U.S. patent application number 11/358197 was filed with the patent office on 2006-08-03 for methods for enhancing expression of recombinant proteins.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Chung Chan, Tracey A. Pownder.
Application Number | 20060172424 11/358197 |
Document ID | / |
Family ID | 34797947 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060172424 |
Kind Code |
A1 |
Chan; Chung ; et
al. |
August 3, 2006 |
Methods for enhancing expression of recombinant proteins
Abstract
The present invention provides two novel E. coli synthetic
promoters, Syn 1 and Syn 2. Syn 1 and Syn 2 are two tight regulated
synthetic promoters that control uninduced, leaky expression of
proteins that are toxic and interfere with production of
recombinant protein from an E. coli expression system.
Inventors: |
Chan; Chung; (Sammamish,
WA) ; Pownder; Tracey A.; (Bellevue, WA) |
Correspondence
Address: |
Shelby J. Walker;ZymoGenetics, Inc.
Patent Department
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
34797947 |
Appl. No.: |
11/358197 |
Filed: |
February 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11054063 |
Feb 9, 2005 |
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11358197 |
Feb 21, 2006 |
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11008853 |
Dec 10, 2004 |
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11054063 |
Feb 9, 2005 |
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60529412 |
Dec 12, 2003 |
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Current U.S.
Class: |
435/471 |
Current CPC
Class: |
C12N 15/67 20130101;
C12N 15/70 20130101; C12N 15/72 20130101; C07K 14/54 20130101; C07K
14/245 20130101 |
Class at
Publication: |
435/471 |
International
Class: |
C12N 15/74 20060101
C12N015/74 |
Claims
1. A tightly regulated synthetic promoter comprising a first lac
operator and a second lac operator, wherein the first lac operator
is positioned at +13 with respect to the transcriptional start site
and the second lac operator is positioned between the -10 and -35
sites.
2. The tightly regulated synthetic promoter of claim 1, wherein the
promoter further comprises an UP element upstream from the second
lac operator.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 11/054,063, filed Feb. 9, 2005, which is a continuation-in-part
of U.S. application Ser. No. 11/008,853, filed Dec. 10, 2004, which
claims the benefit of U.S. Provisional Application Ser. No.
60/529,412 filed Dec. 12, 2003, all of which applications are
herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The increased availability and identification of genes from
human and other genomes has led to an increased need for efficient
expression of recombinant proteins. The expression of proteins in
bacteria is by far the most widely used approach for the production
of cloned genes. For many reasons, expression in bacteria is
preferred to expression in eukaryotic cells. For example, bacteria
are much easier to grow than eukaryotic cells. More specifically,
the availability of a wealth of sophisticated molecular genetic
tools and thousands of mutants make E. coli, as an expression host,
extremely useful for protein production. However, the high-level
production of functional proteins in E. coli., especially those
from eukaryotic sources has often been difficult. Inefficient
translation initiation is the most common reason for poor
expression (Schoner, B. E., et al., Methods Enzymol. 185:94-103,
1990).
[0003] Initiation sites where the enzyme binds to specific
nucleotides sequences are known as promoters. By determining the
nucleotide sequences of protected regions from numerous E. coli
genes, a consensus sequence for the E. coli promoter has been
identified. The most conserved sequence is a hexamer (-10 element)
centered at about the -10 position from the transcription
initiation site. The consensus sequence is TATAAT (SEQ ID NO:).
Upstream sequences around -35 also have a region of sequence
similarity, TTGACA (-35 element; SEQ ID NO:), which is most evident
in efficient promoters. The distance separating the -35 and -10
elements is between 16 and 18 base pairs in 90% of promoters. The
distance is critical for maintaining necessary structural
conformation of the two sites for RNA polymerase binding.
[0004] Regulation of promoter regions in prokaryotic genes contain
multiple binding sites for transcription factors. Interactions
between and among regulatory proteins bound to promoter regions
produce both positive and negative synergistic effects on gene
expression. Regulation of lac operon of E. coli has been studied
extensively and has become a model for negative control of gene
expression at the level of transcription. (See, e.g., Muller-Hill
B., Prog Biophys Mol. Biol., 30(2-3):227-52, 1975; and Sauer R T,
Structure 4(3):219-22, 1996.) Transcription of the lac operon in
Escherichia coli is repressed by the binding of Lac repressor
(LacR) to lac operator O1, a pseudo-palindromic sequence centered
11 base pairs downstream of the transcription start. Repressors
appear to inhibit transcription by either steric hindrance
preventing the binding of DNA polymerase to the promoter, or by
preventing the transition of the closed state of the promoter to
the open state. The lac repressor exists as a tetramer, and full
repression of the wild-type lac promoter by wild-type LacR requires
the presence of at least two other operator sequences that must not
only be in close proximity to the lac operator O1, i.e. located at
base pair 401 and 92 for the auxiliary operators O2 and O3,
respectively, but the operator sequences must also be present on
the same side of the DNA helix. LacR mutants lacking the C-terminal
heptad repeat are only capable of dimer formation, yet still
repress, albeit at a much reduced level. Repression of the lac
promoter by the mutants is comparable to repression by tetrameric
LacR when both auxiliary operators are not present. For a general
review, see, Muller-Hill B. Curr Opin Microbiol. 1(2):145-51,
1998.
[0005] Ideal promoter systems for overproducing recombinant
proteins in E. coli should: (1) provide strong expression of cloned
genes; (2) function only under inducing conditions; and (3) have a
simple method of induction so that large-scale operation is
possible. However, while the available repertoire of E. coli
expression systems usually produce high levels of the corresponding
cloned gene products, in many cases synthesize substantial levels
of cloned gene products in uninduced or repressed conditions.
Generally, these systems include controllable expression vectors
based on the strong inducible promoter, tac and T7. Both promoter
systems have only one lac operon site to regulate the target gene
expression. Full repression of these two strong promoters has been
difficult to achieve. Furthermore, the leaky basal expression of
some toxic proteins can have a detrimental impact on cell growth
and viability.
[0006] Despite advances in the expression of recombinant proteins
in bacterial hosts, there exists a need for improved methods for
higher yields for protein production.
SUMMARY OF THE INVENTION
[0007] The present invention provides two novel E. coli synthetic
promoters, Syn 1 (SEQ ID NO:1) and Syn 2 (SEQ ID NO:2). Syn 1 and
Syn 2 are two tight regulated synthetic promoters that control
uninduced, leaky expression of proteins that are toxic and
interfere with production of recombinant protein from an E. coli
expression system. Thus, these promoters provide an advantage by
controlling basal expression of target genes. Specifically, these
promoters provide a sterically repressed promoter in which an
additional lac operator site is inserted between the -10 and -35
elements, where it can interfere sterically with binding of RNA
Polymerase and thereby prevent the assembly of a poised
transcriptional complex.
