (Ingelheim am Rhein,
activity [11]. What is more, the extract is easily contaminated by
Germany). All other chemicals and reagents were obtained from
multiple components. The enzymes must be purified from the
other commercial sources and were of the highest purity available.
lyophilized powder of the earthworm. As a result, this therapeuti-
cally fibrinolytic drug is expensive. So, high yields of recombinant
lumbrokinases are needed. Plasmid construction
In this study, we cloned and expressed the mature peptide of PI239
in Escherichia coli. The expressed insoluble inclusion body was puri- Reverse transcription-polymerase chain reaction (RT-PCR) was
used to obtain the gene fragment encoding lumbrokinase PI239

* Corresponding author. Address: Department of Geriatrics, First Affiliated
Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Zhejiang
Province, Hangzhou 310003, China. Fax: +86 571 87236178.
E-mail address: gbbf2000@sina.com (Y.-m. Yang).
1
Abbreviations used: IPTG, isopropyl-b-D-thiogalactoside; RT-PCR, reverse tran-
scription-polymerase chain reaction; LB, Luria–Bertani; MALDI-TOF, matrix-assisted
laser desorption/ionization time-of-flight; MS, mass spectrometry; BSA, bovine serum
albumin; PBS, phosphate-buffered saline; ECL, enhanced chemiluminescence.

1046-5928/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.pep.2009.08.013
 Z.-r. Xu et al. / Protein Expression and Purification 69 (2010) 198–203
in L. bimastus. Total RNA was extracted with Trizol reagents (Invit-
rogen Life Technologies, Carlsbad, CA, USA) according to the man-
ufacturer’s protocol. The tissue was homogenized manually with
single-use pellet pestles (Kontes Glass Company, Vineland, NJ,
USA). The concentration and purity of total RNA was estimated
by spectrophotometry. The cDNA was prepared using M-MuLV re-
verse transcriptase (Fermentas Life Science, Canada) and incubated
for 5 min at 70 °C, reverse transcribed for 1 h at 44 °C, and inacti-
vated for 5 min at 75 °C. PCR amplification was performed with
Pyrobest DNA polymerase (TaKaRa, Dalian, China) and the specific
primer sequences used for PI239 mature peptide were 50 -CGG
CATATGATTGTCGGAGGAATTGAAGC-30 (sense) and 50 -GCTCTAGAG
CATTAGTTGTTGGTGATGATGTCTG-30 (antisense) (synthesized by
Invitrogen, USA). NdeI and SalI restriction sites are underlined.
PCR amplification was programmed as follows on an Eppendorf
Mastercycler Gradient PCR System (Eppendorf, Germany): Taq
activation for 5 min at 95 °C, 30 cycles of denaturation for 30 s at
95 °C, an annealing process for 30 s at 55 °C, and an extension for
1.5 min at 72 °C. Finally, rTaq was added and the sample was incu-
bated at 72 °C for further 30 min. The amplified gene was recov-
ered using an agarose gel and cloned into pMD20-T vector. The
recombinant plasmid was named pMDLK. After DNA sequencing,
the lumbrokinase gene was recovered from pMDLK plasmid by
digestion with NdeI and SalI and cloned into the pET-22b prokary-
otic expression vector. The resulting plasmid was designated pET-
LK. All constructs were confirmed by DNA sequencing and E. coli
BL21(DE3) cells were transformed with the plasmid pETLK for pro-
tein expression.
Expression of rLK in E. coli and fermentation
A single transformed colony was inoculated into 10 mL Luria–
Bertani (LB) medium supplemented with ampicillin (100 lg/mL)
and grown while shaking overnight at 200 rpm at 37 °C. Three mil-
liliter bacteria culture was transferred to 300 mL fresh LB medium
in a 500 mL shake flask. The culture was grown at 37 °C until the
OD600 reached 0.5 and IPTG was added to 1 mM to induce the
expression of recombinant lumbrokinase. The culture was agitated
for an additional 4 h, yielding a final OD600 of 4. Cells (1 mL) were
collected by centrifugation and the pellet was resuspended in
100 lL of ddH2O, mixed with 20 lL of 6 SDS loading buffer
(0.35 M Tris, pH 6.8; 10.28% SDS; 36% glycerol; 5% b-mercap-
toethanol and 0.012% bromphenol blue) and heated at 100 °C for
10 min. The sample was centrifuged at 12,000 rpm for 8 min and
10 lL supernatant was analyzed by SDS–PAGE and stained by Coo-
massie blue R-250.
