中国药科大学 学报

374 Journal of China Pharmaceutical University 2004 , 35(4):

374 ～ 378

A Thioredoxin-(His)6 Fusion Protein of Reteplase :Expres-

sion , Purification , and Activity Analysis
ZHANG Xin-Yuan , LIAO Jian-Min , SUN Shi-Jing , SHEN Zi-Long
(School of Biopharmaceutical Sciences , China Pharmaceutical University , Nanjing 210009 , China)

【ABSTRACT】 AIM:
To increase the expression amount of reteplase , a vector was constructed which expressed the fusion pro-
tein thioredoxin-(His)6-rPA.The purification of reteplase was simplified by one-step affinity chromatography in this study.
METHOD:Reteplase gene was inserted into the prokaryotic expression vector pET32a and was fused to the downstream of the
thioredoxin and the (His)6Tag in the same reading frame.The fusion protein was expressed in E.coli BL21 (DE3)induced
by lactose.After refolded in vitro by one-step dilution , the fusion protein was simply purified using Ni2 +-chelating chromatogra-
phy.Fibrin plate assay was used to detect the bioactivity of the fusion protein.RESULT:
A fusion protein of 56 kD was ob-
tained.The amount of the fusion protein was more than 60% of the total bacterial protein.Comparing with the non-fusion
reteplase , the yields of target protein increased about 50%.The purity of the fusion protein reached above 80 % after one-step
purification using Ni 2+-chelating chromatography.The bioactivity of the refolded and purified fusion protein was detected.
CONCLUSION:Expression level of the fusion protein increased distinctively compared with that of the non-fusion protein.
Bioactivity of reteplase was detected even though a fusion peptide was fused to the N-terminal of reteplase.
【KEY WORDS】 Reteplase(rPA); (His)6Tag ;
Thioredoxin; Fusion protein; Purification ;
Expression; Activity
【CLC Number】 R943 【Document code】 A 【Article ID】 1000-5048(2004)04-0374-05

to make reteplase expressed in the periplasm of E.col-

1 Introduction
Reteplase is the third-generation plasminogen acti-
vator in the treatment of acute myocardial infarction
( .It is a 39.6 kDa , single-chain , nonglycosylated
AMI)
peptide.Comparing with human tissue-type plasminogen
activator (tPA), reteplase consists of the kringle 2 and
protease domains while it lacks the finger , epidermal
[ 1]
growth factor , and kringle-1 domains . Researches
demonstrated that binding of tPA to fibrin was affected
most by mutations in the other domains than kringle 2
domain[ 2] .Therefore reteplase has lower affinity for fib-
[ 1]
rin and longer half-life than tPA .The biological activ-
ity of reteplase depends on the correct formation of nine
disulfide bonds.
Reteplase is produced as inactive inclusion bodies
in E.coli and requires in vitro folding to become ac-
tive[ 1] .Schäffner et al used secretion expression vectors
i[ 3] .Manosroi et al used a phagemid vector to express
reteplase in E.coli XL-1 Blue and gained the protein
as an active form both in the periplasm and in the cul-
ture supernatant[ 4] .Although secretion expression yields
native , disulfide-bridged proteins , inclusion bodies have
their own advantages such as high protein yields , pro-
tease-resistant and convenient purification process (sim-
ply by centrifugation to yield highly concentrated and
[ 5-7]
relatively pure protein) .In addition , toxic proteins
may not inhibit cell growth when present in inactive form
as inclusion bodies[ 7] .
This study aims at increasing the expression yield of
reteplase and simplifying the process of purification
based on former research in our laboratory [ 8] .
2 Experimental Procedures
2.
1 Materials