DESCRIPTION OF THE INVENTION
[0008] The most common biotechnological use of a promoter is to
direct synthesis of a heterologous protein. However, a strong
promoter is not necessarily optimal. The ability to turn the
promoter off is as important as the promoter's strength. Promoters
that are constitutively "on" are not generally used for protein
expression in E. coli because of difficulties in the creation and
maintenance of the plasmid construct. Two important reasons for
this are that (i) production of heterologous protein in high yield
is very draining to the resources of the cell, and (ii) many genes
encode "toxic proteins". These factors can result in a strong
selection against cells carrying such plasmid constructs and may be
the cause of plasmid instability or even the inability to transform
cells successfully. The strongest promoters may result in the
accumulation of heterologous protein to more than 50% of the total
cell protein. This reduces the cell's ability to produce proteins
needed for growth. The phenomenon of foreign proteins' toxicity is
well known, even though it is still poorly understood. Some general
rules have been established, however, that can help predict the
toxicity of given proteins. As an example, DNA-binding proteins
tend to be highly toxic when overexpressed in E. coli, as are
proteins that disrupt vital functions such as electron transport or
membrane integrity. These two factors, toxicity and the draining of
cell resources, tend to select against cells carrying plasmids with
genes expressed constitutively from strong promoters. To get around
this problem, an inducible promoter is typically used to produce
recombinant protein in E. coli. Such promoters remain off until a
specific signal is delivered to the cell. Under control of this
signal, the promoter is turned on only when the expression of the
gene is desired. See e.g. Goldstein M A, Doi R H. Prokaryotic
promoters in biotechnology. Biotechnol Annu Rev. 1995;1:105-28; see
also Joseph M. Fernandez, James Hoeffler Gene Expression Systems:
Using Nature for the Art of Expression, Published by Academic Press
(January 1999) pgs. 45-64.
[0009] In 1982, de Boer and his colleagues reported the development
of a hybrid trp/lac promoter. They showed that the -35 region of
trp promoter in combination with the -10 region of lac promoter,
separated by a 16-base pair spacing, resulted in a promoter
stronger than either promoter alone. They designated this hybrid
promoter system as the tac promoter. See e.g. de Boer H A, Comstock
L J, Vasser M., "The tac promoter: a functional hybrid derived from
the trp and lac promoters" Proc. Natl. Acad. Sci. 80(1):21-5
(1983); see also U.S. Pat. No. 4,551,433; see also Amann E, Brosius
J, Ptashne M, "Vectors bearing a hybrid trp-lac promoter useful for
regulated expression of cloned genes in Escherichia coli" Gene
25(2-3):167-78 (1983). However, all known inducible promoter
systems have a residual level of activity or "leakiness" which
leads to the inappropriate transcription and expression of the gene
being cloned under the control of the promoter. In most cases, this
is not a problem because the gene product being produced is well
tolerated by the cell, i.e., the gene product is non-toxic.
However, in instances where the gene product being produced is
toxic or even lethal to the cell, even these small amounts of
expression can be detrimental. In fact, there are certain toxic
genes that have been characterized as "unclonable" because they are
unstable in any cloning vector.
[0010] The present invention addresses and overcomes the problem of
promoter leakiness by providing two novel E. coli synthetic
promoters, Syn 1 (SEQ ID NO:1) and Syn 2 (SEQ ID NO:2). The present
invention is further described below. However, the following
definitions are provided to facilitate understanding of the
invention.
[0011] As used herein, the term "affinity tag" is used herein to
denote a polypeptide segment that can be attached to a second
polypeptide to provide for purification or detection of the second
polypeptide or provide sites for attachment of the second
polypeptide to a substrate. In principal, any peptide or protein
for which an antibody or other specific binding agent is available
can be used as an affinity tag. Affinity tags include a
poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075
(1985); Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione
S transferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu
affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA
82:7952 (1985)), substance P, FLAG peptide (Hopp et al.,
Biotechnology 6:1204 (1988)), streptavidin binding peptide, or
other antigenic epitope or binding domain. See, in general, Ford et
al., Protein Expression and Purification 2:95 (1991). DNA molecules
encoding affinity tags are available from commercial suppliers
(e.g., Pharmacia Biotech, Piscataway, N.J.).
[0012] The terms "amino-terminal" or "N-terminal" and
"carboxyl-terminal" or "C-terminal" are used herein to denote
positions within polypeptides. Where the context allows, these
terms are used with reference to a particular sequence or portion
of a polypeptide to denote proximity or relative position. For
example, a certain sequence positioned carboxyl-terminal to a
reference sequence within a polypeptide is located proximal to the
carboxyl terminus of the reference sequence, but is not necessarily
at the carboxyl terminus of the complete polypeptide.
[0013] The term "ARS" refers to a yeast autonomous replicating
sequence.
[0014] The term "CEN6" refers to a yeast centromeric sequence.
[0015] A "cloning vector" is a nucleic acid molecule, such as a
plasmid, cosmid, or bacteriophage, which has the capability of
replicating autonomously in a host cell. Cloning vectors typically
contain one or a small number of restriction endonuclease
recognition sites that allow insertion of a nucleic acid molecule
in a determinable fashion without loss of an essential biological
function of the vector, as well as nucleotide sequences encoding a
marker gene that is suitable for use in the identification and
selection of cells transformed with the cloning vector. Marker
genes typically include genes that provide resistance to
antibiotic.
[0016] "Complementary DNA (cDNA)" is a single-stranded DNA molecule
that is formed from an mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand. The term "cDNA" also
refers to a clone of a cDNA molecule synthesized from an RNA
template.
[0017] The term "complement of a nucleic acid molecule" refers to a
nucleic acid molecule having a complementary nucleotide sequence
and reverse orientation as compared to a reference nucleotide
sequence.
[0018] The term "contig" denotes a nucleic acid molecule that has a
contiguous stretch of identical or complementary sequence to
another nucleic acid molecule. Contiguous sequences are said to
"overlap" a given stretch of a nucleic acid molecule either in
their entirety or along a partial stretch of the nucleic acid
molecule.
[0019] A "core promoter" contains essential nucleotide sequences
for promoter function, including the start of transcription. By
this definition, a core promoter may or may not have detectable
activity in the absence of specific sequences that may enhance the
activity or confer tissue specific activity.
[0020] An "enhancer" is a type of regulatory element that can
increase the efficiency of transcription, regardless of the
distance or orientation of the enhancer relative to the start site
of transcription.
[0021] The term "expression" refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0022] An "expression vector" is a nucleic acid molecule encoding a
gene that is expressed in a host cell. Typically, an expression
vector comprises a transcriptional promoter, a gene, an origin of
replication, a selectable marker, and a transcriptional terminator.
Gene expression is usually placed under the control of a promoter,
and such a gene is said to be "operably linked to" the promoter.
Similarly, a regulatory element and a core promoter are operably
linked if the regulatory element modulates the activity of the core
promoter. An expression vector may also be known as an expression
construct.
[0023] A "fusion protein" is a hybrid protein expressed by a
nucleic acid molecule comprising nucleotide sequences of at least
two genes.
