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Applied and Environmental Microbiology, August 2000, p. 3166-3173, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Simple and Efficient Method for Heterologous
Expression of Clostridial Proteins
Alexey G.
Zdanovsky* and
Marina V.
Zdanovskaia
Promega Corporation, Madison, Wisconsin
53711-5399
Received 11 April 2000/Accepted 15 May 2000
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ABSTRACT |
Many clostridial proteins are poorly produced in Escherichia
coli. It has been suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in E. coli and the quantities of
the corresponding tRNAs in E. coli are not sufficient to
ensure efficient translation of the corresponding clostridial
sequences. To address this issue, we amplified three E. coli genes, ileX, argU, and leuW, in E. coli; these genes encode tRNAs that
are rarely used in E. coli (the tRNAs for the ATA, AGA, and
CTA codons, respectively). Our data demonstrate that amplification of
ileX dramatically increased the level of production of most
of the clostridial proteins tested, while amplification of
argU had a moderate effect and amplification of
leuW had no effect. Thus, amplification of certain tRNA
genes for rare codons in E. coli improves the
expression of clostridial genes in E. coli, while
amplification of other tRNAs for rare codons might not be needed for
improved expression. We also show that amplification of a particular
tRNA gene might have different effects on the level of protein
production depending on the prevalence and relative positions of the
corresponding codons in the coding sequence. Finally, we describe a
novel approach for improving expression of recombinant clostridial
proteins that are usually expressed at a very low level in E. coli.
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INTRODUCTION |
Clostridial proteins, such as
tetanus toxin and seven serologically distinct botulinum neurotoxins
(botulinum neurotoxin serotype A [BoNT/A], BoNT/B, BoNT/C, BoNT/D,
BoNT/E, BoNT/F, and BoNT/G) that are produced by Clostridium
tetani, Clostridium botulinum, Clostridium
argentiensis, and Clostridium baratti, are powerful tools for studying the mechanisms of synaptic vesicle exocytosis (3, 20-23). These toxins have been also used for
therapeutic purposes, such as the treatment of strabismus,
blepharospasms (24, 25), and many other neurological
conditions, as well as in clinical dermatology (4).
Currently, BoNT/A and other clostridial neurotoxins and their fragments
are purified from native Clostridium strains by using traditional purification protocols. Because these microorganisms are
anaerobes, they pose technical problems. In addition, gene manipulation
methods have not been developed for these microorganisms. Therefore, it
has been difficult to construct Clostridium strains that
produce derivatives of neurotoxins and other proteins. Genes for all
eight clostridial neurotoxins have been cloned, and their sequences
have been identified (2, 6, 9, 18, 30, 31). Many attempts to
express fragments of clostridial neurotoxins in Escherichia
coli have failed because of the unusually high AT content of
clostridial DNA. Makoff et al. successfully expressed a tetanus toxin
fragment in E. coli (12) by optimizing sequences for codon usage in E. coli by complete synthesis of
these sequences de novo. This approach, however, is very laborious and expensive.
Recently, several groups of workers have demonstrated that rarely used
codons can have a pronounced effect on the translation efficiency
of cloned genes in E. coli (5, 8, 26). Molecular studies have shown that the ATA, AGA, and CTA codons are rarely used in E. coli. At the same time, these codons are
abundant in clostridial genes. To investigate the impact of these
codons on translation of clostridial genes in E. coli,
we amplified in E. coli the ileX,
argU, and leuW genes (7, 11, 14, 15), which encode tRNAs that translate the ATA, AGA, and CTA codons, respectively. We demonstrated that amplification of the ileX
gene resulted in dramatic increases in production of most of the
clostridial proteins tested. Indeed, when we examined fragments of
tetanus toxin, BoNT/A, BoNT/B, BoNT/C, BoNT/E, the Ia protein of
Clostridium perfringens iota toxin, and the C3 protein from
C. botulinum, we observed significant increases in
production in E. coli for all of these proteins except C3.
Amplification of the argU gene also had moderate positive
effects on the levels of production of these proteins. Amplification of
the leuW gene, however, did not have a noticeable effect.
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MATERIALS AND METHODS |
Cells and plasmids.