【Received date】 2003-12-22 【＊Corresponding author】 Tel : 025-83271389 E-mai l : zilong_shen @yahoo.com.cn
【Foundation It em】 The project was supported by the Foundation of Sci ence and Technology Office of Jiangsu Province(No.BG2002318)
 No .
4 ZHANG Xin-Yuan , et al:
A thioredoxin-(His)6 fusion protein of reteplase :
Plasmid J1 containing reteplase gene , JM109 , BL21
(
DE3)and pET32a were prepared by our laboratory.
Restriction enzymes Kpn Ⅰ , Hind Ⅲ , Xho Ⅰ and others
®
were purchased from MBI.T4 DNA ligase and pGEM -
T vector were purchased from Promega.Gel DNA Ex-
traction Kit was purchased from Shanghai Sangon.His-
TM
Trap chelating Kit was purchased from Amersham Bio-
sciences.Chemicals were of analytical grade and were
purchased from BBI. Human fibrinogen (FIBRO-
RAAS ®) was purchased from Shanghai RAAS Blood
Products Co., Ltd.
2.2 Construction of Reteplase Fusion Protein Expression
Vector
A pair of primers were designed :
the sense primer
P1 :-CCGGTACCATTGAAGGCCGTTCTTACCAAGGA
5′
AACAGTGACTGCTACTTTGGG-3′ and the anti-sense
primer P2 :-CCAAGCTT TTATCACGGTCGCATGTTG
5′
TCAC-3′ .The sense primer and the anti-sense primer
contain the underlined Kpn Ⅰ and Hind Ⅲ restriction
site respectively.The sense primer also contains a 12-
base sequence following Kpn Ⅰ site which encodes the
cleavage site recognized by Factor Xa.Reteplase gene
was amplified from J1 by PCR.
The PCR products were purified and digested with
Kpn Ⅰ and Hind Ⅲ , electrophoresed on 1 % agarose
gels , and purified from excised gel slices using Gel DNA
Extraction Kit and ligated into Kpn Ⅰ/ Hind Ⅲ digested
pET32a(Novagen)to generate the pET-rPA plasmid.
The overnight ligation products were transformed into
JM109[ 9] .After PCR analysis and restriction endonucle-
ase analysis , the pET-rPA was sequenced (Shanghai
.
Sangon)
2.
3 Expression
Plasmids pET-rPA were extracted , purified and
[ 9]
transformed into E.coli BL21 (DE3) .One clone
was inoculated into 5ml LB medium (with 60 μ g/ ml
ampicillin)and incubated with appropriate shaking at 37
℃overnight.The following morning 2 ml cells were in-
oculated to 200 ml fresh LB medium containing ampi-
[ 7, 9]
cillin (
60 μg/ml)in a 1 000 ml flask .Cells were
incubated with shaking at 37 ℃ until the OD600 was of
6 , and lactose was added to a final concentration
about 0.
expression, purification, and activity analysis 375
of 5.0 mmol/ L.Cells were cultivated for another 3 ～ 4 h
and then were harvested by centrifugation at 8 000 r/min
for 10 min at 4 ℃.The supernatant was saved , and the
pellet was resuspended in 10 ml buffer A (20 mmol/L
Tris-HCl pH 8.0).Cells were disrupted by sonication
on ice.The inclusion bodies were obtained after cen-
trifugation at 12 000 r/min for 15 min.The pellet , con-
taining the inclusion bodies , was resuspended in 10 ml
cold buffer B (20 mmol/ L Tris-HCl , 0.5 mol/L NaCl ,
2 %(v/ v)Triton X-100 pH 8.0)to wash the inclusion
bodies and centrifuged at 12 000 r/min for 15 min.The
pellet was resuspended in 5 ml of buffer C (8 mol/L
urea , 20 mmol/ L Tris-HCl , 0.
5 mol/ L NaCl , 5 mmol/L
imidazole , 2-mercaptoethanol pH 8.
0).The solution was
stirred for 4 h at room temperature.After centrifugation
at 12 000 r/min for 15 min at 4 ℃ the supernatant was
collected for renaturation and purification.Samples of
each step were saved for SDS-PAGE analysis.
2.4 Refolding
1ml solution of inclusion bodies was added into 9
ml refolding buffer (
0.1 mol/ L Tris-HCl , 0.
8 mol/L L-
Arg , 1 mmol/ L GSH(glutathione , reduced form , 1 mmol/
L EDTA , 0. 1 mmol/ L GSSG (glutathione , oxidized
form), pH 8.0).Refolding buffer was stirred slowly
when the solution of inclusion bodies was being added.
The refolding solution of the fusion protein was stored at
4 ℃ for 24 h and then examined by activity assay.
2.
5 Purification
TM
The HisTrap chelating 1 ml column was prepared
as the manual described.After equilibrating the column
with 5 ml buffer C (20 mmol/L Tris-HCl , 0.
5 mol/L
NaCl , 8 mol/ L urea , 5 mmol/ L imidazole , 2-mercap-
0), 2 ml sample was loaded on the col-
toethanol pH 8.
umn.The column was washed with 5 ml buffer C and
then eluted with 6 ml buffer D (20 mmol/ L Tris-HCl ,
0. 5 mol/ L NaCl , 300 mmol/L imidazole , 2-mercap-
.All fractions were collected for SDS-
toethanol pH 8.0)
PAGE and activity assay.
2.
6 Assay of Fusion Protein Activity
Fibrin plates were prepared as the following proce-
dure :
10 ml 1 % agarose , 10 ml 4 mg/ml human fibrino-
gen (FIBRORAAS ®)and 1ml 10 U/ ml thrombin were
 376 中 国 药 科 大 学 学 报 J China Pharm Univ 35 卷

