Novel expression and secretion vector systems
for heterologous protein production in Escherichia coli
Technical Field
The present invention relates to a
recombinant DNA expression/secretion system in
Gram-negative prokaryotes such as Escherichia
coli including but not restricted to E. coli BL21
DE3 and E. coli K12. More particularly, the invention
relates to a system that combines the potential of
signal peptide-based translocation of recombinant
proteins to the periplasmic space of E. coli
with membrane defective mutants of E. coli to
further aid secretion into the extracellular space.
Background Art
Prokaryotes have been widely used for
the production of recombinant proteins. Controlled
expression of the desired polypeptide or protein is
accomplished by coupling the gene encoding the protein
through recombinant DNA techniques behind a promoter,
the activity of which can be regulated by external
factors. This expression construct is carried on a
vector, most often a plasmid. Introduction of the
plasmid carrying the expression construct into a host
bacterium and culturing that organism in the presence
of compounds that activate the promoter results in high
levels of expression of the desired protein. In this
way, large quantities of the desired protein can be produced.
E. coli is the most commonly used prokaryote
for protein production. Many different varieties of
plasmid vectors have been developed for use in E. coli
to build expression systems. The different variations
employ several different types of promoters, selectable
markers and origins of replication where each of the
different configurations imparts a unique property to
the expression vector. In the most common arrangement,
the expressed protein accumulates in the cytoplasm.
While this approach is useful for some proteins, not
all proteins can be accumulated in the cytoplasm in an
active state. Often, when the desired protein is
produced at high levels relative to the host proteins,
the protein accumulates as an insoluble particle also
known as an inclusion body. Proteins which accumulate as
inclusion bodies are difficult to recover in an active form.
Two ways of solving this problem are
either to export the target protein to the periplasm
between the inner and outer membranes or to facilitate
secretion into the extracellular space. There are
several potential advantages to having a cloned gene
product secreted into the periplasmic
space/extracellular medium, including: 1) The protein
product can avoid cytoplasmic proteases; 2) normally
secreted proteins such as hormones, ligninolytic
enzymes, and dextranases may only be able to fold in
their active conformation in E. coli if secreted; 3)
correct formation of disulfide bonds can be
facilitated because the periplasmic space provides a
more oxidative environment than the cytoplasm. 4)
toxic enzymes such as nucleases or proteases cannot be
produced in the cytoplasm due to their potential to
exert a toxic effect on the host; and 5) ease of purification.
To target proteins to the periplasm,
they are expressed as fusions with signal peptide
sequences (e.g. PelB, OmpA, DsbA, TorA and MalE) that
follow different secretion pathways (e.g. Sec, Tat and
SRP). Other strategies include: a. Modification of
signal peptides to enhance translocation. b. Use of
heterologous signal sequences in E. coli. c.
Co-expression of periplasmic chaperones (e.g. Dsb family
proteins) d. Protease-negative mutant strains to
reduce proteolysis.
Export of recombinant proteins to the
periplasm of E. coli is in many cases
preferable to cytoplasmic production. However, when
the protein is overexpressed, export efficiency
decreases significantly and some advantages of the
system are lost. To avoid overloading the host's
translocation machinery following overexpression of
signal peptide-recombinant protein fusions attempts
have been made to supplement the native secretion
machinery with the corresponding translocons from
secretory pathways.
Extracellular production of recombinant
proteins has several advantages over secretion into
the periplasm. Extracellular production does not
require outer-membrane disruption to recover target
proteins, and, therefore, it avoids intracellular
proteolysis by periplasmic proteases and allows
continuous production of recombinant proteins.
A number of methods have been applied to
promote extracellular secretion of recombinant
proteins from E. coli. These include the use of
biochemicals, physical methods (osmotic shock, freezing
and thawing), lysozyme treatment, and chloroform
shock. However, these methods can be applied only
after harvesting cells. E. coli normally does not
secrete proteins extracellularly except for a few
classes of proteins such as toxins and hemolysin. In
general, movement of recombinant proteins from the
periplasm to the culture medium is the result of
compromising the integrity of the outer membrane.