E. coli JM109 cells were used to
propagate plasmids. E. coli BL21(
DE3) was used for
expression of recombinant proteins (29).
The pGEM-T vector (Promega) was used to clone PCR products. The vectors
pETA32-22, pET28b(+), pPhe23-1, and pETSynB53Km were used to construct
expression plasmids encoding clostridial proteins, and the vector
pACYC184 was used to amplify genes for tRNA. Plasmid pET28b(+) is a
commercial vector obtained from Novagen. Plasmid pETA32-22 is a
derivative of pET3b (23) that encodes mutagenized fragment A
of diphtheria toxin (unpublished data). Plasmid pPhe23-1 was
constructed previously and contains a sequence encoding a Pseudomonas exotoxin A derivative (33). Both
pETA32-22 and pPhe23-1 were used in this study because they contain a
promoter of bacteriophage T7 and efficient signals for initiation of
translation. Plasmids pETSynB53Km and pETPA5 were constructed
previously by using plasmid pET28b(+) (unpublished data). Plasmid
pETSynB53Km encodes a soluble portion of rat synaptobrevin 2 (SynB),
and plasmid pETPA5 encodes a fragment of Pseudomonas
exotoxin A.
DNA-modifying enzymes.
Restriction enzymes
Acc65I, BamHI, BglII,
Eco52I, EcoICRI, EcoRI,
HindIII, NcoI, NdeI,
SacI, SalGI, StuI, and
XhoI, as well as T4 DNA polymerase, were produced by
Promega. A rapid DNA ligation kit and an Expand high-fidelity PCR
system were supplied by Boehringer Mannheim.
Oligonucleotides.
The oligonucleotides used for PCR, as well
as the oligonucleotides used for cloning, are listed in Table
1. All of these oligonucleotides were
synthesized by Promega.
Nucleic acids.
Total DNAs from C. botulinum
strains producing serotype A, B, C, and E neuotoxins, as well as DNAs
from C. tetani and C. perfringens, were kindly
provided by Uri Vertiev (Moscow, Russia). Total-RNA preparations were
purified from exponential cultures of E. coli BL21(DE3)
containing either plasmid pACYC184 or plasmid pACYC-IRL10 by an
alternative protocol for rapid isolation of RNA from gram-negative bacteria described previously (1). Then RNA preparations
were treated with RNase-free DNase I for 60 min at 37°C, and RNAs
were purified by phenol-chloroform extraction and precipitation with ethanol.
RT-PCR.
Reverse transcription-PCR (RT-PCR) was
performed by using E. coli total RNA, primers
listed in Table 1 (Ile1 and Ile2 for amplification of the
ileX gene fragment, Arg1 and Arg2 for amplification of the
argU gene fragment, and Leu1 and Leu2 for amplification of
the leuW gene), and an Access RT-PCR system (Promega) as
recommended by the manufacturer.
Construction of plasmids.
pGEM-IleArg7 was constructed by
cloning into vector pGEM-T a PCR-amplified fragment containing the
ileX and argU genes. As shown in Fig.
1, fragments containing the
ileX and argU genes were originally amplified
from E. coli chromosomal DNA as separate DNA fragments by
using primers 5'-Ile-tRNA and 3'-Ile-tRNA for ileX and
primers 5'-Arg-tRNA and 3'-Arg-tRNA for argU (Table 1). These fragments were then combined in a separate PCR mixture by using
primers 5'-Ile-tRNA and 3'-Arg-tRNA.
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FIG. 1.
Construction of plasmids encoding tRNAs. The
ileX, argU, and leuW sequences encode
tRNAs that recognize the ATA, AGA, and CTA codons, respectively.
The Apr, Tetr, and Cmr sequences
encode genes for antibiotic resistance. The T7 and Sp6 sequences encode
promoters from bacteriophages T7 and sp6, respectively. The shaded
areas represent sequences of subB-E tRNA operons other than
leuW. DNA pol Taq, Taq DNA polymerase; DNA polT4,
T4 DNA polymerase.
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pACYC-IleArg10 was constructed by joining the
HindIII-
SalGI fragment of plasmid
pGEM-IleArg7 containing the
ileX and
argU genes
with the large
HindIII-
SalGI fragment of
plasmid pACYC184
(Fig.