mixed at 37 ℃.The recombinant staphylokinase was about 30 % of bacterial total proteins after induced 3 ～ 4
used as a positive control.Samples mentioned above h (Fig 3).Thus cells were harvested 3 ～ 4 h after induc-
were spotted on the fibrin plate (8 μ
l each hole)and in- tion.
cubated at 37 ℃ overnight.

3 Results

3.
1 Construction of pET-rPA Expression Vector
After 25 cycles PCR amplification , a 1 110-bp
product was obtained and demonstrated by 1 % agarose
gel elctrophoresis ( )and was inserted into pET32
Fig 1.
by Kpn Ⅰ/ Hind Ⅲ cleavage sites on 5′and 3′ends in
the correct reading frame generating pET-rPA expression
Fig 2.Restriction analysis of plasmid pET-rPA on agarose gel (1 %)
vector. Lane 1 :
λHin dⅢ/ EcoR Ⅰ 3 Marker (from MBI);
Lane 2 :
pET-rPA digested
with Kpn Ⅰ/ Hind Ⅲ ; pET-rPA digested with Pst Ⅰ ;
Lane 3: Lane 4 :
pET-
rPA digested with Xba Ⅰ/ Xho Ⅰ ;
Lane 5 :
pET-rPA plasmid uncut ;
Lane 6 :
pET32a plasmid

Fig 1.Confirmation of PCR product by agarose gel (1 %)elct rophoregram

Lane 1 :PCR amplificat ion product ;Lane 2 :
λHin d Ⅲ/ Eco R Ⅰ 3 M arker
(f rom M BI). Fig 3.SDS-PAGE (12 %)analysis of fusion protein expression induced by
5.0 mmol/ L lactose
In this vector , the reteplase gene was placed at the tot al proteins of BL21(DE3)without any plasmid ;
Lane 1: Lane 2 :
molecular
downstream of a TrxTag and a (His)6 Tag.The TrxTag mass marker ;Lane 3 :total proteins of BL21(DE3)transformed by pET-

expresses thioredoxin to catalyze correct formation of rPA , before induction ;