Compromising the integrity of the outer
membrane of bacteria can be achieved by a number of
approaches. One method involves fusing the product to
a carrier protein that is normally secreted into the
medium (e.g. hemolysin), or to a protein expressed on
the outer membrane (e.g. OmpF).
Proteins secreted into the E.
coli periplasm can also be released into the
culture medium by co-expression of kil or the gene
coding for the third topological domain of the
transmembrane protein TolA (TolAIII) or the out genes
from E. chrysanthemi EC16. Bacteriocin release protein
(BRP) can also be used in the extracellular production
of recombinant proteins in E. coli.
Another approach to the extracellular
production of target proteins uses L-form cells,
wall-less, or wall-deficient cells. Knockouts of outer
membrane proteins (omp, tol, lpp, env) have been
constructed and the corresponding leaky strains have
been used to facilitate secretory recombinant protein expression.
E. coli K12 is a GRAS organism which makes it a
safe system for the large scale production of
therapeutic proteins. However, K12 is not commonly
regarded as a secretory recombinant protein expression
strain. An approach towards generation of an efficient
secretory E. coli K12 strain as described in
this invention combines the potential of signal
peptide- protein fusions and over-expression of
translocon components with membrane defective mutants.
The present invention utilizes the
power of novel signal peptides whose nucleotide
sequence are chosen such that they can direct the
protein of interest to different cellular compartments
of the E. coli cell. Hence, this invention
offers a platform of seven different signal peptides
that can be tested for determining the best combination
possible for secretory expression of the protein of interest.
Summary of the invention
The present invention relates to a
recombinant DNA expression/secretion system in
Gram-negative prokaryotes such as an Escherichia
coli, including but not restricted to E. coli K12
or E. coli BL21 DE3. The said system combines the
potential of signal peptide-based translocation of
recombinant proteins to the periplasmic space of E.
coli with membrane defective mutants to further aid
secretion into the extracellular space.
Another aspect of the present invention is
an expression vector which optionally includes a helper
plasmid which facilitates the expression of translocons to
facilitate improved periplasmic secretion of the
over-expressed recombinant protein. The system can further
be used for production of specific proteins secreted by
the E. coli host where normally such proteins are not
secreted by the host. In addition, this system also
facilitates efficient production of specific proteins of
interest in E. coli. The expression vector
comprises secretory signal sequence, inducible promoter
and a gene of interest.
Yet another aspect of the present invention
is a method of obtaining a recombinant cell, said method
comprises acts of - (a) obtaining a recombinant vector,
(b) transforming a host cell with the recombinant vector
and (c) optionally co-transforming the host cell with a
helper plasmid to obtain the recombinant cell;
Yet another aspect of the present invention
is a method of obtaining a recombinant peptide, said
method comprising acts of - (a) obtaining recombinant
vector comprising a secretory signal sequence, an
inducible promoter and a gene of interest (b) transforming a
host cell with the recombinant vector and optionally,
co-transforming the host cell with a helper plasmid, (c)
expressing the recombinant vector and secreting the
recombinant peptide into an extracellular medium and (d)
optionally purifying to obtain the recombinant peptide.
Yet another aspect of the present invention
is a kit for obtaining recombinant peptide, said kit
comprising an expression vector, a recombinant cell or
combinations thereof; and a method of assembling a kit for
obtaining recombinant peptide, said method comprising act of
combining expression vector, recombinant cell or
combinations thereof.
Brief Description Of Drawings
In order that the invention be readily
understood and put into practical effect, reference
will now be made to exemplary embodiments as
illustrated with reference to the accompanying figures.
The figures together with a detailed description
below, are incorporated in and form part of the
specification, and serve to further illustrate the
embodiments and explain various principles and
advantages, in accordance with the present invention.