1).
pACYC-IleArgLeu17 was generated by combining the large
Eco52I-
SalGI fragment of plasmid pACYC-IleArg10
with the fragment of
E. coli chromosomal DNA encoding the
subB-E tRNA operon. The fragment
was amplified by PCR
performed with primers Leu-5' and Leu-3'
(Table
1) and was treated with
the
SalGI and
Eco52I restriction
endonucleases
(Fig.
1).
pACYC-IRL10 was generated by treating plasmid pACYC-IleArgLeu17 with
Eco52I and
Acc65I and then with T4 DNA polymerase
and
ligase.
pACYC-Ile7 was constructed by treating plasmid pACYC-IleArg10 with
XhoI and
SalGI endonucleases, T4 DNA polymerase,
and
ligase.
pACYC-Arg34 was generated by treating pACYC-IleArg10 DNA
with
HindIII and
XhoI endonucleases, T4
DNA polymerase, and
ligase.
pACYC-L10 is a derivative of pACYC-IRL10 and was constructed by
treating plasmid pACYC-IRL10 with
SalGI,
HindIII, T4 DNA polymerase,
and ligase (Fig.
1).
pACYC-RL5 was constructed by treating plasmid pACYC-IRL10 with
HindIII and
XhoI endonucleases, T4 DNA
polymerase, and ligase
(Fig.
1).
pETI
10PA10, pETR
10PA25, and
pETL
10PA32 were constructed by joining the large
NdeI-
SacI fragment of plasmid pETPA5 with
synthetic
DNA fragments formed by the oligonucleotide pairs
Ile10-Ile10-comp,
Arg10-Arg10-comp, and Leu10-Leu10-comp (Table
1),
respectively.
pGEM-BoNT/B-L5, pGEM-BoNT/C-L2, pGEM-BoNT/E-L13, pGEM- BoNT/B-H13,
pGEM-BoNT/C-H6, and pGEM-BoNT/E-H10 encoding the light
and heavy chains
of BoNT/B, BoNT/C, and BoNT/E were constructed
by cloning DNA fragments
amplified from corresponding clostridial
genome DNAs into the vector
pGEM-T.
pETBoNT/B-L10, pETBoNT/C-L20, and pETBoNT/E-L31 encoding the light
chains of BoNT/B, BoNT/C, and BoNT/E, respectively, were
constructed by replacing the small
BamHI-
EcoRI
fragment in plasmid
pETA32-22 with the small
BamHI-
EcoRI fragments from plasmids
pGEM-BoNT/B-L5,
pGEM-BoNT/C-L2, and pGEM-BoNT/E-L13,
respectively.
pETBoNT/A-L22Km encoding the light chain of BoNT/A was constructed by
replacing the small
BamHI-
EcoRI fragment in
plasmid
pET28b(+) with the fragment amplified from
C. botulinum by using
primers BoNT/A-N and BoNT/A-LC (Table
1).
pETBoNT/B-H18 was constructed by replacing the small
NcoI-
EcoRI fragment of plasmid pET28b(+) with the
small
NcoI-
EcoRI fragment
from plasmid
pGEM-BoNT/B-H13.
pETBoNT/C-H14 and pETBoNT/E-H10 were generated by subcloning into the
BamHI site of plasmid pET28b(+) light
BamHI-
BglII fragments
from plasmids
pGEM-BoNT/C-H6 and pGEM-BoNT/E-H10,
respectively.
pGEM-C3-20 encoding the C3 protein was generated as a result of
cloning into the pGEM-T vector the DNA fragment amplified
from
C. botulinum DNA with primers N-C3 and 1C3-C (Table
1).
pTSC3-7 encoding the C3 protein was constructed by joining the large
fragment of plasmid pPhe23-1 with the small fragment
of plasmid
pGEM-C3-20. A fragment of plasmid pPhe23-1 was generated
by treating
pPhe23-1 DNA with
HindIII, T4 DNA polymerase, and
BamHI. A fragment of plasmid pGEM-C3-20 was generated by
treating
pGEM-C3-20 DNA with
NdeI, T4 DNA polymerase, and
BglII.