Fermentation was performed using a fermentor (working
volume of 5 L). A single transformed colony was inoculated in
10 mL Luria–Bertani medium supplemented with ampicillin
(100 lg/mL) and grown at 220 rpm and 37 °C overnight. Two
hundred milliliters of semi-defined medium (16 g/L tryptone,
10 g/L yeast extract, 20 mL/L glycerol, and 5 g/L NaCl) with
100 mg/L of ampicillin was inoculated with an overnight culture
and grown at 220 rpm and 37 °C until the culture reached an
OD600 of 2–3. The culture was used to inoculate the fermentor to
a starting OD600 of 0.1–0.2. Five liters of sterile medium (5 g/L tryp-
tone, 5 g/L yeast extract, 10 g/L glycerol, 2 g/L KH2PO4, 4 g/L
K2HPO4, and 3 g/L MgSO4) were aseptically added to the fermentor
and inoculated with the culture as described above. Fermentation
was performed using the following parameters: 37 °C, pH 7.0, air-
flow 5 L pm (1 vvm), agitation 250–850 rpm, and dissolved oxygen
30%. When the OD600 reached 7, cultures were induced by addition
of IPTG (final concentration is 1 mM) at 37 °C for 4–6 h. Cells were
harvested by centrifugation (Beckman J2-HS) at 15,000 rpm for
20 min at 4 °C.
199
Cell lysis and inclusion body washing, solubilization, purification, and
refolding
About 100 g (wet wight) of the harvested cell paste were resus-
pended in 500 mL of STE buffer (10 mM Tris–HCl, pH 7.5, 10 mM
NaCl, 1 mM EDTA, pH 8.0) and sonicated in ice. The pellet was col-
lected by centrifugation. Inclusion body of rLK was washed with
1000 mL of 4 M urea, 1% Triton X-100, 5 mM EDTA, 5 mM
b-mercaptoethanol, 50 mM Tris–HCl, pH 8.0, at room temperature
for 30 min twice, followed with 1000 mL of 5 mM EDTA, 50 mM
Tris, pH 8.0, at room temperature for 30 min. The final pellets of
inclusion bodies (about 250 mg protein) were solubilized by
homogenizing in 15 mL of 10 mg/L DTT, 7 M guanidine hydrochlo-
ride at 4 °C overnight. The supernatant was collected by centrifu-
gation. A chromatography column (2.4  100 cm) was packed
with 500 mL of depyrogenated Sephacryl-300 resin for the purifi-
cation of rLK. The column was equilibrated with 7 M guanidine
HCl. The supernatant of solubilized rLK was applied to the column.
The chromatography was performed at a flow rate of 1 mL/min and
rLK fraction was eluted with equilibration buffer.
The purified protein was diluted with 7 M guanidine HCl to
0.2 mg/mL and refolded by dialyzing against 30 times volume
of10 mM Tris–HCl, pH 8.0, supplement with 14 mM b-mercap-
toethanol, 5 lM CuSO4 at 20 °C for 8 h and finally dialyzed against
PBS at 4 °C for 24 h. The refolded protein was collected by centrifu-
gation at 15,000 rpm for 20–30 min to remove the pellet. The final
renatured protein was lyophilized and stored at 80 °C.
Characterization of rLK
The concentration of recombinant protein was measured using
the BCA protein assay reagent (Pierce, Rockford, IL, USA). The pur-
ity of the protein was evaluated by SEC-HPLC. The sample (5 lg) in
PBS was injected onto a 7.5 mm  300 mm G2000SW column
(TOSOH Corporation) at a flow rate of 0.5 mL/min. Peaks were
detected by monitoring at a wavelength of 275 nm. The purity of
rLK was calculated as a percentage of the total peak area detected.
N-Terminal and C-terminal amino acid sequencings were carried
out by Shanghai GeneCore Biotechnologies Co., Ltd. (Shanghai,
China). The molecular mass of the purified rLK was determined
by matrix-assisted laser desorption/ionization time-of-flight (MAL-
DI-TOF) mass spectrometry (MS).
Western blot analysis
Proteins were transferred to nitrocellulose membranes
(0.22 lm; Invitrogen) after SDS–PAGE using a Bio-Rad Trans-Blot
Semi-Dry electrophoretic cell. The unoccupied sites on the mem-
brane were blocked with 1% (w/v) bovine serum albumin (BSA)
in phosphate-buffered saline (PBS) for 1 h. The membrane was
rinsed with PBS and placed in 1:500 diluted rabbit anti-lumbrokin-
ase antibody (Biosynthesis, China) for 1 h with constant shaking.