In accordance with the embodiments of
the present invention, FIG. 1 depicts the schematic
representation of Plasmid map for the expression
vector, pAEV01 carrying the signal peptide-target
protein fusion under the control of an inducible T5
promoter .
In accordance with the embodiments of
the present invention, FIG. 2 is a schematic
representation of the T7 promoter, signal peptide
sequence, ribosome binding site and transcription start
site of Bacillus stearothermophilus maltogenic amylase
In accordance with the embodiments of
the present invention, FIG. 3A shows 0.1mM IPTG
induction followed by SDS-PAGE analysis of lysates
from strains expressing MalE, TorA and pelB signal
peptide fusions to maltogenic amylase.
In accordance with the embodiments of
the present invention, FIG. 3B shows 0.1mM IPTG
induction studies followed by SDS-PAGE analysis of
lysates from strains expressing DsbA, YcdO, FhuD, MdoD
and pelB signal peptide fusions to maltogenic amylase.
In accordance with the embodiments of
the present invention, FIG. 4 shows 1mM IPTG induction
studies followed by SDS-PAGE analysis of lysates from
strains expressing MalE, YcdO, TorA, FhuD and pelB
signal peptide fusions to maltogenic amylase.
In accordance with the embodiments of
the present invention, FIG. 5A shows Fold induction
with 0.1 mM of maltogenic amylase activity fused to
pelB, MalE, FhuD, DsbA and MdoD compared to
corresponding uninduced cultures.
In accordance with the embodiments of
the present invention, FIG. 5B shows fold induction
with 1 mM of maltogenic amylase activity fused to
MalE, FhuD, TorA, YcdO and pelB compared to
corresponding uninduced cultures.
Brief Description of Tables
In accordance with the embodiments of the
present invention, Table 1 lists amino acid residues of
the signal sequences and respective export pathways.
In accordance with the embodiments of the
present invention, Table 2 lists signal sequences.
In accordance with the embodiments of the
present invention, Table 3 lists signal sequence plasmid information.
Detailed Description of the Invention
In order to more clearly and concisely
describe and point out the subject matter of the claimed
invention, the following definitions are provided for
specific terms which are used in the following written description.
By the term 'Expression'
we mean transcription or translation, or both, as context requires.
By the term an 'Expression
vector' we refer to recombinant DNA molecule
containing the appropriate control nucleotide sequences
(e.g., promoters, enhancers, repressors, operator sequences
and ribosome binding sites) necessary for the expression
of an operably linked nucleotide sequence in a particular
host cell. The expression vector may be self-replicating,
such as a plasmid, and may therefore carry a replication
site, or it may be a vector that integrates into a host
chromosome either randomly or at a targeted site. The
expression vector may contain a selection gene as a
selectable marker for providing phenotypic selection in
transformed cells. The expression vector may also contain
sequences that are useful for the control of translation.
By the term 'operably
linked/linking' or 'in operable
combination' we refer to nucleotide sequence
positioned relative to the control nucleotide sequences to
initiate, regulate or otherwise direct transcription
and/or the synthesis of the desired protein molecule.
By the term 'Nucleotide'
we refer to a ribonucleotide or a deoxyribonucleotide.
'Nucleic acid' refers to a polymer of
nucleotides and may be single-or double-stranded.
'Polynucleotide' refers to nucleic acid
that is twelve or more nucleotides in length.
By the term 'Nucleotide sequence
of interest' we mean nucleotide sequence that
encodes a 'protein, polypeptide or peptide sequence
of interest,' the production of which may be deemed
desirable for any reason, by one of ordinary skill in the
art. Such nucleotide sequences include, but are not
limited to, coding sequences of structural genes, regulatory
genes, antibody genes, enzyme genes, etc., or portions
thereof. The nucleotide sequence of interest may comprise
the coding sequence of a gene from one of many different organisms.
A nucleotide sequence
'encodes' or 'codes for'
a protein if the nucleotide sequence can be translated to
the amino acid sequence of the protein. The nucleotide
sequence may or may not contain an actual translation
start codon or termination codon.