pETiota11Km encoding the iota toxin Ia protein was generated by
replacing the small
BamHI-
XhoI fragment in
plasmid pETSynB53Km
with the fragment that was amplified by using
primers iota/IaN
and iota/IaC from
C. perfringens DNA and
was treated with
BamHI
and
XhoI.
pETTeNT-L12Km and pETTeNT-H4Km encoding the light and heavy chains of
tetanus toxin, respectively, were generated by direct
cloning of
fragments amplified from
C. tetani DNA into expression
vector pET28b(+). A fragment encoding the light chain of tetanus
toxin
after amplification was treated with
NdeI and
HindIII restriction
endonucleases and was joined with a
large
NdeI-
HindIII fragment
of plasmid
pET28b(+). A fragment encoding the heavy chain of tetanus
toxin after
amplification was treated with
StuI and
HindIII restriction
endonucleases and joined with the
large
HindIII-
EcoICR fragment
of plasmid
pET28b(+).
Expression and purification of recombinant proteins.
When
cell cultures were at an absorbency at 600 nm of 0.4 to 0.5, protein
expression was induced by adding
isopropyl-
-D-thiogalactopyranoside (IPTG). Cells were
harvested 90 min later. Light chains of BoNT/B and BoNT/E were
recovered after inclusion bodies were dissolved in 7 M guanidine
hydrochloride and renatured in 10 mM Tris-HCl-1 mM EDTA-300 mM
arginine (pH 7.0). Proteins were further purified by using ion-exchange chromatography.
Proteolytic assay.
Two recombinant proteins,
SynB-receptor-associated protein (RAP) and 25-kDa
synaptosome-associated protein (SNAP25)-RAP (unpublished data), which
contained RAP (27, 28) fused with SynB and SNAP25, respectively, were used to detect the enzymatic activities of light
chains of clostridial neurotoxins. The light chains of BoNTs were incubated with the appropriate substrate proteins in buffer containing 10 mM Tris-HCl (pH 6.8) and 1 mM ZnSO4 at
37°C for 1.5 h. After incubation, the proteins were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by
4 to 20% Tris-glycine gels from Novex and were visualized by staining
with Coomassie blue.
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RESULTS |
Amplification of tRNAs recognizing codons ATA, AGA, and CTA
rarely used in E. coli.
To investigate the impact of ATA,
AGA, and CTA codon usage on translation of clostridial genes
in E. coli, we amplified the ileX,
argU, and leuW (as part of the
subB-E tRNA operon) genes in E. coli. This was
done by amplifying known sequences of interest (7, 11, 14,
15) by PCR and subsequently cloning the sequences into a
multicopy plasmid. Plasmid pACYC184 was chosen as an appropriate vector
because it is compatible with the pBR-based vectors that we used for
cloning and expression of clostridial proteins in our studies. To
investigate the role of each of the three rarely used codons
(ATA, AGA, and CTA) on expression of clostridial genes in
E. coli, plasmids encoding the ileX
(pACYC-Ile7), argU
(pACYC-Arg34), and leuW
(pACYC-L10) genes separately or in the combinations
ileX-argU (pACYC-IleArg10),
argU-leuW (pACYC-RL5), and
ileX-argU-leuW (pACYC-IleArgLeu17 and
pACYC-IRL10) were constructed (Fig. 1) and introduced
into E. coli BL21(DE3). We did not observe any decrease
in the growth rate of cells containing the amplified ileX
gene, which is different than the results reported previously (19,
32). Indeed, cells containing plasmid pACYC-Ile7
grew at a rate as similar to the rate of growth of cells
containing plasmid pACYC184 (data not shown).
Similar results were obtained with cells containing plasmids pACYC-Arg34, pACYC-L10, and
pACYC-IRL10. We observed an almost 50% decrease in the
growth rate of cells containing plasmid
pACYC-IleArgLeu17. Because plasmid pACYC-IRL10 is
a derivative of plasmid pACYC-IleArgLeu17 and because the
growth rate of cells containing plasmid pACYC-IRL10 was
normal, we concluded that the decrease in the growth rate in the case
of plasmid pACYC-IleArgLeu17 was related to amplification of the part of subB-E operon, which is missing in plasmid
pACYC-IRL10 (Fig. 1) and is different from the
leuW gene.
