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http://www.abbs.info e-mail:[email protected] ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(4): 405-410 CN 31-1300/Q |
Cloning, Expression of the Abrin-a A-chain in Escherichia
coli and Measurement of the Biological Activities in vitro
(
College of Animal Sciences, Huazhong Agricultural University, Wuhan
430070, China;
1Institute
of Virology, College of Life Sciences, Wuhan University, Wuhan 430072,
China )
There
has been considerable interest in RIPs (e.g. abrin, ricin and similar plant toxins) in
recent years due to their potential use in the development of therapeutic
agents, such as toxin-antibody
conjugates targeted against tumor cells or parasites. In addition, RIPs have long been known to exhibit
antiviral activity. The currently best characterized example is the type-I RIP--trichosanthin, which has been shown to have a potent
inhibitory activity against HIV-1-infected T-cells and macrophages[4].
As for clinical application,
immunotoxins consisting of native whole abrin or abrin A-chain linked to
specific targeting ligands or monoclonal antibodies directed specific cells
have been generated and exhibited selective anti-tumor effects in experimental
animal tumor models[5]. However, there are many inherent problems in the use of native toxins
for therapy such as the limitation of the plant resources, the complication of purification
procedure and so on. Therefore,
the possibility of generating whole toxin or toxin A-chain by
recombinant techniques may for several reasons be of great advantage in such
applications.
In
the present work, we reported on
the molecular cloning of ABRaA coding sequence, as well as the expression of
recombinant protein in Escherichia coli and demonstrated that the
purified recombinant protein inhibited protein biosynthesis by modifying rat
liver 28 S rRNA. The production of large quantities of active homogeneous
proteins is desirable in the study and the development of immunotoxins as
therapeutic agents made with such recombinant proteins.
1.1
Materials
l-[3H]-leucine
was purchased from Amersham International. The rabbit reticulocyte lysate
cell-free system for protein biosynthesis, Taq DNA polymerase, pGEM-T easy vector,
RNasin inhibitor, the
molecular mass markers, the
restriction endonucleases and T4 DNA ligase were obtained from Promega. The
expression vector pET28b, E.coli
host strain BL21, and pET system
components came from Invitrogen. The deoxyribonucleotide primers were
synthesized in an Applied Biosystem automated DNA synthesizer by Sangon Inc., Shanghai. The Trizol reagent for total
RNA isolation and the MESSENGER MAKERTM mRNA
isolation system were purchased from the Life Technologies, Gibco BRL. The TALON nickel affinity
resin column for protein purification is the product of Clontech. The native ABRaA
was purchased from Sigma. All other chemicals were of analytical grade.
1.2
Molecular cloning of the ABRaA coding sequence by RT-PCR
Maturing
Abrus precatorius, about 1
month after flowering, were
obtained from a local source. Total RNA was isolated from the endosperm of the
seeds by using Trizol reagent according to the manufacturer's directions. The
poly(A)+ RNA was purified with the MASSENGER MAKERTM
mRNA isolation system which uses oligo(dT) cellulose chromatography. The
poly(A)+ RNA was used for cDNA synthesis of ABRaA coding
region. Briefly, 2 mg
of poly(A)+ RNA samples were reverse transcribed at 42 ℃
for 1 h in a 20 mL
reaction mixture containing 2 mmol/L
oligo(dT)16 primer, 1 mmol/L dNTPs
mixture, 1 mmol/L
dithiothreitol, 20 u RNasin
ribonuclease inhibitor, 100 u
moloney murine leukaemia virus (MMLV) reverse transcriptase, and 4 mL
of 5×cDNA synthesis buffer. After the heat
inactivation (95 ℃, 5 min) of the reverse
transcriptase, the synthesized
single-strand cDNA products (2 mL)
were then subjected to PCR with the following primers to obtain the coding
region of ABRaA: sense primer, 5′-GGGATCC-
GAAGATAGGCCCATCAAGTTT-3′,
which encodes the first seven N-terminal amino acid residues of ABRaA
behind a BamHI restriction site; antisense primer, 5′-AGAATTCTTAATTTGGCGGATTGC-AGAC-3′,
which encodes the last seven C-terminal amino acid residues of ABRaA
with a stop codon following the KpnI restriction site. The expected
RT-PCR product was 750 bp containing the complete open reading frame of ABRaA
gene. The PCR reaction mixture (25 mL)
contained 2 mL
of the first-strand cDNA, 2.5 mL
10×PCR buffer, 200 mmol/L
of each dNTP, 1.5 mmol/L MgCl2, 2 u Taq DNA polymerase and 0.5 mmol/L
of each primer, respectively.