Lane 4 :
1 h after induction ;
Lane 5 :
2 h after induc-
tion ;
Lane 6 :
3 h af ter induction ;
Lane 7 :
4 h after induction ;
Lane 8 :
5 h af-
disulfide bonds and the (
His)6Tag makes the fusion pro-
ter induction
tein easy to be purified and detected via western blot-
ting.Thus the molecular mass of the fusion protein Different concentrations (1 ～ 10 mmol/ L)of lac-
thioredoxin-( 6-rPA is about 56 kD.
His) tose were tested and 5 mmol/ L was chosen.This con-
The correct insertion of reteplase gene was verified centration of lactose could induce the expression of the
by restriction analysis with Kpn Ⅰ/ Hind Ⅲ , Pst Ⅰ , Xba fusion protein effectively but was not the highest (data
Ⅰ/ Xho Ⅰ (Fig 2.), and PCR analysis.The plasmid not shown) .
pET-rPA was sequenced by Shanghai Sangon and deter- 3.
3 Refolding and Purification of the Fusion Protein
mined to be correct. Bioactivity of the fusion protein was detected after
3.
2 Expression of the Fusion Protein refolding and purification.Eluted protein was collected
The fusion protein expression was induced by lac- by fractions (3 ml/ fraction).After being purified by
tose.After adding lactose , 1ml culture was collected per HisTrapTM chelating column , the purity of the fusion pro-
hour for SDS-PAGE analysis.The results showed that tein could reach more than 80 %(Fig 4) .Active fusion
the amount of the fusion protein reached a peak and was protein was obtained after in vitro refolding (Fig 5).
 No .
4 ZHANG Xin-Yuan , et al:
A thioredoxin-(His)6 fusion protein of reteplase :
The work described above was only a preliminary experi-
ment to prove the activity of the refolded and purified fu-
sion protein.More detailed work to quantify the activity
of the fusion protein will be done in the near future.
Fig 4.SDS-PAGE (12 %) analysis of purification results by HisTrapTM
chelating column
Lane 1:molecular mass marker ;
Lane 2:refolding solution before purifica-
tion ;
Lane 3 :
f raction1 of the elution ;
Lane 4 :
f raction 2 of the elution
Fig 5.Fibrin plate analysis of reteplase activity
A.positive cont rol (staphylokinase 3 mg/ ml);B.positive cont rol (staphy-
lokinase 0.03 mg/ml);
C.positive control (staphylokinase 0.003 mg/ ml);
D.refolding solution of pET-rPA before purification ;E.fraction1of elution
(0.27 mg/ml);
F.fraction 2 of elution (0.74 mg/ ml);
G.refolding buffer
as negative control ;H.refolding solution of BL21 ;I.refolding solution of
pET32a
4 Discussion
Reteplase was expressed as non-fusion protein in
BL21(DE3)using expression vector pET28a in our lab-
oratory before.Two main problems were that expression
level was low (less than 20 %)and purification was dis-
commodious and no purified single component reteplase
protein was got by using gel chromatography.In this
study we inserted reteplase gene into the downstream of
the thioredoxin gene and a (His)6 Tag of the pET32a
generating the expression vector , pET-rPA , to express the
expression, purification, and activity analysis 377
fusion protein thioredoxin-(His)6-rPA , and the results
demonstrated that high expression level of the fusion pro-
tein was achieved.Latest data showed that the expres-
sion amount of the fusion protein was more than 50 %
and in some conditions was more than 80 %.In order to
get purified reteplase L-lysine-Sepharose affinity chro-
matography was normally used[ 14] .However L-lysine-
2+
Sepharose was not economical.In this study Ni -NTA
column was used and it was more economical than L-ly-
sine-Sepharose.
Many proteins that are normally produced in an in-
soluble form in E.coli tend to become more soluble
when fused with the N-terminal thioredoxin (Trx ·Tag)
sequence[ 7] .Members of the thioredoxin superfamily
share two features in common :
they contain a short se-
quence motif that includes a Cys-X1-X2-Cys sequence
(the active site)and an overall structure containing this
motif that corresponds to what is called a thioredoxin-like
fold[ 10] .The actual role of each of these proteins in the
cell is partly determined by the redox potential of the
protein and partly by the direction of the electron trans-
[ 10, 11]
port pathway it participates in .In this study sever-
al induction conditions were tested (e.g.induction time
and temperature , kinds of inducer , different concentra-
tions of lactose), and the results showed that reteplase
fusion protein existed mainly as insoluble form (data not
shown).More detailed work will be done to determine
whether thioredoxin would help refolding of the fusion
protein in vitro.Protein disulfide isomerase (PDI)is a
resident foldase of the endoplasmic reticulum , which con-
sists of five discrete domains[ 12, 13] .It catalyzes the in
vitro oxidative refolding of reduced , disulfide-containing
[ 13]
proteins .We shall use PDI in further study to in-
crease the efficiency of refolding.
In this study we used lactose instead of IPTG (iso-
propyl-β-D-thiogalactopyranoside)as the inducer.The
expression level of the fusion protein induced by lactose
was as high as by IPTG while lactose was much more e-
conomical.
In order to increase the expression amount of the
fusion protein , several measures will be taken such as
changing the component of medium and the codons of
 378 中 国 药 科 大 学 学 报 J China Pharm Univ 35 卷

