全 文 ：ISSN 0582-9879
生 物 化 学 与 生 物 物 理 学 报
ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003 , 35(1):13-17 CN 31-1300/Q
Received:July 22 , 2002　　Accepted:September 6 , 2002
This work was supported by grants f rom the National Nature S cience
Foundation of China (No.39770875 and 39425005) and the Foundation of
Shanghai Science and Technology Commission(No.97XD14022)
＊ Corresponding author: Tel , 86-21-64747035; Fax , 86-21-
64338357;E-mai l , boliang@server.shcnc.ac.cn
Expression , Purification and in vitro N-myristoylation of
Human Src N-terminal Region
MA Han-Hui , YANG Li , LI Bo-Liang＊
(State Key Laboratory of Molecular Biology , Institute of Biochemistry and Cell Biology ,
Shanghai Institutes for Biological Sciences , the Chinese Academy of Sciences , Shanghai 200031, China )
Abstract　　The DNA fragment encoding N-terminal region of human c-Src was amplified from Caco-2 cell total
RNA by RT-PCR and cloned into vector pMFHT to obtain His-tag fusion expression plasmid pMF-SrcHT , which
was based on T7 expression system.The fusion protein SrcHT was highly expressed in E .coli BL21(DE3)
harboring the pMF-SrcHT and purified from bacterial lysate by Ni-IDA affinity chromatography.The assays using
[ 3H]-labeled substrate demonstrate that the purified fusion protein SrcHT can be effectively N-myristoylated by
recombinant human myristoyl-CoA :protein N-myristoyltransferase (NMT) in vitro.This work is a basis for
further biochemical studies and development of new anti-cancer chemotherapeutic drugs based on specific
inhibition of N-myristoylation of human Src.
Key words　　Src;His-tag;fusion expression;N-myristoylation
　　V-Src , a transforming product of Rous sarcoma
virus , is a tyrosin kinase.Its cellular homologue(c-Src)
has a widespread cellular distribution.The family of Src-
related protein tyrosine kinases includes nine members.
There are common multiple regulatory domains for the Src
family proteins to respond to a number of receptor-
mediated signals with changes of both kinase activity and
intracellular localization[ 1] .It has been shown that the
membrane association of Src protein requires addition of
myristic acid to the N-terminal glycine via an amide
linkage[ 2] .
N-myristoylation is a biochemical modification of
proteins in which the myristic acid (C14:0) is co-
translationally linked to NH2-terminal glycine residues of
various cellular and viral proteins
[ 3] .The enzyme
responsible for transferring myristate onto the N-terminus
of the protein substrates is myristoyl-CoA :protein N-
myristoyltransferase[ 4] (NMT , EC 2.3.2.97).A large
number of cellular N-myristoylproteins with diverse
functions have been identified.With the essential role of
N-myristoylproteins in many physiological and pathological
events such as signal transduction , carcinogenesis and
viral replication and assembly , N-myristoylation of
proteins by NMT , therefore , has been recognized as
possible chemotherapeutic target for anti-viral , anti-fungal
and anti-neoplastic therapy[ 5] .
Replacement of the N-terminal glycine in Src with
either alanine or glutamic acid preventing myristoylation
and morphological transformation indicates that myristoyl
moiety is essential for transforming activity of Src protein
kinase[ 6] , which is activated in human colon carcinoma ,
compared with that in normal colon tissues or cultures of
normal colon mucosal cells[ 7] .Moreover , increased NMT
activity has also been observed in rat and human colonic
tumors[ 8] .Accordingly , N-myristoylation of Src has been
proposed as a target for developing chemotherapeutic drugs
against colon cancer.The N-terminal 16 residues of Src
have been synthesized for studying N-myristoylation and
its correlated functions[ 9] .In this paper , His6-tagged N-
terminal region of Src was expressed and purified from E.
coli as a substrate of NMT , which provides a basis for
biochemical studies and exploration of new anti-cancer
chemotherapeutic drugs based on specific inhibition of N-
myristoylation of Src.
1　Materials and Methods
1.1　Materials
1.1.1 　Plasmid and bacteria 　　Expression plasmid
pMFHT[ 10] containing His-tag coding sequence is
constructed in our lab.E.coli XL1-Blue and BL21
(DE3) are used as hosts for cloning and protein
expression.
1.1.2　Enzymes and reagents　　Restriction enzymes
and T4 DNA ligase were purchased from New England
Biolabs , Boehringer Mannheim or Gibco BRL Company.
Agarose was purchased from Gibco BRL Company.