A 'protein, polypeptide or peptide
sequence of interest' is encoded by the
'nucleotide sequence of interest.' The protein,
polypeptide or peptide may be a protein from any organism,
including but not limited to, mammals, insects,
micro-organisms such as bacteria and viruses. It may be
any type of protein, including but not limited to, a
structural protein, a regulatory protein, an antibody, an
enzyme, an inhibitor, a transporter, a hormone, a
hydrophilic or hydrophobic protein, a monomer or dimer, a
therapeutically-relevant protein, an industrially-relevant
protein, or portions thereof.
A 'peptide' is polymer of
four to 20 amino acids, a 'polypeptide' is a
polymer of 21 to 50 amino acids and a 'protein' is
a polymer of more than 50 amino acids.
By the term 'Portion'
when used in reference to a protein we refer to fragments
of that protein. The fragments may range in size from four
amino acid residues to the entire amino acid sequence of the
protein, minus one amino acid.
By the term 'Purified' or
'to purify' we refer to removal of
undesired components from a sample. For example, to purify
the secreted protein from growth medium, may mean to remove
other components of the medium (i.e., proteins and other
organic molecules), thereby increasing the percentage of
the secreted protein. The terms 'modified',
'mutant' or 'variant' are
used interchangeably herein, and refer to: (a) a
nucleotide sequence in which one or more nucleotides have
been added or deleted, or substituted with different
nucleotides or modified bases or to (b) a protein, peptide
or polypeptide in which one or more amino acids have been
added or deleted, or substituted with a different amino
acid. A variant may be naturally occurring, or may be
created experimentally by one of skilled in the art. A
variant may be a protein, peptide, polypeptide or
polynucleotide that differs (i.e., an addition, deletion
or substitution) in one or more amino acids or nucleotides
from the parent sequence.
By the term 'Periplasm'
we refer to gel-like region between the outer surface of
the cytoplasmic membrane and the inner surface of the
lipopolysaccharide layer of gram-negative bacteria.
By the term 'Secretion'
we refer to the excretion of the recombinant protein that
is expressed in a bacterium to the periplasm or
extracellular growth medium.
In accordance with preferred embodiments,
the present invention relates to an expression vector
comprising secretory signal sequence, inducible promoter
and gene of interest. The present invention further relates
to a recombinant cell comprising said vector, optionally
alongwith helper plasmid, wherein, said recombinant cell
is a membrane defective cell.
Another preferred embodiment of the present
invention relates to a method of obtaining recombinant
cell, said method comprising steps of: a. obtaining
recombinant vector, b. transforming host cell with the
recombinant vector; and c. optionally co-transforming the
host cell with helper plasmid to obtain the recombinant cell.
Another embodiment relates to a method of
obtaining recombinant peptide, said method comprising
steps of: a. obtaining recombinant vector comprising
secretory signal sequence, inducible promoter and gene of
interest; b. transforming host cell with the recombinant
vector and optionally, co-transforming the host cell with
helper plasmid; c. expressing the recombinant vector and
secreting the recombinant peptide into extracellular
medium; and d. optionally purifying to obtain the
recombinant peptide.
In an embodiment of the present invention,
the said secretory signal sequence is codon optimized
sequence selected from group comprising SEQ ID NO 1 to SEQ
ID NO 7; and the inducible promoter is T5 promoter.
In an embodiment of the present invention,
said gene of interest is selected from group comprising
prokaryotic and eukaryotic genes.
In yet another embodiment of the present
invention, said cell is a prokaryotic cell, preferably an
E.coli K12; and the helper plasmid is a plasmid
carrying chaperons or translocons from prokaryotic
secretory system.
Another embodiment of the present invention
relates to a kit for obtaining recombinant peptide, said
kit comprising expression vector, recombinant cell or a
combination thereof.
The present invention further includes a
method of assembling a kit for obtaining recombinant
peptide, said method comprising act of combining
expression vector, recombinant cell or combinations thereof.