To confirm that amplification of tRNA genes resulted in increased
accumulation of the corresponding tRNAs, we performed an
RT-PCR
analysis of total RNA isolated from BL21(
DE3) cells carrying
either pACYC184 or pACYC-IRL10. Our analysis revealed
that in
order to obtain equal concentrations of PCR-amplified fragments
corresponding to products of the
ileX,
argU, and
leuW genes, three
or four additional PCR cycles were needed
for RNA from cells carrying
pACYC184 than for RNA from cells carrying
pACYC-IRL10 (data not
shown). Furthermore, to confirm
that the amplified genes encode
functional tRNAs, we constructed three
plasmids that encode a
fragment of
Pseudomonas exotoxin A,
pETI
10PA10, pETR
10PA25, and
pETL
10PA32. The N-terminal regions of the proteins encoded
by
these plasmids contained stretches of 10 isolucine (codon ATA),
arginine (codon AGA), and leucine (codon CTA) residues,
respectively.
Figure
2 shows data for
expression of
Pseudomonas exotoxin A derivatives
encoded
by these plasmids in BL21(
DE3) cells containing either
plasmid pACYC184 or plasmid pACYC-IRL10. Production
of exotoxin
A derivatives encoded by plasmids pETI
10PA10,
pETR
10PA25, and
pETL
10PA32 was more efficient
in cells containing plasmid pACYC-IRL10
than in
cells containing control plasmid pACYC184. Protein encoded
by parent
plasmid pETPA5 was produced with the same efficiency
in cells
containing pACYC184 and in cells containing pACYC-IRL10.
These data confirm that amplification of the
ileX,
argU, and
leuW genes increased the functional
levels of the corresponding tRNAs
in
E. coli.
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FIG. 2.
Effect of amplification of the ileX,
argU, and leuW genes in E. coli
BL21(DE3) on production of different derivatives of
Pseudomonas exotoxin A. BL21(DE3) cells were
cotransformed with a plasmid encoding a derivative of
Pseudomonas exotoxin A (lanes 1 and 2, pETPA5; lanes 3 and
4, pETI10PA10; lanes 5 and 6, pETR10PA25;
lanes 7 and 8, pETL10PA32) and with either pACYC184 (lanes
1, 3, 5, and 7) or pACYC-IRL10 (lanes 2, 4, 6, and 8).
Cells were induced with IPTG and lysed, and cell proteins were
separated by 4 to 20% gradient SDS-PAGE and visualized by Coomassie
blue staining. Lane M contained the Mark12 wide-range protein standard
from Novex.
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Construction of plasmids encoding fragments of BoNTs and expression
of these fragments in E. coli.
As described above, we
constructed a set of plasmids encoding the light chains of BoNT/A,
BoNT/B, BoNT/C, and BoNT/E, as well as the heavy chains of BoNT/B,
BoNT/C, and BoNT/E. Fragments encoding the light and heavy chains of
BoNTs were amplified from clostridial genomic DNA by using primers
listed in Table 1. Amplified fragments were cloned into expression
vectors to create BoNT fragment-encoding genes whose
transcription was under control of the efficient bacteriophage T7
promoter, and the region around the start codon was also optimized to ensure efficient initiation of translation. To analyze expression of
our recombinant genes, we introduced these plasmids into
BL21(DE3) cells that simultaneously were transformed with either
pACYC184 or derivatives of this plasmid containing the
ileX, argU, and leuW genes, and
the proteins produced were analyzed by SDS-PAGE. As shown in Fig.