Pseudomonas acyl CoA synthetase , LiCoA , acrylamide ,
bisacrylamide , IPTG (isopropylthio-β-D-galactoside),
PMSF (phenylmethylsulfonyl fluoride), imidazole and
iminoacetic acid (IDA)-Sepharose 6B were purchased
from Sigma Company.[ 9 , 10 (n)-3H] myristic acid ,
AmplifyTM and Hyperfilm were obtained from Amersham
Company.Trizol reagent was from Gibco BRL Company.
M-MLV Reverse Transcriptase was from Promega
Company.Taq DNA polymerase was from Sino-American
Biotechnology Company.
1.2　Methods
1.2.1　Cell culture　　Caco-2 cells are propagated in
DMEM(Gibco BRL)containing 20%fetal bovine serum ,
100 u/ml penicillin and 100 mg/L streptomycin at 37°C
and in 10%CO2.
1.2.2　RT-PCR 　 　 primer 1 (5′-GGACCATGG-
GTAGCAACAAG-3′)as the forward primer with a NcoI
site (underlined) and primer 2 (5′-AGGGAAT- TC
GCCTGGATGGAGTCG-3′)as the reverse primer with an
EcoRI site (underlined)were designed for amplifying
DNA fragment encoding N-terminal region of human Src
(147 amino acids), and synthesized in Institute of
Biochemistry and Cell Biology , Shanghai Institutes for
Biological Sciences , the Chinese Academy of Sciences.
The total RNA from Caco-2 cells was prepared according
to single step acid guanidinium thiocyanate phenol
chloroform method (Trizol Regent , Life Technologies ,
Inc).The first-strand cDNA synthesis was performed by
annealing 4 μg of total RNA from Caco-2 cells with 0.5
μg of oligo(dT)(12-18 in length)in a total volume of
20 μl and reverse transcribing with 200 u of M-MLV
Reverse Transcriptase at 37 °C for 60 min.After this
reaction , the DNA fragment encoding N-terminal region of
human Src was amplified from 1 μl aliquot of the mixture
in a PCR reaction containing 10 mmol/L Tris-HCl
(pH 8.3), 50 mmol/L KCl , 1.5 mmol/L MgCl2 , 0.2
mmol/L for each dNTP , 0.4 μmol/L of primer 1
(forward primer)and primer 2 (reverse primer)and 1 u
of Taq polymerase.The reaction mixture was denatured at
94 °C for 45 s , annealed at 50 °C for 45 s , and
polymerized at 72 °C for 45 s.Thirty cycles were
performed and followed by a 10-min extension at 72°C.
1.2.3　Construction of expression plasmid pMF-SrcHT　
　The amplified DNA fragments encoding N-terminal
region of human Srcwere inserted into upstream of His-tag
sequence on an expression vector of pMFHT under the
control of T7 promoter.The expression plasmid pMF-
SrcHT was obtained by analysis of restriction digestion
and DNA sequencing.All of the DNA manipulation or
identification including the digestion with restriction
enzymes , agarose gel electrophoresis , purification of DNA
fragments and ligation with T4 DNA ligase were performed
as described by Sambrook et al .[ 11] .
1.2.4　Expression of the fusion protein SrcHT 　　E.
coli BL21(DE3)harboring the fusion expression plasmid
pMF-SrcHT was grown to A600 = 0.4 -0.6 in LB(Luria-Bertani medium)containing 100 mg/L ampicillin
at 37°C , and then induced to produce the fusion SrcHT
by adding IPTG to a final concentration of 0.5 mmol/L
and the incubation was extended for additional 3 h.The
cells were harvested centrifugation for 10 min at 5000 g
and samples were analyzed by 15%SDS-PAGE according
to the methods of Laemmli[ 12] .
1.2.5　Purification of the fusion protein SrcHT　　The
recombinant SrcHT was purified using one-step Ni-IDA
affinity chromatography[ 13] .Briefly , the induced E.coli
BL21(DE3)cells harboring the expression plamid pMF-
SrcHT were harvested by centrifugation for 10 min at 5000
g , resuspended in buffer A (20 mmol/L Tris-HCl
pH 7.9 , 0.5 mol/L NaCl , 10% glycerol , 1 mmol/L
PMSF , 40mmol/L imidazole)and sonicated on ice for 30
min for 30 times (with 1 min interval per time).The
supernatant fraction was obtained from the sonicated
bacterial lysate by centrifugation for 30 min at 10 000 g
and applied to Ni-IDA agarose column at a constant flow-
rate of 4 ml per min.Then , the non-specific bound
proteins were removed with buffer A (20 mmol/L Tris-
HCl pH 7.9 , 0.5mol/L NaCl , 10% glycerol , 1 mmol/
L PMSF , 40 mmol/L imidazole) and the recombinant
SrcHT was eluted with buffer B(20mmol/L Tris-HCl pH
7.9 , 0.5mol/L NaCl , 10%glycerol , 1 mmol/L PMSF ,
200 mmol/L imidazole).The eluted proteins were
identified by 15% SDS-PAGE analysis described as
above.