In yet another embodiment of the present
invention, the helper plasmid is selected from group
comprising of plasmids carrying any component of the
chaperones or translocons from the bacterial secretory
machinery; illustratively, SEC and TAT.
The present invention furthermore relates
to a method for producing a recombinant protein,
polypeptide or peptide of interest through secretion of
the recombinant protein, polypeptide or peptide to the
extracellular growth medium.
In an embodiment of the present invention,
the method utilizes expression vectors carrying particular
codon optimized variations of native E. coli secretory
signal sequences to direct the secretion of the
recombinant protein, polypeptide or peptide to the periplasm
via the SEC, TAT or SRP export pathways alone or in
combination (Table 1).
In an embodiment of the present invention,
the expression vector carries a signal sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6 or
SEQ ID NO: 7; downstream of an inducible T5 promoter (Table 2).
In one embodiment, the expression vector is
selected from the group consisting of plasmids pAEV01,
pAEV02, pAEV03, pAEV04, pAEV05, pAEV06, pAEV07 (Table 3).
In one embodiment, the expression vector is
for use in a prokaryotic host cell, for example,
Escherichia coli or a strain thereof.
In yet another embodiment of the present
invention, an isolated host cell transformed by any of the
expression vectors is provided, such that the cell
expresses and secretes a protein, polypeptide or peptide of
interest encoded by the nucleic acid. In one embodiment, the
host cell is a prokaryotic host cell, for example,
Escherichia coli or a strain thereof.
In still another embodiment of the present
invention, the use of signal peptides corresponding to
sequence ID. No. 2, 3 and 5 that harness the features of
two periplasmic secretion signals in a single nucleotide
sequence is described. Thus the use of the corresponding
expression vectors can avoid the clogging of a particular
secretion pathway and improve protein yields. The
expression vectors described in this invention will carry
the signal-peptide target protein fusion under the control
of an inducible T5 promoter thus making them amenable to
use in an E. coli K12 host for inducing heterologous
protein expression.
In still another embodiment of the present
invention, the use of helper plasmids to co-express
translocons belonging to the SEC and TAT secretory
pathways is described. In particular one of these helper
plasmids will encode the genes secY, secE and secG as a
single operon. Another helper plasmid will encode tatA,
tatB and tatC as a single operon. Both these translocon
encoding operons will be under the control of an inducible
T5 promoter thus making them amenable to use in an E. coli
K12 host for inducing heterologous protein expression.
In still another embodiment of the present
disclosure, an E. coli strain that has a defective outer
membrane co-transformed with the signal
peptide-recombinant protein fusion vector and the translocon
encoding plasmid. This E. coli strain will not only target
the recombinant protein to the periplasmic space but will
also facilitate passive diffusion of its leaky protein
across the outer membrane into the extracellular medium.
EXAMPLES
In order that this invention to be more
fully understood the following preparative and testing
examples are set forth. These examples are for the purpose
of illustration only and are not to be construed as
limiting the scope of the invention in any way.
EXAMPLE 1
CLONING OF MALTOGENIC AMYLASE
The following example illustrates the
cloning of maltogenic amylase coding gene into pET20b+ and
replacement of the pelB signal peptide in pET20b+
Maltogenic amylase (MA) with seven other signal peptides
described in this invention SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7.
Maltogenic amylase gene from Bacillus
stearothermophilus was amplified using primers carrying
NcoI and BamHI sites. The primer sequences are: MANcoI fwd
primer: 5'-
gatcgtaccatgggaATGAGCAGTTCCGCAAGCGT-3' and MABglII rev
primer: 5'- gatcgtacagatctTCTAGACTAGTTTTGCCACG-3'.