Amplification was performed in a Perkin Elmer thermal cycler under the
following conditions. After a hot-start step (94 ℃, 5 min), samples were subjected to 35 cycles of denaturation at 94 ℃
for 1 min, annealing at 50 ℃
for 1 min, extension at 72 ℃
for 1.5 min, and a final cycle of
elongation for 8 min at 72 ℃.
Control reaction without cDNA was carried out in parallel to ensure that
reagents were not contaminated and were consistently negative. The PCR product
was analyzed by electrophoresis on 1.5% agarose gels with DNA visualized by
ethidium bromide staining,
subsequently purified on low-melting agarose gels and subcloned into the
pGEM-T easy vector to form pGEM-ABRaA for amplification and nucleotide
sequencing analysis.
1.3
Construction of the expression plasmid
The
coding region of ABRaA was expressed with the 6×His
fusion vector, pET28b. The
expression plasmid was constructed by ligating the 750 bp BamHI-KpnI
fragment, which contained the entire
ABRaA coding sequence derived from RT-PCR as described above, in frame into the pET28b expression
vector linearized by the same enzymes. The resulting construct, pET-ABRaA, was confirmed by sequencing and the
relative restriction enzymes digestion.
1.4
Expression of ABRaA in Escherichia coli
The
E.coli host strain BL21 cells were transformed with the expression
plasmid pET-ABRaA by CaCl2-mediated transformation. The
transformants were grown at 37 ℃
in LB medium (100 g/L trypeptide,
50 g/L yeast extract, 100
g/L NaCl) supplemented with kanamycin (100 mg/L) to an optical absorbance of
0.4-0.6
at 600 nm. IPTG was then added to a final concentration of 1 mmol/L and the
cells were further incubated for 2-5
h at 37 ℃.
Cells were harvested by centrifugation at 10 000 g for 10 min and resuspended
in buffer A (140 mmol/L NaCl, 2.7
mmol/L KCl, 10 mmol/L Na2HPO4, 1.8 mmol/L KH2PO4, pH 7.3) containing 1 mmol/L EDTA, 5 mmol/L dithiothreitol, 500 mmol/L
PMSF, and 100 mg/L lysozyme. After
an incubation for 30 min at 4 ℃, the resuspended cells were disrupted by
sonication (five times of 30 s pulses separated by 30 s periods of cooling).
The solution added with 1% Triton X-100 was gently stirred for 1 h at 4 ℃.
Proteins samples were analyzed by 12% SDS-PAGE under denaturing conditions
according to reference [6]. The gel was stained with Coomassie brilliant blue
to detect the protein bands.
1.5
Purification of the recombinant ABRaA
Aliquotes
of 5 mL overnight culture of transformants containing ABRaA expression
plasmids were added into aliquots of 1 L LB medium and cultured to an
absorbance of 0.6 at 600 nm in 37 ℃.
After induction by IPTG, the
bacteria cells were harvested by centrifugation and the cell pellet was
resuspended in 10 volumes of buffer A. The resuspended cells were lysed in
buffer A by sonication for 3-4
min. After centrifugation at 10 000 g for 15 min, the supernatant was loaded onto a 2 mL
TALON nickel affinity resin column. The column was washed with 20 mL buffer A
followed by 20 mL buffer A containing 1 mol/L NaCl. After reequilibration the
column with 20 mL buffer A, the
bound proteins were eluted with buffer A containing 250 mmol/L imidazole. The
eluted proteins were diluted 10-fold in buffer A and loaded onto a Mono Q
column equilibrated with buffer A containing 60 mmol/L NaCl. The column was
eluted with a 50 mL linear gradient from 60 mmol/L to 250 mmol/L NaCl, both in buffer A. The recombinant ABRaA
was thus yielded.
1.6
Measurement of the biological activities of recombinant ABRaA in
vitro
The
biological activities of recombinant ABRaA were studied by measuring its
protein biosynthesis inhibition and RNA N-glycosidase activities in
vitro. The inhibition effect of the purified recombinant protein on
translation in vitro was determined by measuring the incorporation of l-[3H]-leucine
into protein in a rabbit reticulocyte lysate cell-free system. Briefly, various amounts of recombinant or
native (as positive control) ABRaA were mixed with 11.5 mL
of rabbit reticulocyte lysate in 20 mmol/L Tris-HCl (pH 7.8) containing 4 mCi/L
l-[3H]-leucine,
1.5 mmol/L MgCl2,
5 mmol/L dithiothreitol,
and 50 mmol/L KCl, followed
by incubation at 30 ℃
for 90 min. The reactions were precipitated with 25% trichloroacetic acid and
collected on glass microfiber filters by filtration with Whatman GF/C, and the radioactivities of the filters
were determined with a liquid scintillation counter. Each reported inhibition
point is calculated as the mean of triplicate individual tests.