[0024] "Heterologous DNA" refers to a DNA molecule, or a population
of DNA molecules, that does not exist naturally within a given host
cell. DNA molecules heterologous to a particular host cell may
contain DNA derived from the host cell species (i.e., endogenous
DNA) so long as that host DNA is combined with non-host DNA (i.e.,
exogenous DNA). For example, a DNA molecule containing a non-host
DNA segment encoding a polypeptide operably linked to a host DNA
segment comprising a transcription promoter is considered to be a
heterologous DNA molecule. Conversely, a heterologous DNA molecule
can comprise an endogenous gene operably linked with an exogenous
promoter. As another illustration, a DNA molecule comprising a gene
derived from a wild-type cell is considered to be heterologous DNA
if that DNA molecule is introduced into a mutant cell that lacks
the wild-type gene.
[0025] A "heterologous" peptide or polypeptide means a peptide or
polypeptide encoded by a non-host DNA molecule.
[0026] An "isolated nucleic acid molecule" is a nucleic acid
molecule that is not integrated in the genomic DNA of an organism.
For example, a DNA molecule that encodes a growth factor that has
been separated from the genomic DNA of a cell is an isolated DNA
molecule. Another example of an isolated nucleic acid molecule is a
chemically-synthesized nucleic acid molecule that is not integrated
in the genome of an organism. A nucleic acid molecule that has been
isolated from a particular species is smaller than the complete DNA
molecule of a chromosome from that species.
[0027] "Linear DNA" denotes non-circular DNA molecules with free 5'
and 3' ends. Linear DNA can be prepared from closed circular DNA
molecules, such as plasmids, by enzymatic digestion or physical
disruption.
[0028] "Nucleic acid" or "nucleic acid molecule" refers to
polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic
acid (RNA), oligonucleotides, fragments generated by the polymerase
chain reaction (PCR), and fragments generated by any of ligation,
scission, endonuclease action, and exonuclease action. Nucleic acid
molecules can be composed of monomers that are naturally-occurring
nucleotides (such as DNA and RNA), or analogs of
naturally-occurring nucleotides (e.g., .alpha.-enantiomeric forms
of naturally-occurring nucleotides), or a combination of both.
Modified nucleotides can have alterations in sugar moieties and/or
in pyrimidine or purine base moieties. Sugar modifications include,
for example, replacement of one or more hydroxyl groups with
halogens, alkyl groups, amines, and azido groups, or sugars can be
functionalized as ethers or esters. Moreover, the entire sugar
moiety can be replaced with sterically and electronically similar
structures, such as aza-sugars and carbocyclic sugar analogs.
Examples of modifications in a base moiety include alkylated
purines and pyrimidines, acylated purines or pyrimidines, or other
well-known heterocyclic substitutes. Nucleic acid monomers can be
linked by phosphodiester bonds or analogs of such linkages. Analogs
of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like. The term "nucleic acid molecule" also includes so-called
"peptide nucleic acids," which comprise naturally occurring or
modified nucleic acid bases attached to a polyamide backbone.
Nucleic acids can be either single stranded or double stranded.
[0029] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides."
[0030] A "promoter" is a nucleotide sequence that directs the
transcription of a structural gene. Typically, a promoter is
located in the 5' non-coding region of a gene, proximal to the
transcriptional start site of a structural gene. Sequence elements
within promoters that function in the initiation of transcription
are often characterized by consensus nucleotide sequences. These
promoters include, for example, but are not limited to,
IPTG-inducible promoters, bacteriophage T7 promoters and
bacteriophage .lamda.P.sub.L. See Sambrook et al., Molecular
Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 2001. A typical
prokaryotic promoter will have three components, consisting of
consensus sequences at -35 and -10 with a sequence of between 16
and 19 nucleotides between them (Lisset, S. and Margalit, H.,
Nucleic Acids Res. 21: 1512, 1993). Promoters of this sort include
the lac, trp, trp-lac (tac) and trp-lac(trc) promoters. If a
promoter is an inducible promoter, then the rate of transcription
increases in response to an inducing agent. In contrast, the rate
of transcription is not regulated by an inducing agent if the
promoter is a constitutive promoter. Repressible promoters are also
known.
[0031] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein. Proteins are
defined herein in terms of their amino acid backbone structures;
substituents such as carbohydrate groups and non-peptidic groups
are generally not specified, but may be present nonetheless.
[0032] An "isolated polypeptide" is a polypeptide that is
essentially free from contaminating cellular components, such as
carbohydrate, lipid, or other proteinaceous impurities associated
with the polypeptide in nature. Typically, a preparation of
isolated polypeptide contains the polypeptide in a highly purified
form, i.e., at least about 80% pure, at least about 90% pure, at
least about 95% pure, greater than 95% pure, or greater than 99%
pure. One way to show that a particular protein preparation
contains an isolated polypeptide is by the appearance of a single
band following sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis of the protein preparation and Coomassie Brilliant
Blue staining of the gel. However, the term "isolated" does not
exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0033] The term "RBS II" refers to a synthetic ribosomal binding
site sequence.
[0034] A "recombinant host" is a cell that contains a heterologous
nucleic acid molecule, such as a cloning vector or expression
vector.
[0035] A "regulatory element" is a nucleotide sequence that
modulates the activity of a core promoter. For example, a
eukaryotic regulatory element may contain a nucleotide sequence
that binds with cellular factors enabling transcription exclusively
or preferentially in particular cells, tissues, or organelles.
These types of regulatory elements are normally associated with
genes that are expressed in a "cell-specific," "tissue-specific,"
or "organelle-specific" manner. Bacterial promoters have regulatory
elements that bind and modulate the activity of the core promoter,
such as operator sequences that bind activator or repressor
molecules.
[0036] The term "RNAP" refers to RNA polymerase.
[0037] The term "rrnB" refers to an E. coli ribosomal RNA
operon.
[0038] The term "secretory signal sequence" denotes a DNA sequence
that encodes a peptide (a "secretory peptide") that, as a component
of a larger polypeptide, directs the larger polypeptide through a
secretory pathway of a cell in which it is synthesized. The larger
polypeptide is commonly cleaved to remove the secretory peptide
during transit through the secretory pathway.
[0039] The term "tac promoter" refers to the hybrid E. coli
promoter which contains the -10 region of the lac UV5 promoter and
the -35 region of the trp promoter.
[0040] The term "URA3" refers to the yeast orotidine-5'-phosphate
decarboxylase gene.
[0041] Due to the imprecision of standard analytical methods,
molecular weights and lengths of polymers are understood to be
approximate values. When such a value is expressed as "about" X or
"approximately" X, the stated value of X will be understood to be
accurate to .+-.10%.
Design of Syn 1 and Syn 2
[0042] The present invention addressed the need for preventing
leaky expression of toxic genes in an E. coli expression system.
The present invention is based on mechanistic information about the
functioning of the lac promoter. This information was used to
design a new promoter with a surprisingly improved efficiency of
the repression of expression of toxic genes in an expression
system. This high level of repression can be utilized to reduce or
remove overproduction of toxic proteins in E. coli.
[0043] The tac promoter system has a lac operator centered at +13
with respect to the transcriptional start site. Binding of the lac
repressor to the operator blocks transcriptional elongation.
However, this interaction does not prevent the formation of a
transcriptionally competent complex. In vitro footprinting and
cross-linking studies demonstrated that the lac repressor and RNAP
could occupy the promoter/operator region simultaneously. This may
contribute to inadequate repression of promoter activity. See e.g.