Next, this PCR product was digested with
NcoI and BglII enzymes and cloned into the NcoI and BamHI
digested pET-20b(+) vector. The resulting ligation mix was
transformed into DH5α E. coli cells. The plasmid
was sequence verified to ascertain correct maltogenic
amylase coding gene and was then digested with NdeI and
NcoI to remove pelB signal peptide by gel elution. Seven
signal peptides (SEQ. ID. No. 1, 2, 3, 4, 5, 6 and 7) were
synthesized with NdeI and NcoI overhangs and cloned into
this vector. The resulting vectors retained the reading
frame defined by the ATG start codon from pET-20b(+) (FIG.
2). These seven plasmids were transformed into DH5α E.
coli cells. Plasmid was isolated from DH5α E.
coli cells, validated and then used to transform BL21
DE3 (the producer strain in which heterologous gene
expression can be induced using IPTG).
EXAMPLE 2
This example illustrates the induction
studies on the different maltogenic amylase signal peptide
fusions using SDS- PAGE and maltogenic amylase activity assays.
All Eight BL21(DE3) strains were grown in
minimal medium supplemented with glucose as the carbon
source and 100 ug/ml ampicillin kept overnight in an
incubator shaker 37 °C, 200 rpm. This culture was diluted
1:100 into a fresh 250 ml flask with 50 ml yeast extract
media containing ampicillin and grown at 37°C in a shaker
incubator at 200 rpm. 0.1mM IPTG was added to the culture
when the OD at 600 nm reached 0.6. Culture was then
incubated at 26°C for 16 h at 200 rpm. The induced and
un-induced cultures (grown the same way as the induced
cultures except no IPTG was added) were pelleted down at
3500 rpm for 15 minutes. The pellet was re-suspended in
sample buffer containing 10mM NaCl, pH 5. This pellet was
sonicated to release the soluble protein, cell debris was
pelleted out and the supernatant was analyzed on an
SDS-PAGE. Similarly induction was carried out by adding 1 mM
IPTG to the cultures and induction temperature was
maintained at 30°C.
Following induction with 0.1mM IPTG and
grown for 16 h at 26°C strains carrying pAEV01, pAEV05,
pAEV06 and pET-20b(+) construct revealed a significant
protein band at 70kDa, the expected size of maltogenic
amylase on a 12% SDS- PAGE. Induction of maltogenic amylase
protein levels in pAEV06 and pAEV01 was comparable to that
of the parent vector and that of pAEV05 was higher than
the parent (FIG. 3A and 3B). There was no maltogenic
amylase produced in strains carrying pAE04 and pAEV07.
pAEV03 plasmid carrying strain showed
induction of a truncated protein and the pAEV02 strain
showed significant leaky expression of maltogenic amylase
i.e. there was no difference in the amount of maltogenic
amylase produced with and without induction (FIG. 3A and 3B).
Experiments to determine the localization
of the maltogenic amylase protein will be carried out.
This will shed light on the secretory nature of the signal
peptide fusions. Our data suggests that fusions to
different signal peptides would contribute differently to
the expression secretion of different proteins of interest.
Following induction with 1mM IPTG and
grown for 6 h at 30°C strains carrying pAEV01, pAEV02,
pAEV04, pAEV05 and pET-20b(+) construct revealed a
significant protein band at 70kDa, the expected size of
maltogenic amylase on a 12% SDS-PAGE (FIG. 4). This data
indicates that different IPTG concentrations and
temperature post induction play an important role in
heterelogous protein expression.
The sonicated supernantant was also
subjected to determination of maltogenic amylase activity
using the glucose oxidase method. Higher fold induction in
terms of maltogenic amylase activity was observed in the
pAEV01 construct compared to parent when the cultures were
induced with 0.1mM IPTG (FIG. 5A).
Both the pAEV05 and pAEV01 construct
transformed strains showed higher fold induction in terms
of maltogenic amylase activity compared to parent when the
cultures were induced with 1mM IPTG (FIG. 5B). Strains
carrying other constructs that showed maltogenic amylase
expression on an SDS-PAGE exhibited similar functional
maltogenic amylase activity as the parent (FIG. 5A and 5B).