The
N-glycosidase activities of recombinant or native (as positive control)
ABRaA were determined by treating
rat liver ribosomes with certain protein on a reaction buffer (113 mmol/L
KCl, 10 mmol/L MgCl2, 0.05% b-mercaptoethanol, 2 u RNasin). After adding the toxic
protein, the reaction mixture was
incubated at 37 ℃
for 15 min, then the reaction was
terminated by adding 5 g/L SDS. The reaction products were extracted with
phenol, precipitated with alcohol, then treated with 0.8 mol/L
aniline, pH 4.5, to selectively cleave the 28 S rRNA at
the depurinated site by b-elimination.
The reaction products were analyzed by using 7 mol/L urea-35 g/L PAGE and the
gel was stained with ethidium bromide. For each experiment, each concentration of the ABRaA was
measured in triplicate.
2.1
Cloning of the coding sequence of ABRaA cDNA
From
the seeds of Abrus precatorius,
total RNA and poly(A)+ RNA were isolated, and the coding region of ABRaA
cDNA was synthesized by RT-PCR by the application of the gene specific
primers, which are based on the
genomic DNA sequence of intact abrin and cDNA sequences of abrin A-chains
reported. The amplified full-length open reading frame of ABRaA cDNA was
750 bp (as shown in Fig.1) and encoded an expected protein of 27.5 kD.
Fig.1 Identification of the amplified ABRaA
coding sequence and the expression plasmid pET-ABRaA
M,
lDNA/HindIII
molecular weight markers. 1,
negative control of RT-PCR; 2,
the amplified coding sequence of ABRaA cDNA (BamHI- KpnI
fragment) by RT-PCR; 3, pET28b
vector linearized by BamHI /KpnI enzymes; 4, the pET-ABRaA plasmid confirmed
by BamHI/KpnI digestion.
2.2
Expression and purification of recombinant ABRaA
The
E.coli host strain BL21 cells transformated with the expression plasmid
pET-ABRaA produced recombinant fusion protein of the expected molecular
mass (Mr), which
was majored as soluble pattern. The fusion protein was purified from the cell
lysate by a single-step affinity chromatography on a TALON nickel affinity
resin column. After elution, the
free recombinant ABRaA was obtained. The yield of the soluble recombinant
protein was about 4 mg/L of induced culture. The recombinant ABRaA was
homogeneous upon analysis by 12% SDS-PAGE, with an estimated Mr of approximately 28
kD (see Fig.2)
Fig.2 SDS-PAGE analysis of ABRaA
proteins
The samples of expressed and purified
recombinant ABRaA fusion proteins as well as the native ABRaA (as positive
control) were analyzed by 12% SDS-PAGE and Coomassie blue staining. M
represents protein molecular mass markers. Proteins extracted from BL21 cells
without plasmid transformation (lane 1),
transformed with empty plasmid pET28b (lane 2) or transformed with
expression plasmid pET-ABRaA but without IPTG induction (lane 3) were
served as negative control. Lanes 4-7, proteins extracted from BL21 cells
transformed with expression plasmid pET-ABRaA at different IPTG
induction time period (2, 3, 4, 5 h); lane 8,
purified recombinant ABRaA; lane 9, native ABRaA (as positive control).
2.3
Inhibition of protein biosynthesis by recombinant ABRaA in vitro
Recombinant
or native (as positive control) ABRaA was tested for their biological
activities by measuring their
abilities to inhibit the protein biosynthesis of rabbit reticulocyte cell-free
system. The recombinant ABRaA was the potent inhibitor of protein biosynthesis in certain system, and the ability of recombinant ABRaA to
inhibit protein biosynthesis is comparable with that of native ABRaA. As shown
in Fig.3, the IC50
values of recombinant and native ABRaA were determined as 0.08 and 0.06 nmol/L, respectively.
Fig.3 Inhibitory effects of recombinant or
native ABRaA on protein biosynthesis in a cell-free rabbit reticulocyte
lysate system
Recombinant or native ABRaA was tested
for their inhibitory effects on the protein biosynthesis of cell-free rabbit
reticulocyte lysate system in vitro. The incorporation of radioactivity
in l-[3H]-leucine into protein was measured. Each point
denotes the mean for triplicate assays.