3 and 4,
cells cotransformed with both a BoNT fragment-encoding plasmid and
plasmid pACYC184 produced proteins of interest in such low quantities
that they were not detectable on Coomassie blue-stained gels. Also, we
were not able to detect substantial amounts of BoNT fragments in the
cells containing plasmid pACYC-Arg34,
pACYC-L10, or pACYC-RL5 instead of
pACYC184. In contrast, production of recombinant BoNT fragments was
substantially greater in cells cotransformed with either
pACYC-Ile7, pACYC-IleArg10, or
pACYC-IRL10 and BoNT fragment-encoding plasmids. Thus,
amplification of the ileX gene plays a major role in
increasing the production of BoNT fragments. Also, cells
containing plasmid pACYC-IleArg10 or
pACYC-IRL10 produced proteins of interest at slightly (up
to twofold) higher levels than cells containing plasmid
pACYC-Ile7 produced these proteins. This improved
production effect was observed with cells that were grown for 1.5 h after induction of expression with IPTG but not in cells grown for
16 h after induction of expression when no significant
accumulation of proteins of interest was observed (data not
shown).
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FIG. 3.
Effect of amplification of the
ileX, argU, and leuW genes in E. coli BL21(DE3) on production of light chains of BoNT/A (A),
BoNT/B (B), BoNT/C (C), and BoNT/E (D). The expression of each protein
was evaluated in the presence or absence of amplified ileX,
argU, or leuW genes, as follows: lane 1, no
amplification (pACYC184); lane 2, pACYC-Ile7; lane 3, pACYC-Arg34; lane 4, pACYC-L10; lane 5, pACYC-IleArg10; lane 6, pACYC-RL5; and
lane 7, pACYC-IRL10. Cells were induced with IPTG and
lysed, and the total cell proteins were separated by 4 to 20% gradient
SDS-PAGE and visualized by Coomassie blue staining. The arrows
indicate the locations of the various neurotoxin proteins. The
molecular weight markers used (lane M) were phosphorylase b
(molecular weight, 97,400), bovine serum albumin (66,200), glutamate
dehydrogenate (55,000), ovalbumin (42,700), aldolase (40,000), carbonic
anhydrase (31,000), soybean trypsin inhibitor (21,500), and lysozyme
(14,400).
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FIG. 4.
Effect of amplification of the
ileX, argU, and leuW genes in E. coli BL21(DE3) on production of heavy chains of BoNT/B (A)
and BoNT/E (B). The expression of each protein was evaluated in the
absence or presence of the amplified ileX, argU,
or leuW genes, as follows: lane 1, no amplification
(pACYC184); lane 2, pACYC-Ile7; lane 3, pACYC-Arg34; lane 4, pACYC-L10; lane 5, pACYC-RL5; lane 6, pACYC-IleArg10; and
lane 7, pACYC-IRL10. Cells were induced with IPTG and
processed for SDS-PAGE as described in the legend to Fig. 2. The arrows
indicate the locations of the expressed proteins BoNT/B-H and BoNT/E-H.
The molecular weight markers used (lane M) were the molecular weight
markers described in the legend to Fig. 3.
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The identities of the proteins were confirmed with specific antibodies.
Furthermore, to ensure the functional integrity of
the toxin products
as proteases, we carried out enzymatic activity
tests as described
above. Recombinants BoNT/A-L, BoNT/B-L, and
BoNT/E-L were recovered
from inclusion bodies by using the denaturation-renaturation
procedure described above, and their enzymatic activities were
tested. Figure
5 shows that BoNT/B-L was
active in cleavage of
the SynB-RAP fusion protein but was not active
when the SNAP25-RAP
fusion protein was the substrate. In contrast,
BoNT/A-L (Fig.
6) and BoNT/E-L (data not
shown) cleaved SNAP25-RAP and but did
not cleave the SynB-RAP fusion
protein. The activities of recombinant
light chains were completely
inhibited by EDTA. The specificities
of BoNT/B-L and BoNT/E-L were also
confirmed in vivo by using
previously described systems (
10,
13).
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FIG. 5.
Effect of amplification of the
ileX, argU, and leuW genes on
production of the botulinum C3 protein (A), the iota toxin Ia protein
(B), and the light chain of tetanus toxin (C) in BL21(DE3)
cells. The expression of each protein was evaluated in the absence or
in the presence of amplified ileX, argU, or
leuW genes, as follows: lane 1, no amplification (pACYC184);
lane 2, pACYC-Arg34; lane 3, pACYC-Ile7; lane 4, pACYC-L10; lane 5, pACYC-IleArg10; lane 6, pACYC-RL5; and
lane 7, pACYC-IRL10. Cells were induced with IPTG and
processed for SDS-PAGE as described in the legend to Fig. 2. The arrows
indicate the locations of proteins C3 and Ia and the light chain of
tetanus toxin (TeNT-L). The molecular weight markers used (lane M) were
the molecular weight markers described in the legend to Fig. 3.