Straney S B, Crothers D M., "Lac repressor is a transient
gene-activating protein" Cell. 1987 Dec. 4;51(5):699-707; see also
Lee J, Goldfarb A. "Lac repressor acts by modifying the initial
transcribing complex so that it cannot leave the promoter" Cell.
1991 Aug. 23;66(4):793-8; see also Mosrin-Huaman, C. "Translocation
of Escherichia coli RNA polymerase against a protein roadblock in
vivo highlights a passive sliding mechanism for transcript
elongation" Mol Microbiol. 2004 March;51(5):1471-81.
[0044] The present invention overcomes this problem by the addition
of a second lac operator at a site where binding of the lac
repressor would sterically block access of RNAP to the promoter.
Syn 1 was constructed with two lac operator sites, one in the
original location found in the tac promoter and the other located
between the -10 and -35 sites. The expression vector containing Syn
1 promoter was derived from the expression vector containing the
tac promoter, pTAP237, and was named pTAP399 (described in Example
2 below).
[0045] An attempt was made to improve the properties of Syn 1 by
increasing its strength. The A+T rich region upstream of -40 in the
rRNA rrnB P1 promoter, the UP element, is known to increase
transcription 30- to 70-fold by binding RNAP. See e.g. Estrem S T,
"Identification of an UP element consensus sequence for bacterial
promoters" Proc Natl Acad Sci USA. 1998 Aug. 18;95(17):9761-6.
Similar A+T rich sequences have been identified upstream of many
bacterial and phage promoters, but they are not as highly conserved
as -10 and -35 elements. A consensus UP element sequence was
generated by selection in vitro for upstream sequences that promote
rapid RNAP binding to rrnB P1 promoter, followed by an in vivo
screen for high promoter activity. The consensus UP element
consists of alternating A- and T-tracts. Gourse and colleagues
found that the effects of UP elements on transcription correlated
generally with the degree of similarity to the UP element consensus
sequence. Id; see also Ross W., "Escherichia coli promoters with UP
elements of different strengths: modular structure of bacterial
promoters" J. Bacteriol. 1998 October;180(20):5375-83. Thus, Syn 2
was constructed with two lac operator sites, as in Syn 1. However,
Syn 2 contains the UP element in the rRNA rrnB P1 promoter. The
expression vector containing Syn 2 promoter was derived from the
expression vector containing Syn 1, pTAP399, and was named pTAP413
(described in Example 3 below).
[0046] Accordingly, the present invention is based on two novel
promoters, Syn 1 and Syn 2, both of which contain an additional lac
operator between the -10 and -35 elements of the tac promoter. Syn
2 also has an UP element from the rrnB P1. The use of these two
novel promoters resulted in more efficient repressed transcription
without affecting the expression level of a gene after induction.
Syn 2, as compared with Syn 1 has no additional impact on promoter
strength, but does have a further increased efficiency of
repression.
Expression Vectors
[0047] An expression vector can be introduced into host cells using
a variety of standard techniques including calcium phosphate
transfection, liposome-mediated transfection,
microprojectile-mediated delivery, electroporation, and the like.
The transfected cells can be selected and propagated to provide
recombinant host cells that comprise the expression vector stably
integrated in the host cell genome. Techniques for introducing
vectors into eukaryotic cells and techniques for selecting such
stable transformants using a dominant selectable marker are
described, for example, by Ausubel (1995) and by Murray (ed.), Gene
Transfer and Expression Protocols (Humana Press 1991).
Selectable Marker Genes
[0048] A wide variety of selectable marker genes are available
(see, for example, Kaufman, Meth. Enzymol. 185:487 (1990); Kaufman,
Meth. Enzymol. 185:537 (1990)). In the present context, a suitable
selectable marker is "titratable," in that the resistance of a cell
to a high dose of toxic drug will be related to the number of
selectable marker proteins produced by the cell. This
characteristic is lacking when the selectable marker is an enzyme
that can neutralize a high number of toxic drug molecules per
enzyme.
[0049] As one skilled in the art would know selectable markers for
bacterial expression include markers that confer antibiotic
resistance. Antibiotics such as ampicillin, tetracycline,
chloramphenicol, and kanamycin are commonly used. An expression
vector can carry more than one such antibiotic resistance gene. See
also, Sambrook et al., ibid. Other selectable markers can be used,
as well, and in some cases it may be preferable to make use of a
selectable marker that does not require the use of an antibiotic.
One example of this sort of selectable marker uses the hok/sok
system from plasmid R1. The hok gene encodes the toxic Hok protein
of 52 amino acids and the sok gene encodes an antisense RNA, which
is complementary to the hok mRNA leader sequence. This selectable
marker is known to one skilled in the art and is described in more
detail by Gerdes, K. et al., Genetic Engineering, 19:49-61,
1997.
Suitable E. coli Hosts
[0050] Suitable prokaryotic hosts include E. coli strains of E.
coli include BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I,
DH5, DH5I, DH5IF', DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101,
JM101, JM105, JM119, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451,
and ER1647 (see, for example, Brown (ed.), Molecular Biology Labfax
(Academic Press 1991)). However, suitable hosts are not limited to
these strains.
Bacterial Expression
[0051] When expressing a polypeptide in bacteria such as E. coli,
the polypeptide may be retained in the cytoplasm, typically as
insoluble granules, or may be directed to the periplasmic space by
a bacterial secretion sequence. In the former case, the cells are
lysed, and the granules are recovered and denatured using, for
example, guanidine isothiocyanate or urea. The denatured
polypeptide can then be refolded and dimerized by diluting the
denaturant, such as by dialysis against a solution of urea and a
combination of reduced and oxidized glutathione, followed by
dialysis against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic space in a
soluble and functional form by disrupting the cells (by, for
example, sonication or osmotic shock) to release the contents of
the periplasmic space and recovering the protein, thereby obviating
the need for denaturation and refolding.
[0052] Methods for expressing proteins in prokaryotic hosts are
well-known to those of skill in the art (see, for example, Williams
et al., "Expression of foreign proteins in E. coli using plasmid
vectors and purification of specific polyclonal antibodies," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
page 15 (Oxford University Press 1995), Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc.
1995), and Georgiou, "Expression of Proteins in Bacteria," in
Protein Engineering: Principles and Practice, Cleland et al.
(eds.), page 101 (John Wiley & Sons, Inc. 1996)). Standard
methods for introducing expression vectors into bacterial cells are
provided, for example, by Ausubel (1995).
[0053] The invention is further illustrated by the following
non-limiting examples.
EXAMPLE 1
Construction of Plasmid pTAP237
[0054] Plasmid pTAP237 was generated by inserting a PCR-generated
linker into the SmaI site of pTAP186 by homologous recombination.