Then, the membrane was washed with washing buffer (50 mM
phosphate, pH 7.0, 0.05% Triton X-100) three times (5 min each)
and incubated with 1:1000 diluted HRP-labeled goat anti-rabbit
IgG (Boster, China). Immune complexes were detected by en-
hanced chemiluminescence (ECL) according to the manufacturer’s
specifications (Santa Cruz, CA, USA).
Fibrinolytic activity assay of rLK
Fibrinolytic activity was measured with plasminogen-rich fibrin
plates using the method of Nakajima and Zhang et al. [12] with
t-PA as a standard. Samples were added to the plate. Total bacterial
protein without IPTG induction was used as the negative control
and t-PA of several concentrations (2, 4, 6, and 8 IU/ml or 0.2,
200 Z.-r. Xu et al. / Protein Expression and Purification 69 (2010) 198–203
0.4, 0.6, and 0.8 lg) served as positive controls. Plates were then
incubated at 37 °C for 6 h and the results were measured.
Animals
Male Sprague–Dawley (SD) rats were purchased from the
National Rodent Laboratory Animal Resource (Shanghai, China)
and housed in a room with controlled temperature (23 ± 2 °C)
and humidity (60% ± 5%); they were provided alternating 12-h
periods of light and darkness. The rats were housed in the depart-
mental holding room for 1 week and fasted for 24 h prior to the
experiments. All procedures involving animals were approved by
the Ethical Committee of the Institute and performed according
to the Principles of Laboratory Animal Care (China). All rats were
randomly divided into different groups (10 each group) and rLK
(2, 4, and 8 kIU/kg) and t-PA (2 kIU/kg) were administered orally
and intravenously, respectively (twice a day for 3 days). Thrombus
formation was induced 10 min after the last administration.
Thrombosis induction in vitro
Two milliliters of blood samples were collected from the
abdominal aorta of rats and rapidly placed in 4-mm-diameter sili-
cone tubes. The tubes were then rotated in a thrombosis instru-
ment at 17 rpm and 37 °C for 15 min. Finally, the thrombus was
harvested and the remnant blood was absorbed with a Whatman
No. 1 filter paper. The wet weight and length of the thrombus were
measured. Then the thrombus was dried by incubating at 64 °C for
30 min and weighed again.
Artery–vein bypass thrombosis model induction in rats
Thrombus formation was induced by the method described by
Xu and Ou [13]. The rats (250–300 g) were anesthetized with so-
dium pentobarbital (50 mg/kg, ip) and supine fixed. The right
common carotid artery and left external jugular vein were exposed
and dissected free from the surrounding tissues. Two polyethylene
tubes (10 mm diameter  100 mm length) were linked with a
weighted silk (120 mm in length) contained tube (20 mm diame-
ter  80 mm length). Tubes filled with heparin (50 IU/ml) were
connected with the left external jugular vein and the right com-
mon carotid artery. The artery–vein loop was ligated and blood
flow was restored for 15 min, and then the silk line was carefully
removed together with the thrombus. The weight and length of
the thrombus were measured.
Statistical analysis
All data were expressed as the mean ± SEM. Statistical evalua-
tion was performed by the two-tailed Dunnett’s test for multiple-
group comparisons or Student’s t test for two-group comparisons.
Differences were considered significant when the p value was less
than 0.05.
Results
Cloning and expression of rLK
RT-PCR was carried out to obtain the DNA fragment encoding
recombinant LK. After 30 cycles of reaction, the result of electro-
phoresis showed that LK gene had been amplified. The DNA frag-
ment encoding lumbrokinase was cloned into the prokaryotic
expression plasmid pET-22b() (Fig. 1A), which was confirmed
by restriction enzyme digestion with NdeI and SalI and DNA
sequencing (Fig. 1B). The pETLK was transformed into E. coli
Fig. 1. Schematic diagrams of pET-22b()-rLK expression vector. (A) The recombi-
nant gene encoding rLK was cloned into the pET-22b() vector and expressed in
E. coli BL21(DE3) strain under the control of T7 promoter. (B) Restriction analysis of
recombinant plasmid pET-22b()-rLK. Lane M—DL2000 DNA marker; lane 1—
plasmid pET-22b()-rLK digested with NdeI and SalI. Arrowhead indicates the
target gene.