Results indicate that a much higher amount
of functional maltogenic amylase is getting targeted into
the periplasmic space compared to the parent plasmid in
the case of pAEV01 and pAEV05. MalE and FhuD appear to
represent improved signal peptides compared to pelB
for maltogenic amylase localization to the E. coli
periplasmic space.
Table 1
Signal sequence |
Pathway |
Amino acid
sequence for each signal sequence |
Resulting plasmid |
MalE |
SEC |
MKIKTGARILALSALTTMMFSASALApAEV01 |
pAEV01 |
YcdO |
TAT+SEC |
MTINFRRNALQLSVAALFSSAFMANApAEV02 |
pAEV02 |
MdoD |
TAT+SEC |
MDRRRFIKGSMAMAAVCGTSGIASLFSQAAFA |
pAEV03 |
TorA |
TAT |
MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAAQAA |
pAEV04 |
FhuD |
TAT+SEC |
MSGLPLISRRRLLTAMALSPLLWQMNTAHA |
pAEV05 |
DsbA |
SRP |
MKKIWLALAGLVLAFSASAAQ |
pAEV06 |
OmpA |
SEC |
MKKTAIAIAVALAGFATVAQA |
pAEV07 |
Table2
Seq ID no.1 |
atgaaaattaaaaccggcgcgcgcattctggcgctgagcgcgctgaccaccatgatgtttagcgctagcgcgctggcc |
“MalE”
|
Seq ID no.2 |
atggatcgccgccgctttattaaaggcagcatggcgatggcggcggtgtgcggcaccagcggcattgctagcctgtttagccaggcggcgtttgcc |
“YcdO”
|
Seq ID no.3 |
atggatcgccgccgctttattaaaggcagcatggcgatggcggcggtgtgcggcaccagcggcattgctagcctgtttagccaggcggcgtttgcc |
“MdoD”
|
Seq ID no.4 |
atgaacaacaacgatctgtttcaggcgagccgccgccgctttctggcgcagctgggcggcctgaccgtggcgggcatgctggggcccagcctgctgaccccgcgccgcgcgaccgcggcgcaggcc |
“TorA”
|
Seq ID no.5 |
atgagcggcctgccgctgattagccgccgccgcctgctgaccgcgatggcgctgagcccgctgctgtggcagatgaacaccgcgcatgcc |
"FhuD"
|
Seq ID no.6 |
atgaaaaaaatttggctggcgctggcgggcctggtgctggcgtttagcgctagcgcc |
"DsbA"
|
Seq ID no.7 |
atgaaaaaaaccgcgattgcgattgcggtggcgctggcgggctttgcgaccgtggcgcaggcc |
"OmpA"
|
Plasmid Name
|
Secretion Pathway
|
Total base pair (bp)
|
Signal Sequence
|
pAEV01 |
SEC |
5,784 |
atgaaaattaaaaccggcgcgcgcattctggcgctgagcgcgctgaccaccatgatgtttagcgctagcgcgctggcc |
pAEV02 |
TAT+SEC |
5,784 |
atgaccattaactttcgccgcaacgcgctgcagctgagcgtggcggcgctgtttagcagcgcgtttatggcgaacgcc |
pAEV03 |
TAT+SEC |
5,802 |
atggatcgccgccgctttattaaaggcagcatggcgatggcggcggtgtgcggcaccagcggcattgctagcctgtttagccaggcggcgtttgcc |
pAEV04 |
TAT |
5,832 |
atgaacaacaacgatctgtttcaggcgagccgccgccgctttctggcgcagctgggcggcctgaccgtggcgggcatgctggggcccagcctgctgaccccgcgccgcgcgaccgcggcgcaggcc |
pAEV05 |
TAT+SEC |
5,796 |
atgagcggcctgccgctgattagccgccgccgcctgctgaccgcgatggcgctgagcccgctgctgtggcagatgaacaccgcgcatgcc |
pAEV06 |
SRP |
5,763 |
atgaaaaaaatttggctggcgctggcgggcctggtgctggcgtttagcgctagcgcc |
pAEV07 |
SEC |
5,769 |
atgaaaaaaaccgcgattgcgattgcggtggcgctggcgggctttgcgaccgtggcgcaggcc |
Thus, the present invention utilizes the
power of novel signal peptides whose nucleotide sequence
has been optimized to support efficient translation. These
sequences are chosen such that they can direct the protein
of interest to different cellular compartments of the E.
coli cell. Hence, this invention offers a platform of
seven different signal peptides that can be tested for
determining the best combination possible for secretory
expression of the protein of interest.