Plasmid pTAP186 was derived from the plasmids pRS316 (a
Saccharomyces cerevisiae shuttle vector) and pMAL-c2, an E. coli
expression plasmid derived from pKK223-3 and comprising the tac
promoter and the rmB terminator. Plasmid pTAP186 contains a
kanamycin resistance gene in which the Sma I site has been
destroyed and has NotI and SfiI sites flanking the yeast ARS-CEN6
and URA3 sequences, facilitating their removal from the plasmid by
digestion with NotI. The PCR-generated linker replaced the
expression coupler sequence in pTAP186 with the synthetic RBS II
sequence. It was prepared from 100 pmoles each of oligonucleotides
zc29,740 (SEQ ID NO: 3) and zc29,741 (SEQ ID NO: 4), and
approximately 5 pmoles each of oligonucleotides zc29,736 (SEQ ID
NO: 5) and zc29,738 (SEQ ID NO: 6). These oligonucleotides were
combined by PCR for ten cycles of 94.degree. C. for 30 seconds,
50.degree. C. for 30 seconds, and 72.degree. C. for 30 seconds,
followed by 4.degree. C. soak. The resulting PCR products were
concentrated by precipitation with two times the volume of 100%
ethanol. The pellet was resuspended in 10 .mu.L water to be used
for recombining into the recipient vector pTAP186 digested with
SmaI to produce the construct containing the synthetic RBS II
sequence. Approximately 1 .mu.g of the PCR-generated linker and 100
ng of pTAP186 digested with SmaI were mixed together and
transformed into competent yeast cells (S. cerevisiae
SF838-9D.alpha.). The yeast was then plated onto -URA DS plates and
left at room temperature for about 72 hours.
[0055] The Ura+ yeast transformants from a single plate were
resuspended in 2-3 ml H.sub.2O and spun briefly to pellet the yeast
cells. The cell pellet was resuspended in 1 ml of lysis buffer (2%
Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA).
Five hundred microliters of the lysis mixture was added to an
Eppendorf tube containing 300 .mu.l acid washed glass beads and 500
.mu.l phenol-chloroform and vortexed for 1 minute. The mixture was
then spun for 5 minutes in an Eppendorf centrifuge at maximum
speed. Three hundred microliters of the aqueous phase was
transferred to a fresh tube, and the DNA precipitated with 600
.mu.l 100% ethanol, followed by centrifugation for 10 minutes at
4.degree. C. The DNA pellet was resuspended in 100 .mu.l
H.sub.2O.
[0056] One microliter of the recovered DNA was transformed into E.
coli MC1061. Clones were screened by colony PCR as disclosed above
but using 25 cylces and using 20 pmoles each of oligonucleotide
zc29,740 (SEQ ID NO: 3) and zc29,741 (SEQ ID NO: 4). Clones
displaying the correct size band on an agarose gel were subject to
sequence analysis. The correct plasmid was designated pTAP237.
EXAMPLE 2
Construction of Plasmid pTAP399 Containing Syn 1
[0057] The plasmid pTAP399 was derived from pTAP237 (described in
Example 1). The objective of this study was to use mechanistic
information about the functioning of the lac promoter to design a
new promoter with improved efficiency of repression, which could be
employed in overproduction of toxic proteins in E. coli. The tac
promoter system has a lac operator centered at +13 with respect to
the transcriptional start site. Binding of the lac repressor to the
operator blocks transcriptional elongation. However, this
interaction does not prevent the formation of a transcriptionally
competent complex. In vitro footprinting and cross-linking studies
demonstrated that the lac repressor and RNAP could occupy the
promoter/operator region simultaneously. This may contribute to
inadequate repression of promoter activity. .sup.12, 13, 14, 15, 16
In an attempt to circumvent this problem, we decided to add an
additional lac operator at a site where binding of the lac
repressor would sterically block access of RNAP to the promoter.
This design was inspired by the mechanism that regulates the lambda
pL and recA promoters. Syn 1, was constructed with two lac operator
sites, one in the original location found in the tac promoter and
the other located between the -10 and -35 sites. The expression
vector containing Syn 1 promoter was derived from the expression
vector containing the tac promoter, pTAP237, and was named
pTAP399.
[0058] Specifically, the PCR-generated linker replaced the tac
promoter in pTAP237 (described in Example 1) with the synthetic
promoter Syn 1 (SEQ ID NO:1). The Syn 1 promoter has two lac
operator sites. One of lac operator is positioned between the
consensus -10 and -35 element sequences, where it can interfere
sterically with RNA polymerase and thereby prevent assembly of a
poised transcriptional complex. The other lac operator site is
inserted 10 bp downstream from the novel promoter, where can block
the initiation of mRNA synthesis. It was prepared from 100 pmoles
each of oligonucleotide zc42,180 (SEQ ID NO:7) and zc42,167 (SEQ ID
NO:8), and approximately 5 pmoles each of oligonucleotide zc42,181
(SEQ ID NO:9) and zc42,179 (SEQ ID NO:10). These oligonucleotides
were combined by PCR for ten cycles of 94.degree. C. for 30
seconds, 50.degree. C. for 30 seconds, and 72.degree. C. for 30
seconds, followed by 4.degree. C. soak. The resulting PCR products
were concentrated by precipitation with two times the volume of
100% ethanol. The pellet was resuspended in 10 .mu.L water to be
used for recombining into the recipient vector pTAP237 digested
with BseRI to produce the construct containing the synthetic
promoter (Syn 1) sequence. Approximately 1 .mu.g of the
PCR-generated linker and 100 ng of pTAP237 digested with BseR I
were mixed together and transformed into competent SF838-9D.alpha.
yeast cells (S. cerevisiae). The yeast was then plated onto -URA DS
plates and left at 30 C for about 72 hours.
[0059] The Ura+ yeast transformants from a single plate were
resuspended in 2-3 ml H.sub.2O and spun briefly to pellet the yeast
cells. The cell pellet was resuspended in 1 ml of lysis buffer (2%
Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA).
Five hundred microliters of the lysis mixture was added to an
Eppendorf tube containing 300 .mu.l acid washed glass beads and 500
.mu.l phenol-chloroform and vortexed for 1 minute. The mixture was
then spun for 5 minutes in an Eppendorf centrifuge at maximum
speed. Three hundred microliters of the aqueous phase was
transferred to a fresh tube, and the DNA precipitated with 600
.mu.l 100% ethanol, followed by centrifugation for 10 minutes at
4.degree. C. The DNA pellet was resuspended in 100 .mu.l
H.sub.2O.
[0060] One microliter of the recovered DNA was transformed into E.
coli MC1061. Clones were screened by colony PCR as disclosed above
but using 25 cycles and using 20 pmoles each of oligonucleotide
zc42,180 (SEQ ID NO:7) and zc42,167 (SEQ ID NO:8). Clones
displaying the correct size band on an agarose gel were subject to
sequence analysis. The correct plasmid was designated pTAP399 and
was deposited with the American Type Culture Collection in
Manassas, Va. and assigned ATCC Accession No. PTA-5700.