2.4
N-glycosidase activity of recombinant ABRaA
The
observed inhibition of protein biosynthesis resulted from catalytic activity of
ABRaA on 28 S rRNA substrate. The RNA N-glycosidase activities of recombinant or native (as positive
control) ABRaA were examined by incubating rat liver ribosomes with various
amounts of certain proteins, and the extracted rRNAs were analysed by gel
electrophoresis. The recombinant and native ABRaA depurinated rat liver 28 S
rRNA at A4324, and after aniline-catalyzed hydrolysis of the phosphodiester
bonds on either side of the modified site, a new small fragment of approximately 420 ribonucleotides
was generated from the 28 S rRNA. As shown in Fig.4, recombinant and native ABRaA
at the concentration of 1 nmol/L, released the small RNA fragment of
approximately 420 nt by their N-glycosidase activities.
Fig.4 N-glycosidase activities of
recombinant and native ABRaA
Rat liver ribosomes were treated with
10-fold serial dilution of recombinant or native ABRaA for 15 min at 37 ℃.
The RNAs were extracted and treated with 0.8 mol/L aniline (+) for 10 min at 4 ℃.
The samples were analyzed by 7 mol/L urea- 35 g/L PAGE and staining with
ethidium bromide. Rat liver ribosomes samples without treatment (negative
control) (lanes 1 and 2), treated
with recombinant ABRaA (lanes 3-6)
or native ABRaA (lanes 7-10)
were shown. The arrow indicates the released small RNA fragment cleaved with
aniline from rRNAs of treated ribosomes.
3
Discussion
On
the basis of the genomic DNA sequence of intact abrin and cDNA sequences of
isoabrin A-chains reported, a pair
of primers specific for the coding sequence of ABRaA was designed, and the open reading frame of ABRaA
was cloned by RT-PCR. The nucleotide sequencing of PCR product confirmed the
correct amplification of ABRaA coding sequence (data not shown). The
coding region of ABRaA cDNA was expressed in E.coli as a 6×His
fusion protein. The expression of the recombinant protein produced an expected
band of approximately 28 kD,
clearly visable against the background of total protein on a stained gel
(as shown in Fig. 2). The expressed ABRaA protein purified on a TALON
nickel affinity resin column was homogenous with high yield (4 mg/L of induced
culture).
We
studied levels of ABRaA expression under different concentrations of IPTG for
induction and the various induction time period, respectively. Over a range of concentrations between 2
mmol/L and 100 nmol/L for induction,
optimal ABRaA expression was obtained at 1 mmol/L of IPTG. As for the
induction time, the optimal time
is for 5 h span after addition of 1 mmol/L IPTG (data not shown).
Previously, the Mirabilis
antiviral protein was shown to inhibit protein
synthesis in an E.coli in vitro translation system and was
detrimental to the host during expression[7]. In the present
work, the A-chain of abrin-a was
expressed at high level without any inhibition of growth of the E.coli
host as determined by A600 measurements compared to a nonrecombinant
vector control (data not shown),
which is consistent with the previous finding that abrin is only
specifically toxic to eukaryotic cells[8].
The measurement of the biological activities of
recombinant ABRaA demonstrated that the recombinant protein was biologically
active. The result of measuring the inhibitory activity of protein biosynthesis
showed that the IC50 of recombinant ABRaA (0.08 nmol/L) for the
inhibition of protein synthesis of rabbit reticulocyte lysate was similar to
that of native (0.06 nmol/L). The recombinant ABRaA was also found to inhibit
protein synthesis of the free-cell system in a dose-dependent manner. The
recombinant ABRaA at the concentration of 1 nmol/L cleaved the N-glycosidic
bond at the A4324 of rat liver 28 S rRNA,
then released an approximately 420 nucleotides RNA fragment after
treatment with aniline, as did
native ABRaA. Taken together,
these results suggested that the recombinant ABRaA was as biologically
active as native.
A major drawback in using type-II RIPs to
inhibit the growth of tumor cells is that they also bind to d-galactose
present on the surface of normal cells by the cell-binding B-chain.
Recently, there has been a growing
interest in using the A-chain of type-II RIPs or the toxophoric A-chain only
type-I RIPs as an alternative in immunotoxin preparations for the reason that
they offer many advantages in the treatment of several clinical diseases[9].
When these A-chains of type-Ⅱ
RIPs (e.g. abrin,
recin, etc.) were
conjugated to tumor-associated monoclonal antibodies, they exhibited several hundreds fold higher inhibitory
activities on the growth of tumor cells and with no cytotoxic to the normal
cells[5]. The selective toxicity of these immunotoxins encourages
further studies in view of a potential use in clinical application for the
therapy of human diseases. Therefore,
the generation of high quantities and high yield of ABRaA by recombinant
techniques will facilitate an exploration of this protein for therapeutic
purposes.
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Received：December
4, 2001Accepted：February
5, 2002
This
work was supported by the Natural Science Foundation of Hubei Province
(No.4006-016031 )
*Corresponding
author: Tel, 86-27-87282091; Fax, 86-27-87280408; e-mail,
[email protected]