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FIG. 6.
Enzymatic activities of recombinant light chains of
BoNT/A and BoNT/B. Substrate proteins SynB-RAP (lane 1) and SNAP25-RAP
(lane 2) and light chains of BoNT/A (lane 3) and BoNT/B (lane 7) were
included. Also included were a mixture of BoNT/A-L and SNAP25 in the
absence (lane 4) and in the presence (lane 5) of EDTA (lane 5) and a
mixture of BoNT/B-L and SynB-RAP in the absence (lane 8) and in the
presence (lane 9) of EDTA. Lanes 6 and 10 contained a mixture of
BoNT/A-L and SynB-RAP and a mixture of BoNT/B-L and SNAP25,
respectively. Proteolytic activity was determined in the presence of 10 mM Tris-HCl (pH 6.8) and 1 mM ZnSO4 for 1.5 h at
37°C. Proteolytic products were separated by 4 to 20% gradient
SDS-PAGE, and proteins were visualized with Coomassie blue as described
in the text. The arrows on the left indicate the positions of molecular
weight markers (lane M) obtained from the Mark12 wide-range protein
standard (Novex), and the arrows on the right indicate the locations of
substrate proteins and their products.
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Construction and expression in E. coli of plasmids
encoding proteins from different representatives of the genus
Clostridium.
We hypothesized that amplification of tRNAs
that recognize rarely used codons may be a useful strategy which
has general applicability for improving the efficiency of heterologous
expression in E. coli. To study this, we tested the
applicability of this procedure for expression of other clostridial
toxins. We used the C3 protein (16) from C. botulinum, the light and heavy chains of tetanus toxin from
C. tetani, and the iota toxin Ia protein from C. perfringens (17) as prototypes. The corresponding
sequences were amplified from the total DNAs of the corresponding
microorganisms by using PCR and the specific primers listed in Table 1
and were placed under control of a bacteriphage T7 promoter with an
efficient translation initiation site as described above. The
resulting plasmids, pTSC3-7 (encoding the C3 protein), pETTeNT-L12Km
and pETTeNT-H4Km (encoding the light and heavy chains of tetanus toxin, respectively), and pETiota11Km (encoding the iota toxin Ia protein), were introduced into BL21(DE3) cells containing either pACYC184 or tRNA-encoding derivatives of this plasmid, and production of the
corresponding recombinant proteins was analyzed by SDS-PAGE. Our
analysis revealed that unlike plasmids encoding fragments of BoNTs,
plasmids pTSC3-7, pETiota11Km, pETTeNT-L12Km (Fig. 5), and pETTeNT-H4Km
(data not shown) gave relatively efficient production of recombinant
proteins in E. coli cells that contained normal quantities
of tRNA genes. Amplification of either ileX,
argU, or leuW separately did not result in
increased production of the C3 protein. Simultaneous amplification of
the ileX and argU genes did slightly improve
production of this protein. In the cases of the pETiota11Km,
pETTeNT-L12Km (Fig. 5), and pETTeNT-H4Km (data not shown)
plasmids, amplification of the ileX gene had a positive effect on production of recombinant proteins. Amplification of the
argU gene in addition to amplification of the
ileX gene also allowed us to improve production of the heavy
chain of tetanus toxin encoded by pETTeNT-H4Km.