612 .
reteplase to E.coli bias.Reteplase gene manipulated in
[ 6] Carrió MM , Villaverde A .
Const ruction and deconst ruction of bacterial
this study was derived from human , the arginine codons
inclusion bodies[ J] .
J Biotechnol , 2002 , 96(1):
3-12 .
AGA and AGG were rare in E.coli while the gene for [ 7] pET System Manual.The 10th edition .
July 2002.
reteplase contained seven of these rare arginine [ 8] Liao J , Zhang J , Shen Z .
Cloning and expression of tissue type plas-

codons[ 3] . minogen activator mutant reteplase(rPA)in E .coli [ J] .Pharma-

ceut ical Biotechnology , 2002 , 9(2):
95-98 .
The next step is to cleavage the fusion protein.We [ 9] Sambrook J , Russell DW .
In :
Molecular Cloning:
A Laboratory Manu-
conceive to perform this work on column and this work is al , 3rd Ed .
Cold Spring Harbor Laboratory Press .
[ 10] slund F , Beckwith J .The thioredoxin superfami ly :redundancy ,
now ongoing.
specificity , and gray-area genomics[ J] .J Bacteriol , 1999 , 181(5):
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[ 1] Noble S , M cTavish D .Reteplase.A review of its pharmacological bA deficiency with secreted thioredoxin variants reveals the crucial
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589-605 . bacterial periplasm[ J] .EMBO J , 1999, 18 :
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[ 2] Bennett WF, Paoni NF , Keyt BA , et al .
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[ J] .
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硫氧还蛋白-(His)6 融合的瑞替普酶的表达 、纯化和活性检测

张新元 , 廖建民 , 孙石静 , 沈子龙

(中国药科大学生物制药学院 , 南京 210009)

【摘 要】 目的 :
构建硫氧还蛋白-(His)6-瑞替普酶融合表达载体 , 提高 瑞替普酶在 大肠杆 菌中的 表达量 , 简化 rPA 的
分离纯化 。 方法 :
将 rPA 基因克隆 于原核表达载体 pET32a 硫氧还 蛋白-(组 氨酸)6 标签下游 , 在大肠 杆菌 BL21(DE3)中用乳
2+
糖进行诱导表达 。 采用一步稀释法对融合蛋白体外复性后 , 使用 Ni 亲和层析柱 , 对包涵体复性液进 行初步纯 化 。 采用体
外溶圈法对复性后和纯化后的融合蛋白进行生物活性的测定 。 结果 :
得到分子量约为 56 kDa 的融合蛋白 , 表达量 达到总蛋
白的 30%以上 。 比本实验室构建的非 融合表 达载 体表 达量提 高了 约 50%。 经过 一步 Ni2 +亲和 层析 , 融 合蛋 白纯度 达到
80 %以上 。 体外溶圈法实验表明融合蛋白复性后和纯化后具有溶栓活性 。 结 论 :
与非 融合表达 相比 , 融 合表达的 表达量明
显提高 。 即使 N 端额外融合一段融合多肽 , rPA 仍然具有生物学活性 。
【关键词】 硫氧还蛋白 ;
(组氨酸)6标签 ;
瑞替普酶 ;
融合蛋白 ;
表达 ;
纯化 ;
活性

【基金项目】 江苏省科技厅基金资助项目(No .
BG2002318)