EXAMPLE 3
Construction of Plasmid pTAP413 Containing Syn 2
[0061] The plasmid pTAP413 was derived from pTAP399 (described in
Example 2). The PCR-generated linker replaced the Syn1 (SEQ ID
NO:1) promoter sequence in pTAP399 with the Syn 2 sequence (SEQ ID
NO:2). The Syn 2 promoter contains two lac operons and shares
similar basic structure as Syn 1 promoter. However, Syn 2 contains
a 40 bp long DNA sequence that is located six bases upstream from
-35 element. The sequence of the Syn 2 promoter in this region was
derived from the UP element sequence of the rrnB1P1 promoter. It
was prepared from 100 pmoles each of oligonucleotides zc42,734 (SEQ
ID NO:11) and zc42,733 (SEQ ID NO:12), and approximately 5 pmoles
each of oligonucleotides zc42,721 (SEQ ID NO:13) and zc42,718 (SEQ
ID NO:14). These oligonucleotides were combined by PCR for ten
cycles of 94.degree. C. for 30 seconds, 50.degree. C. for 30
seconds, and 72.degree. C. for 30 seconds, followed by 4.degree. C.
soak. The resulting PCR products were concentrated by precipitation
with two times the volume of 100% ethanol. Pellet was resuspended
in 10 .mu.L water to be used for recombining into the recipient
vector pTAP399 digested with EcoRI to produce the construct
containing the synthetic promoter Syn 2 (SEQ ID NO:2).
Approximately 1 .mu.g of the PCR-generated linker and 100 ng of
pTAP399 digested with EcoRI were mixed together and transformed
into competent SF8389F.alpha. yeast cells (S. cerevisiae). The
yeast was then plated onto -URA DS plates and left at room
temperature for about 72 hours.
[0062] The Ura+ yeast transformants from a single plate were
resuspended in 2-3 ml H.sub.2O and spun briefly to pellet the yeast
cells. The cell pellet was resuspended in 1 ml of lysis buffer (2%
Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA).
Five hundred microliters of the lysis mixture was added to an
Eppendorf tube containing 300 .mu.l acid washed glass beads and 500
.mu.l phenol-chloroform and vortexed for 1 minute. The mixture was
then spun for 5 minutes in an Eppendorf centrifuge at maximum
speed. Three hundred microliters of the aqueous phase was
transferred to a fresh tube, and the DNA precipitated with 600
.mu.l 100% ethanol, followed by centrifugation for 10 minutes at
4.degree. C. The DNA pellet was resuspended in 100 .mu.l
H.sub.2O.
[0063] One microliter of the recovered DNA was transformed into E.
coli MC1061. Clones were screened by colony PCR as disclosed above
but using 25 cycles and using 20 pmoles each of oligonucleotide
zc42,733 (SEQ ID NO:12) and zc42,734 (SEQ ID NO:11). Clones
displaying the correct size band on an agarose gel were subject to
sequence analysis. The correct plasmid was designated pTAP413 and
was deposited at the American Type Culture Collection in Manassas,
Va. assigned ATCC Accession No. PTA-5701.
EXAMPLE 4
Construction of Plasmids pTAP411, pTAP412 and pTAP414 Containing
Different UP Element Sequences
[0064] The plasmids pTAP411, pTAP412 and pTAP414 were all derived
from pTAP399 (described in Example 2). The PCR-generated linker
replaced the Syn1 (SEQ ID NO:1) promoter sequence in pTAP399 with
the Syn 2 sequence (SEQ ID NO:2). Furthermore, PCR-generated
linkers replaced the upstream sequence (from position -40 to -60)
in pTAP399 with different UP element sequences from other sources.
These linkers were prepared from different sets of partially
overlapping oligonucleotides as follows:
A) pTAP411
[0065] pTAP411 contains a Syn 1 with an UP element consensus
sequence (SEQ ID NO:24) and was prepared from 5 pmoles of
oligonucleotides zc42718 (SEQ ID NO:14) and zc42719 (SEQ ID NO:15)
and 100 pmoles of oligonucleotides zc42733 (SEQ ID NO:12) and
zc42734 (SEQ ID NO:11).
B) pTAP412
[0066] pTAP412 contains a Syn 1 with rrnD P1 UP element sequence
(SEQ ID NO:25) and was prepared from 5 pmoles of oligonucleotides
zc42718 (SEQ ID NO:14) and zc42720 (SEQ ID NO:16) and 100 pmoles of
oligonucleotides zc42733 (SEQ ID NO:12) and zc42734 (SEQ ID
NO:11).
C) pTAP414
[0067] pTAP414 contains a Syn 1 with rrnB P2 UP element sequence
(SEQ ID NO:26) and was prepared from 5 pmoles of oligonucleotides
zc42718 (SEQ ID NO: 14) and zc42727 (SEQ ID NO:17) and 100 pmoles
of oligonucleotides zc42733 (SEQ ID NO:12) and zc42734 (SEQ ID
NO:11).
[0068] The oligonucleotides in each set (as described above) were
combined by PCR in ten cycles of 94.degree. C. for 30 seconds,
50.degree. C. for 30 seconds, and 72.degree. C. for 30 seconds,
followed by 4.degree. C. soak. The resulting PCR products were
concentrated by precipitation with two times the volume of 100%
ethanol. The pellet was resuspended in 10 .mu.L H.sub.2O and used
for recombination into the recipient vector, pTAP399, digested with
BseRI to produce constructs containing the Syn 1 promoter sequence
coupled with appropriate UP element. Approximately 1 .mu.g of the
PCR-generated linker and 100 ng of pTAP399 digested with BseRI were
mixed together and transformed into competent yeast cells (S.
cerevisiae). The cells were then plated onto -URA DS plates and
left at room temperature for about 72 hours.
[0069] The Ura+ yeast transformants from a single plate were
resuspended in 2-3 ml H.sub.2O and spun briefly to pellet the yeast
cells. The cell pellet was resuspended in 1 ml of lysis buffer (2%
Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA).
Five hundred microliters of the lysis mixture was added to an
Eppendorf tube containing 300 .mu.l acid washed glass beads and 500
.mu.l phenol-chloroform and vortexed for 1 minute. The mixture was
then spun for 5 minutes in an Eppendorf centrifuge at maximum
speed. Three hundred microliters of the aqueous phase was
transferred to a fresh tube, and the DNA precipitated with 600
.mu.l 100% ethanol, followed by centrifugation for 10 minutes at
4.degree. C. The DNA pellet was resuspended in 100 .mu.l
H.sub.2O.
[0070] One microliter of the recovered DNA was transformed into E.
coli MC1061. DNA was recovered and transformed into E. coli MC1061.
Clones were screened by colony PCR using the reaction conditions
described above but using 25 cycles and 20 pmoles each of
oligonucleotides zc42,180 and zc42,167. Clones displaying a band of
the correct size on an agarose gel were subjected to DNA sequence
analysis. The resulting plasmids were designated pTAP411 (Syn 1
with UP element consensus sequence), pTAP412 (Syn 1 with rrnD P1 UP
element sequence) and pTAP414 (Syn 1 with rrnB P2 UP element
sequence).