SEQUENCE LISTING
GENERAL INFORMATION :
NUMBER OF SEQUENCES: 7
INFORMATION FOR SEQ ID NO : 1:
SEQUENCE CHARACTERISTICS:
LENGTH: 78 BASE PAIRS
TYPE: NUCLEIC ACID
MOLECULE TYPE: DNA
SEQUENCE DESCRIPTION: SEQ ID NO:1:
atgaaaatta aaaccggcgc gcgcattctg
gcgctgagcg cgctgaccac catgatgttt 60
agcgctagcg cgctggcc 78
INFORMATION FOR SEQ ID NO : 2:
SEQUENCE CHARACTERISTICS:
LENGTH: 78 BASE PAIRS
TYPE: NUCLEIC ACID
MOLECULE TYPE: DNA
SEQUENCE DESCRIPTION: SEQ ID NO:2:
atgaccatta actttcgccg caacgcgctg
cagctgagcg tggcggcgct gtttagcagc 60
gcgtttatgg cgaacgcc 78
INFORMATION FOR SEQ ID NO : 3:
SEQUENCE CHARACTERISTICS:
LENGTH: 96 BASE PAIRS
TYPE: NUCLEIC ACID
MOLECULE TYPE: DNA
SEQUENCE DESCRIPTION: SEQ ID NO:3:
atggatcgcc gccgctttat taaaggcagc
atggcgatgg cggcggtgtg cggcaccagc 60
ggcattgcta gcctgtttag ccaggcggcg tttgcc 96
INFORMATION FOR SEQ ID NO : 4:
SEQUENCE CHARACTERISTICS:
LENGTH: 126 BASE PAIRS
TYPE: NUCLEIC ACID
MOLECULE TYPE: DNA
SEQUENCE DESCRIPTION: SEQ ID NO:4:
atgaacaaca acgatctgtt tcaggcgagc
cgccgccgct ttctggcgca gctgggcggc 60
ctgaccgtgg cgggcatgct ggggcccagc
ctgctgaccc cgcgccgcgc gaccgcggcg 120
caggcc 126
INFORMATION FOR SEQ ID NO : 5:
SEQUENCE CHARACTERISTICS:
LENGTH: 90 BASE PAIRS
TYPE: NUCLEIC ACID
MOLECULE TYPE: DNA
SEQUENCE DESCRIPTION: SEQ ID NO:5:
atgagcggcc tgccgctgat tagccgccgc
cgcctgctga ccgcgatggc gctgagcccg 60
ctgctgtggc agatgaacac cgcgcatgcc 90
INFORMATION FOR SEQ ID NO : 6:
SEQUENCE CHARACTERISTICS:
LENGTH: 57 BASE PAIRS
TYPE: NUCLEIC ACID
MOLECULE TYPE: DNA
SEQUENCE DESCRIPTION: SEQ ID NO:6:
atgaaaaaaa tttggctggc gctggcgggc
ctggtgctgg cgtttagcgc tagcgcc 57
INFORMATION FOR SEQ ID NO : 7:
SEQUENCE CHARACTERISTICS:
LENGTH: 63 BASE PAIRS
TYPE: NUCLEIC ACID
MOLECULE TYPE: DNA
SEQUENCE DESCRIPTION: SEQ ID NO:7:
atgaaaaaaa ccgcgattgc gattgcggtg
gcgctggcgg gctttgcgac cgtggcgcag 60
gcc 63