1.2.6 　 In vitro N-myristoylation assay 　　Firstly ,
[ 3H] -labeled myristoyl-CoA was synthesized as described
by Towler et al.[ 14] and the reaction mixture containing
20 mmol/L Tris-HCl (pH 7.4), 1 mmol/L
dithiothreitol , 10 mmol/L MgCl2 , 0.1mmol/L EGTA , 5
mmol/L ATP , 10 mmol/L LiCoA , 1μmol/L [ 9 , 10-3H]
myristic acid (5.2 μCi), and 0.3 u/ml pseudomonas
acyl CoA synthetase was allowed to incubate at 30°C for
30 min.Then , the NMT assay was carried out in a final
20μl volume of NMT buffer containing 30 mmol Tris-HCl
(pH 7.4), 0.5 mmol/L EDTA , 0.45 mmol/L 2-
mercaptoethanol , 10 μmol/L peptide , 1%Triton X-100 ,
and the purified recombinant human NMT.The NMT
reaction was performed by adding 10μl synthesized [ 3H] -
myristoyl-CoA in each reaction firstly , then with
14 　　　ACTA BIOCHIMICA et BIOPHYSICA SINICA Vol.35 , No.1　　　　　
Fig.1　Construction of expression plasmid for the fusion protein SrcHT
(A)Diagram of human Src protein and its N-terminal region (147 amino acids , arrowed oppositely)containing the Gly residue at N-end.(B)Separation of
amplified DNA fragment encoding N-terminal region of human Src.DNA fragment encoding N-terminal region of human Src(lane 2)was amplif ied from Caco-2
cell total RNA by RT-PCR and separated by 1.5% agarose gel electrophoresis with the size control of molecular weight markers(lane 1).(C)Construct for
expression of the fusion protein SrcHT.DNA fragment encoding N-terminal region of human Srcwas inserted into upstream of His-tag sequence in expression vector
cont rolled under T7 promoter and terminator.
incubation at 30 °C and extension for 30 min and was
stopped by boiling for 5 min.Finally , the boiled NMT
reaction mixture was subjected to 15% SDS-PAGE and
the [ 3H] -myristoyl-peptides were analyzed by
autoradiography.
2　Results and Discussion
2.1　Construct for expression of the fusion protein
SrcHT
N-terminal region (147 amino acids)of human Src
containing N-end of Gly residue [ Fig.1(A)] was
designed to use as substrate of NMT.The relative DNA
fragment was amplified from Caco-2 cell total RNA by RT-
PCR with a set primers specific for human Src gene
(GenBank accession No.AH002989).The 458 bp DNA
fragment thus obtained was separated by 1.5% agarose
gel electrophoresis [ Fig.1(B)] .DNA fragment encoding
N-terminal region of human Src was fused to upstream of
His-tag sequence on expression vector pMFHT to yield
expression plasmid pMF-SrcHT [ Fig.1(C)] .The
construct is used to express a 182-amino acid fusion
protein SrcHT which consists of N-terminal region of
human Src(2-148 amino acids)and an extension of 34-
amino-acid C-terminus ( 149 -
1 8 2 ) containing His-tag (152-157 amino acids)
Fig.2　Sequences of the DNA fragment inserted into expression
vector and the amino acids of human Src N-terminal region
The DNA fragment encoding human Src N-t erminal region(2-148 amino
acids)was inserted into the expression vector by NcoI and EcoRI site
(underlined).T7 promoter sequence(bolded and underlined), transcription
initiation(TI , arrowed)and Shine Dalgarno sequence (S.D., underlined)
are the vector sequences presented at upstream of coding sequence for fusion
protein SrcHT.The amino acids of human Src N-terminal region(2-148
amino acids) are bolded.The 34-amino-acid (149 - 182)C-terminal
extension containing His-tag(152-157 amino acids , underlined)i s encoded
by vector sequence.
encoded by vector sequence (Fig.2).
2.2 　Expression and purification of the fusion
protein SrcHT
　 　 After the expression plasmid pMF-SrcHT was
15Jan., 2003 MA Han-Hui et al.:Expression , Purification and in vitro N-myristoylation of Human Src N-terminal Region
Fig.3 　SDS-PAGE analysis of expression and purification of
fusion protein SrcHT
The bacterial lysates were prepared from un-induced (lane 1)and induced
(lane 2)E .coli BL21(DE3) cells harboring pMF-SrcHT by sonication.