EXAMPLE 5
Construction of Expression Constructs Containing Human IL-20 and
Comparison of Promoter Strength
[0071] A series of plasmids was constructed to express the human
IL-20 (SEQ ID NO:18; amino acid sequence SEQ ID NO:19) which was
codon optimized for E. coli. A DNA fragment of human IL-20 was
isolated using PCR with two primers, primer zc29,957 (SEQ ID NO:20)
and primer zc23,993 (SEQ ID NO:21). Primer zc29,957 (SEQ ID NO:20)
contained 41 bp of flanking vector sequence and 24 bp corresponding
to the amino terminus of human IL-20 (SEQ ID NO:18). Primer
zc23,993 (SEQ ID NO:21) contained 38 bp corresponding to the 3' end
of the vector which contained the human IL-20 insert. The PCR
conditions were as follows: 25 cycles at 94.degree. C. for 30
seconds, 50.degree. C. for 30 seconds, and 72.degree. C. for 1
minute; followed by a 4.degree. C. soak. A small aliquot (2-4
.mu.L) of the PCR sample was run on a 1% agarose gel with
1.times.TBE buffer for analysis and the expected 500 bp fragment
was observed. The remaining volume of the 100 .mu.L reaction was
precipitated with 200 .mu.L absolute ethanol. Pellet was
resuspended in 10 .mu.L H.sub.2O and used for recombination into
SmaI cut recipient the six vectors, as described in Examples 1-4
above, to produce the constructs encoding human IL-20.
[0072] All six constructs were then cut with NotI restriction
enzyme (New England Biolabs) to remove the yeast sequence from the
vector backbone, thus streamlining the vector. Ten microliters
plasmid DNA was incubated with 15 .mu.l H.sub.2O, 3 .mu.l buffer 3
(NEB) and 2.0 .mu.l NotI enzyme at 37.degree. C. for one hour. The
reaction was then immediately religated. Seven microliters of the
restriction digest was incubated with 2 .mu.l of 5.times. buffer
and 1 .mu.l of ligase for 30 minutes at room temperature. One
microliter of the ligation reaction was transformed into E. coli.
The transformed bacteria were selected for by plating on LB agar
containing appropriate antibiotic.
[0073] Two colonies from each ligation reaction were selected from
the transformation plates and grown overnight in LB plus
appropriate antibiotic. Plasmid DNA was prepared using QIAprep Spin
Miniprep Kit (Qiagen). Aliquots of the DNA were digested with PvuII
and PstI to confirm the absence of the yeast sequence. Individual
clones harboring the correct expression construct for the human
IL-20 were identified by restriction digest to verify the presence
of the human IL-20 insert. The resulting clones were designated as
pTAP255 (derived from pTAP237), pTAP407 (derived from pTAP399),
pTAP426 (derived from pTAP411), pTAP427 (derived from pTAP412),
pTAP428 (derived from pTAP413) and pTAP429 (derived from pTAP414).
The Not I digested plasmids were re-ligated and transformed into E.
coli host strain W3110.
[0074] Transformation of electrocompetent Ecoli W3110 cells was
done with 1 .mu.l ligated plasmid DNA and 50 .mu.l of W3110 cells
(competent cells were made in-house). The cells were electropulsed
in 0.1 cm cuvettes at 1.75 kV, 25 .mu.F and 100.OMEGA.. Following
electroporation, 250 .mu.l SOC (2% Bacto Tryptone (Difco, Detroit,
Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM
MgCl2, 10 mM MgSO.sub.4, 20 mM glucose) was added to each sample
and outgrown at 37.degree. C. for 1 hour. The entire 250 .mu.l
sample was plated in one aliquot on an LB kanamycin plate [LB broth
(Lennox), 1.8% Bacto Agar (Difco), 25 mg/L kanamycin (Sigma)].
Plates were incubated at 37.degree. C. overnight. Six clones for
each construct were picked for follow-up analysis. They were grown
in 2.0 ml Superbroth II (Becton Dickinson) containing 25 .mu.g/ml
kanamycin at 37.degree. C. overnight. The following day, 1.0 ml of
the overnight digest was used to confirm presence of plasmid DNA.
The Qiagen Spin Miniprep Kit (Qiagen) was used to make plasmid DNA,
following manufacturer's instructions. 1 .mu.l of DNA was digested
with EcoRI (Gibco BRL)/NotI (New England Biolabs) to verify the
correct plasmid restriction digest pattern.
[0075] Induction of cells were done as follows. 37.5 ml of
Superbroth 2+kan 30 .mu.g/ml were inoculated with 375 .mu.l of
starter culture. Cultures grew at 37 C for 1 hour and 50 minutes.
They were then induced with 1 mM IPTG. 12.5 ml of culture was grown
at 37 C and 12.5 ml was grown at 30 C of the expressed protein at
lower temperature. 12.5 mls was not induced for a control. Cultures
were harvested at 3 hours post-induction. 250 .mu.l of culture was
mixed with 250 .mu.l glass beads and 250 .mu.l of Thorner Buffer/5%
dye/5% .beta.ME. Samples were vortexed, then boiled for 5 minutes,
and run on a PAGE gel. 20 .mu.l were loaded per lane on a 4%-12%
NuPAGE Bis-Tris gel (NOVEX). Gels were run in 1.times.MES
buffer.
EXAMPLE 6
Comparison of Gene Expression between the Syn 1 and tac
Promoters
[0076] In order to compare the strength of gene expression between
the promoters of the present invention, the expression level of
human IL-20 was used to compare gene expression driven by the Syn 1
and the tac promoter. pTAP237 and pTAP399 are closely related in
structure except in their promoter region; hence, differences in
the expression of IL-20 should be attributable to differences in
promoter activity. Mature human IL-20 coding sequence (SEQ ID
NO:18) was subcloned into pTAP237 and pTAP399, creating constructs
pTAP255 and pTAP407, respectively (described above). The expression
level of IL-20 after IPTG induction in E. coli transformed with
either vector was then compared. The amount of human IL-20 present
in a lysate from a culture harboring pTAP407 (pTAP399/hIL-20) is
similar to that observed in an extract from a culture harboring
pTAP255 (pTAP237/hIL-20). This demonstrates that these two
promoters have comparable strength, suggesting that insertion of an
additional lac operator between the -35 and -10 regions of the tac
promoter has no negative impact on the promoter strength.
EXAMPLE 7
Basal Expression of Human IL-20 Using tac, Syn 1, Syn 2 and Other
Sterically Repressed Promoters in E. coli
[0077] Numerous studies have indicated that the tac promoter is
leaky. A substantial level of IL-20 was produced in the absence of
induction with IPTG. To address differences in pre-induction
leakage, the expression levels of human IL-20, controlled by the
tac and srp promoters, were compared by Western blot analysis using
an anti-IL20 monoclonal antibody as the probe. Thus, a NuPAGE 4-12%
Bis Tris gel (Invitrogen) was run using 1.times.MES buffer. 2.5
.mu.l of uninduced culture was loaded per lane (5 .mu.l of culture
and buffer). A human IL-20 standard (designated A309F) was loaded
as 25 ng and 50 ng. After the gel was run, the DNA was transferred
to a nitrocellulose membrane via a Novex transfer box and protocol.
The membrane was then blocked in 5% milk and TTBS (160 mM NaCl,
0.1% Tween 20, 20 mM Tris pH7.4) for 30 minutes. It was then
incubated at room temperature with an anti-human IL-20 monoclonal
antibody (designated E0582) as a 1:5000 dilution. The blot was then
washed twice in TTBS for 5-10 minutes each. The washed blot was
incubated at room temperature for one hour in a 1:5000 dilution of
goat anti-mouse antibody (Bio-Rad Laboratories, Hercules, Calif.).