BL21(DE3) for target protein expression. The expressed rLK was
about 26 kDa and the expression level was approximately 10% of
the total bacteria proteins. SDS–PAGE analysis revealed that most
target proteins existed in insoluble fractions. This indicated that
the target proteins were mainly expressed as inclusion bodies in
E. coli.
Solubilization, refolding, and purification of rLK
After inclusion bodies were washed three times they were sol-
ubilized with 7 M guanidine hydrochloride, 10 mg/L DTT at 4 °C
overnight. Approximately 90% of the inclusion bodies were dis-
solved. The soluble rLK was purified by one-step Sephacryl S-300
column (Fig. 2A). Finally, rLK was refolded by dialysis against
10 mM Tris–HCl (pH 8.5) supplement with 14 mM b-mercap-
toethanol, 5 lM CuSO4 and finally dialyzed against PBS. The final
yields were 1.2 mg purified rLK per gram of cell paste (Table 1).
Characterization of rLK
The N-terminal analysis of rLK showed the sequences of amino
acid residues as followed: MIVGGIEARPYEFPW. It confirmed that
the sequences of the N-terminus of purified rLK were in agreement
with the sequences that we designed. The purity of final purified
rLK was analyzed by HPLC and found to be over 95% (Fig. 3).
In vitro activity assay (fibrinogenolytic assays)
The fibrinolytic activity of the purified rLK was analyzed using
fibrin assay plates. The lytic area generated by the recombinant
protease in the medium was 100 mm2/10 lg in the plasminogen-
rich plate. According to the standard curve of t-PA, the lytic activity
of rLK was about 1500 IU/mg protein. The negative control had no
fibrinolytic activities (Fig. 4).
Anti-thrombotic effect of rLK
Two kinds of thrombosis models were used in this study. In the
artery–vein bypass thrombosis model, when rLK and t-PA were
administered for 3 days (twice a day), the thrombus wet weights
were significantly decreased compared with the model group
(Fig. 5A). The thrombus weight of the negative control group was
19.5 mg. The treatment with rLK significantly decreased the
thrombus weights. In the high rLK dose group (8 kIU/kg), the
 Z.-r. Xu et al. / Protein Expression and Purification 69 (2010) 198–203 201

Fig. 2. Purification and identification of rLK. (A) The samples were taken after each washing. Ten microliters of inclusion body protein was loaded. Lane 1—low molecular
weight protein marker (kDa); lane 2—lysate of inclusion bodies before washing; lane 3—lysate of inclusion bodies after washing with 4 M Urea, 1% Triton X-100, 5 mM EDTA,
5 mM b-mercaptoethanol, 50 mM Tris–HCl, pH 8.0, for the first time; lane 4—lysate of inclusion bodies after washing with 4 M Urea, 1% Triton X-100, 5 mM EDTA, 5 mM b-
mercaptoethanol, 50 mM Tris–HCl, pH 8.0, for the second time; lane 5—lysate of inclusion bodies after washing with 5 mM EDTA, 50 mM Tris–HCl (pH 8.0); lane 6—the
purified rLK. (B) Western blot analysis of rLK. Samples were prepared as described under Materials and methods and analyzed via 12% SDS–PAGE followed by Western
blotting with a lumbrokinase-specific goat polyclonal antibody. Lane 1—whole cell lysate of pET-22b-rLK before induction; lane 2—whole cell lysate of pET-22b-rLK after IPTG
induction. (C) Bacteria growth and rLK expression in fed-batch culture.

Table 1
Purification of recombinant lumbrokinase.

Batch Wet cell paste (g) Expression level (%) Inclusion body (g) Total protein (mg) Purified protein (mg) Specific activity (IU/mg) Final yield (%)
20060308 110 10.5 3.2 250 138 1500 55
20060506 100 11.0 3.5 280 140 1600 50
20060708 120 10.0 3.0 240 125 1500 52

Fig. 3. SEC-HPLC analysis of the purity of rLK. Five micrograms of purified rLK were
analyzed on a G2000SW column, detected at OD275.
thrombus was decreased to the size of about 30% that of the neg-
ative control group. The positive drug t-PA also decreased the
thrombus sizes about 20%. In the thrombosis in vitro animal model,
both rLK and t-PA apparently ameliorated the thrombosis. The
thromboses of the rLK group were shorter than that of the model
group and both the wet and dry weight of thrombosis were signif-
icantly reduced. What is more, the protective effects of rLK on the
animal model showed a dose-dependent relationship. The highest
dosages showed significant effect compared with model group
(p < 0.01).