| |
DISCUSSION |
Low levels of expression of clostridial neurotoxins in traditional
organisms such as E. coli may explain why our understanding of the mechanism of action of such an important class of toxins is
lagging behind our understanding of the mechanism of action of other
toxins. The fact that efficient expression of a tetanus toxin fragment
was achieved after complete de novo synthesis of the coding sequence
and adjustment of codons in the sequence on the basis of E. coli codon usage indicated the importance of the codons used in the sequence for efficient production of this
protein (12). Whether this was fully attributable to
codon usage or to other factors, such as the mRNA secondary
structure, was not clear. Furthermore, this approach is expensive and
requires a substantial amount of preliminary work before each protein
can be expressed. In this study, we examined whether rarely used
codons play a major role in decreasing the efficiency of production
of clostridial neurotoxins in E. coli. By amplifying tRNA
genes whose products recognize rarely used codons, we were able to
significantly improve production of clostridial neurotoxin
fragments in E. coli. We did this without
changing the coding sequences of neurotoxin fragments, which confirmed
that rarely used codons play a major role in efficient expression
of clostridial neurotoxin-encoding genes in E. coli. Table
2 shows the frequencies of rarely used codons in the sequences and the effect of amplification of
tRNA-encoding genes on the level of expression. The data show that
there is a definite correlation between the frequency of a particular
codon in the reading frame and the effect of amplification of the
corresponding tRNA-encoding gene on the production of the corresponding
protein. Indeed, of the three codons examined (ATA, AGA, and CTA),
the ATA codon encoding isoleucine is the most frequently used
codon in neurotoxin-encoding sequences, and amplification of the
ileX gene has the most dramatic effect on the level of
expression of this codon. The AGA codon is less prevalent than
the ATA codon, and thus the effect of argU gene
amplification is more modest. Nevertheless, there is not a strong
correlation between the frequencies of rarely used codons in each
gene and the effects of the corresponding tRNA amplification on the
efficiency of expression. Indeed, although ATA occurs more frequently
with the genes encoding the light chain of tetanus toxin (4.8%) and
the iota toxin Ia protein (5.1%) than with the genes encoding the
light chains of BoNT/A (3.8%) and BoNT/E (4.0%), amplification of the
ileX gene had more dramatic effect on production of the last
two proteins than on production of the first two. In addition, even
though all of the clostridial genes examined in this study contain the
CTA codon, expression of neither of them was improved by
amplification of the leuW gene.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Codon usage in recombinant genes and effect of
amplification of tRNA-encoding genes on expression
in E. coli
|
|
It is noteworthy that the disruptive effect of rarely used codons
on translation efficiency depends not only on the prevalence of these
codons but also on their relative locations in the gene. Indeed, in
recombinant genes encoded by plasmids pETBoNT/E-L31 and
pETR10PA25, the frequencies of the AGA codon are
practically the same (2.4 and 2.5%, respectively). Nevertheless,
amplification of the argU gene had a more pronounced effect
on expression of the recombinant gene from plasmid
pETR10PA25, in which all 10 AGA codons were clustered
together, than on expression of the recombinant gene from plasmid
pETBoNT/E-L31, in which 11 AGA codons were randomly spreaded
throughout the coding sequence.
The lack of an effect of leuW gene amplification on
expression of clostridial proteins suggests that the disruptive effect of rarely used codons becomes noticeable only when the total number and relative frequency of these codons in the gene are higher than
certain minimum values. Our results for expression of exotoxin A
derivatives encoded by plasmids pETI10PA10,
pETR10PA25, and pETL10PA32 also suggest that
the effective number of codons varies with each type of codon.
Indeed, of the three types of codons tested, the ATA codon had
the most dramatic effect on production of proteins in E. coli cells. A stretch of 10 of these codons was sufficient to
completely inhibit production of an exotoxin A derivative in E. coli cells that contained normal level of tRNAs. When the same
protein was encoded by a gene that contained 10 CTA codons instead
of ATA codons, production of the protein was significantly
enhanced in the same E. coli cells. Our results also
demonstrate that amplification of tRNA-encoding genes for rare
codons can be used for optimization of protein production and may
be applicable to many other genes for production of proteins that have
great commercial value.
| |
ACKNOWLEDGMENTS |
We thank Said Goueli and Josephine Grosh (Promega Corporation)
for critical reviews of the manuscript and Uri Vertiev (Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia) for providing clostridial DNAs.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Promega
Corporation, 2800 Woods Hollow Road, Madison, WI 53711-5399. Phone:
(608) 298-4658. Fax: (608) 274-4330. E-mail:
azdanovs{at}promega.com.
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Applied and Environmental Microbiology, August 2000, p. 3166-3173, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