Then , the supernatant(lane 3)and pellet (lane 4)were obtained from the
above induced bacterial lysate by centrifugation.Finally , fusion protein
SrcHT (lane 5) is purif ied from the supernatant by Ni-IDA affini ty
chromatography.All of the above samples were directly subjected to 15%
SDS-PAGE with the size control of molecular weight marker(lane M).
Fig.4　In vitro N-myristoylation assay
(A) Autoradiography of [ 3H] -labeled-myristoyl-peptides. In vitro N-
myristoylation assay of the purified SrcHT was carried out by using mCα4H as
positive control.[ 3H] -myristoyl peptides were separated by 15% SDS-PAGE
and then exposed by autoradiography , which indicated the negative control
without NMT substrate (lane 1), expressed SrcHT (lane 2)and positive
cont rol with mCα4H (lane 3).(B)N-terminal amino acids of SrcHT and
mCα4H as NMT substrates.
transformed into E.coli BL21(DE3), SrcHT was highly
expressed after induced by IPTG (Fig.3 , lane 1 and 2).
In order to determine whether the fusion protein is soluble
or not , the cell pellet was resuspended by sonication on
ice with sonication buffer.It is found that SrcHT was
mainly in soluble form(Fig.3 , lane 3 and 4).The lysate
supernatant was applied to Ni-IDA agrose column for
affinity chromatography , and SrcHT was purified in one
step.Thirty four milligram of the fusion protein SrcHT has
been successfully purified from 1 liter of induced bacteria
by one-step affinity chromatography.SDS-PAGE results
indicate that purity of the purified SrcHT is more than
95%(Fig.3 , lane 5).The expressed and purified SrcHT
is a good preparation for its N-myristoylation.
2.3　In vitro N-myristoylation of the fusion protein
SrcHT
In vitro N-myristoylation assay of the purified SrcHT
was performed with αsubunit of mouse cAMP-dependent
protein kinase (mCα4H)[ 15] as positive control.The
results illustrate that the purified SrcHT [ Fig.4(A), lane
2] can be efficiently N-myristoylated by NMT as same as
the control of protein substrate mCα4H [ Fig.4(A)lane
3] , suggesting that they contains the N-end-glycine [ Fig.
4(B)] which is essential for N-myristoylation of
proteins[ 16] and C-terminal fusion His-tag sequence do not
affect its N-myristoylation.Since myristoylation of Src has
been proposed as a potential target for developing
chemotherapeutic drugs against colon cancer
[ 7 ,8] , the fact
that the fusion protein SrcHT has been successfully
expressed and purified as an effective substrate of N-
mystoyltransferase will provide a good basis for studying
the biochemical function and specific inhibitors for N-
myristoylation of human Src.
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人 Src蛋白 N端区段的表达 、纯化和体外豆蔻酰化底物活性
马涵慧　杨　力　李伯良＊
(中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子生物学国家重点实验室 , 上海 200031)
摘要　　利用 RT-PCR技术 , 从来源于人结肠癌 Caco-2 细胞总 RNA中 , 扩增得到编码人Src蛋白N端 147 氨基酸的 DNA 序列
片段。进而构建 T7启动子控制下的 C 端 His-tag融合的表达质粒 pMF-SrcHT , 并转化大肠杆菌 BL21(DE3)。通过 SDS-PAGE 等
分析结果显示 , 在 37°C培养条件下经 IPTG 诱导 , C 端 His-tag融合的人Src蛋白 N端区段(命名为 SrcHT , 21 kD)得到高效表
达 , 并且主要以可溶性形式存在。进一步利用 Ni-IDA亲和层析分离 , 从表达菌裂解上清液中一步纯化获得重组蛋白 SrcHT ,
SDS-PAGE分析纯度达 95%以上。在此基础上 , 以[ 3H]豆蔻酰-CoA为同位素标记底物进行 SrcHT 的体外NMT豆蔻酰化反应测
定。SDS-PAGE 分离和放射自显影分析结果表明 , SrcHT蛋白可被 NMT有效豆蔻酰化而具有 NMT的底物活性。这些为深入详
细研究 Src蛋白豆蔻酰化作用和构建以 Src蛋白豆蔻酰化为靶标的分子筛药体系等打下了重要基础。
关键词　　Src蛋白;His-tag;融合表达;豆蔻酰化
收稿日期:2002-07-22　　接受日期:2002-09-06
国家自然科学基金(No.39770875)及上海市科委(No.97XD14022)资助项目
＊联系人:Tel , 021-64747035;Fax , 021-64338357;e-mail , boliang@server.shcnc.ac.cn
17Jan., 2003 MA Han-Hui et al.:Expression , Purification and in vitro N-myristoylation of Human Src N-terminal Region