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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 ℃ and in 10% CO2.
1.2.2 RT-PCR Primer 1(5′-GGACCATGGGTAGCAACAAG-3′)
as the forward primer with a NcoI site (underlined) and primer 2(5′-AGGGAATTCGCCTGGATGGAGTCG-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 ℃ 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 ℃ for 45 s,
annealed at 50 ℃ for 45 s,
and polymerized at 72 ℃ for 45 s. Thirty cycles were performed and followed by a 10-min
extension at 72 ℃.
1.2.3 Construction
of expression plasmid pMF-SrcHT The
amplified DNA fragments encoding N-terminal region of human Src were 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 ℃, 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, 40 mmol/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, 0.5 mol/L NaCl, 10% glycerol, 1 mmol/L PMSF, 40
mmol/L imidazole) and the recombinant SrcHT was eluted with buffer B (20 mmol/L
Tris-HCl pH 7.9, 0.5 mol/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, 0.1 mmol/L EGTA, 5
mmol/L ATP, 10 mmol/L LiCoA, 1 μmol/L [9,10-3H] myristic acid (5.2
μCi), and 0.3 u/mL pseudomonas acyl CoA synthetase was allowed to incubate at
30℃ for 30 min.
Then, the NMT assay was carried out in a final 20 μl 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 10 μl
synthesized [3H]-myristoyl-CoA in each reaction firstly, then with
incubation at 30 ℃ 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
(A) Diagram of human Src protein and its
Fig.1 Construction
of expression plasmid for the fusion protein SrcHT
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 amplified 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 Src was inserted into upstream of
His-tag sequence in expression vector controlled under T7 promoter and
terminator.
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 – 182) containing
His-tag (152 – 157 amino acids) encoded by vector sequence (Fig.2).
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-terminal 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) is encoded by vector sequence.
2.2 Expression
and purification of the fusion protein SrcHT
After the
expression plasmid pMF-SrcHT was 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.
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 purified from the supernatant by Ni-IDA affinity
chromatography. All of the above samples were directly subjected to 15%
SDS-PAGE with the size control of molecular weight marker (lane M).
2.3 In
vitro N-myristoylation of the fusion protein SrcHT
In vitro N-myristoylation assay of the purified SrcHT was performed with α
subunit 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.
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
control with mCα4H (lane 3). (B) N-terminal amino acids of SrcHT and mCα4H as
NMT substrates.
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Received: July 22, 2002
Accepted: September 6, 2002
This work was supported by grants from the National Nature Science 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-mail,
[email protected]