Discussion
Lumbrokinase capsules have been made from the extract of
earthworms for many years. The capsules of the extracts of
lumbrokinase, which are commercially referred to as Panford or
Fig. 4. Fibrin plate assay of the expression and activity of rLK. All samples were
spotted onto the fibrin plate and incubated at 37 °C for 6 h. Wells 1–4—positive
control t-PA with the concentration of 8, 6, 4, and 2 IU/mL (0.8, 0.6, 0.4, and 0.2 lg
respectively); well 5—negative control (total bacterial protein without IPTG
induction); well 6—purified and renatured rLK.
Boluoke, have been used in Southeast-Asian countries like in China,
Japan, and Korea, and also in North American countries such as in
Canada and the United States [14–17]. But substrate specificity and
inhibition studies of lumbrokinase indicated that it is a member of
the serine protease family. This means that the earthworms-deriv-
ative extract is a mixture of different components in which 4–6 dif-
ferent proteins are included. Although most of them are considered
to exert the same fibrinolytic effect in the human body and are
non-toxic [18], there has been efforts to isolate and prepare one-
component lumbrokinase agents so as to get rid of the contami-
nants that may cause side-effects. However, the preparation pro-
cess is complex and time consuming [11]. Fortunately, the
successful expression of the recombinant lumbrokinase provides
a way to obtain a single component with fibrinolytic activity.
202 Z.-r. Xu et al. / Protein Expression and Purification 69 (2010) 198–203

Fig. 5. Effects of rLK and t-PA on the thrombus formation in rats. (A) Thrombus weight changes were determined after the administration of the drugs into an artery–vein
bypass thrombosis rat model. The rLK and t-PA were orally and intravenously administered, respectively, twice a day for 3 days. (B–D) Thrombus length and weight changes
were determined in the thrombosis in vitro animal model. Data were expressed as the mean ± SEM. *p < 0.05, **p < 0.01; significantly different from the control group.

Several groups have reported successful expression of lumbro-
kinase in E. coli with prokaryotic fusion expression vectors (such
as pGEX-4T), but the fusion tag (GST) needed to be removed and
the purification procedure is complicated. In order to express
lumbrokinase without fusion tags, we designed two primers and
cloned the gene of lumbrokinase PI239 from L. bimastus by RT-
PCR according to the amino acid sequence of the PI239 lumbrokin-
ase [19]. Then the gene was optimized according to the E. coli
codon preference and the secondary structure of mRNA without
changing the amino acids it encodes (patents are being applied).
The recombinant lumbrokinase can be expressed in E. coli as inclu-
sion bodies when induced with IPTG. Therefore, an efficient and
convenient refolding system that would help the purification of
rLK is needed. Renaturation of rLK is very crucial because it is clo-
sely related with final yields and fibrinolytic activity. Many meth-
ods have been reported for protein refolding, such as dilution [20],
dialysis [21], and gel filtration [22]. In this work, we chose dialysis
for protein refolding. We tested three different protein initial con-
centrations (0.1 mg/ml, 0.5 mg/ml, and 1.0 mg/ml) for dialysis
refolding. The results showed that there was a lot of precipitation
at 1.0 mg/ml, a little at 0.5 mg/ml, and little at 0.1 mg/ml. Finally,
we chose 0.1 mg/ml of rLK as final concentration for the refolding
process. This provided the opportunity to generate more soluble
refolded rLK and increase the yield of the protein.
Considering that lumbrokinase is apt to be absorbed in intesti-
nal tract [2], some researchers have tried to express it in physiolog-
ical secretions such as milk so that LK can conveniently be given
orally. But the yield is limited and the processing of milk may de-
struct the activity of LK. E. coli is the best host for bio-drug produc-
tion. In this study, the rLK has the same fibrinolytic activity as t-PA.
Two kinds of thrombosis models were induced and rLK and t-PA
were administered orally and intravenously, respectively. Results
showed that both can apparently inhibit the formation of thrombo-
sis. It indicated that this process for production of lumbrokinase is
suitable and effective. However, further and more detailed studies
should be done before it can play a key role in thrombotic-associ-
ated diseases therapy.
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