The blot was then washed again in TTBS under the same conditions.
The washed blot was then exposed to ECL reagent (Amersham) and
exposed to film.
[0078] The amount of human IL-20 present in a lysate from a culture
harboring pTAP407 (pTAP399/Syn 1/hIL-20) is approximately five-fold
lower than that observed in a lysate from a culture harboring
pTAP255 (pTAP237/tac/hIL-20). The results confirmed that tac
promoter was leaky and produced substantial levels of IL-20 in
uninduced conditions. Syn 1 promoter was capable to reduced the
IL-20 leaky expression by 80%.
[0079] Syn 1 was altered in an attempt to improve its strength by
inserting different UP elements upstream of the promoter region. We
compared the effects of the UP elements on the expression of IL-20
in E. coli after IPTG induction. The amounts of IL-20 present in
lysates from cultures harboring expression constructs with
different UP element sequences were comparable to that observed in
a lysate from a culture harboring pTAP407 (pTAP399/hIL-20), which
lacks an UP element. This result demonstrates that addition of
different UP element sequences to Syn 1 had no impact on the
promoter strength. However, the expression level of IL-20 under
uninduced conditions was also examined. It was discovered that
different UP element sequences had different impacts on basal
expression of IL-20. Most strikingly, it was observed that Syn 2,
which contains the rnB P1 UP element, was able to suppress the
basal expression of IL-20 to a level that could not be detected by
Western blotting techniques.
EXAMPLE 8
Evaluation of Pre-Induction Leakage of Syn 1 and Syn 2 in DNaseI
Expression Vectors
[0080] To test pre-induction leakage of the promoters of the
present invention, a gene coding for a protein known to be toxic to
E. coli, DnaseI, was cloned into pTAP237, pTAP399 and pTAP413. A
DNA fragment of human DNaseI was isolated using PCR with two
primers, primer zc47,073 (SEQ ID NO:22) and primer zc47,074 (SEQ ID
NO:23). Primer zc47,073 contained 41 bp of flanking vector sequence
and 24 bp corresponding to the amino terminus of human DNaseI.
Primer zc47,074 contained 38 bp corresponding to the 3' end of the
vector which contained the human DNaseI insert. The PCR conditions
were as follows: 30 cycles at 94.degree. C. for 1 min, 50.degree.
C. for 1 min, and 72.degree. C. for 1 minute; followed by a
4.degree. C. soak. A small aliquot (2-4 .mu.L) of the PCR sample
was run on a 1% agarose gel with 1.times.TBE buffer for analysis
and the expected 500 bp fragment was observed. The remaining volume
of the 100 .mu.L reaction was precipitated with 200 .mu.L absolute
ethanol. Approximately 1 .mu.g of the PCR product and 100 ng of
SmaI cut recipient vectors (pTAP237, pTAP399 and pTAP413) were
mixed together and transformed into competent SF8389D.alpha. yeast
cells (S. cerevisiae). The cells were then plated onto -URA DS
plates and left at room temperature for about 72 hours.
[0081] The Ura+ yeast transformants from a single plate were
resuspended in 2-3 ml H.sub.2O and spun briefly to pellet the yeast
cells. The cell pellet was resuspended in 1 ml of lysis buffer (2%
Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA).
Five hundred microliters of the lysis mixture was added to an
Eppendorf tube containing 300 .mu.l acid washed glass beads and 500
.mu.l phenol-chloroform and vortexed for 1 minute. The mixture was
then spun for 5 minutes in an Eppendorf centrifuge at maximum
speed. Three hundred microliters of the aqueous phase was
transferred to a fresh tube, and the DNA precipitated with 600
.mu.l 100% ethanol, followed by centrifugation for 10 minutes at
4.degree. C. The DNA pellet was resuspended in 100 .mu.l
H.sub.2O.
[0082] One microliter of the recovered DNA was transformed into E.
coli DH10B. As expected, the tac-controlled DNaseI expression
construct could not be established in E. coli host DH10B. In
contrast, viable clones could be established with both of the Syn 1
and Syn 2-controlled vectors. Induction of DNaseI expression in the
resulting clones led to cell clumping and lysis in liquid culture,
indicating that active DNase was expressed after IPTG induction and
demonstrating the utility of both Syn 1 and Syn 2 for production of
toxic genes in E. coli.
EXAMPLE 9
Evaluation of Pre-Induction Leakage of Syn 1 and Syn 2 in T7
Bacteriophage 0.7 Gene Expression Vectors
[0083] To test pre-induction leakage of the promoters of the
present invention, a gene coding for a protein known to be toxic to
E. coli, the 0.7 gene, was cloned into promoters of the present
invention. The full length 0.7 gene was subcloned into pTAP237
(under the control of tac promoter), pTAP399 (under the control of
Syn 1 promoter) and pTAP413 (under the Syn 2 promoter). The 0.7
gene was isolated using PCR with two primers, primer zc48069 (SEQ
ID NO:27) and primer zc48070 (SEQ ID NO:28). Primer zc48069
contained 41 bp of flanking vector sequence and 24 bp corresponding
to the amino terminus of T7 bacteriophage 0.7 gene (SEQ ID NO:29).
Primer zc48070 contained 38 bp corresponding to the 3' end of the
vector which contained the T7 bacteriophage 0.7 gene insert. The
PCR conditions were as follows: 30 cycles at 94.degree. C. for 1
min, 50.degree. C. for 1 min, and 72.degree. C. for 1 minute;
followed by a 4.degree. C. soak. A small aliquot (2-4 .mu.L) of the
PCR sample was run on a 1% agarose gel with 1.times.TBE buffer for
analysis and the expected 1 kb fragment was observed. The remaining
volume of the 100 .mu.L reaction was precipitated with 200 .mu.L
absolute ethanol. Approximately 1 .mu.g of the PCR product and 100
ng of SmaI cut recipient vectors pTAP237, pTAP399 and pTAP413
(described in Examples 1, 2 and 3) were mixed together and
transformed into competent SF8389D.alpha. yeast cells (S.
cerevisiae). The cells were then plated onto -URA DS plates and
left at room temperature for about 72 hours. The Ura+ yeast
transformants from a single plate were resuspended in 2-3 ml
H.sub.2O and spun briefly to pellet the yeast cells. The cell
pellet was resuspended in 1 ml of lysis buffer (2% Triton X-100, 1%
SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundred
microliters of the lysis mixture was added to an Eppendorf tube
containing 300 .mu.l acid washed glass beads and 500 .mu.l
phenol-chloroform and vortexed for 1 minute. The mixture was then
spun for 5 minutes in an Eppendorf centrifuge at maximum speed.
Three hundred microliters of the aqueous phase was transferred to a
fresh tube, and the DNA precipitated with 600 .mu.l 100% ethanol,
followed by centrifugation for 10 minutes at 4.degree. C. The DNA
pellet was resuspended in 100 .mu.l H.sub.20.
[0084] One microliter of the recovered DNA was transformed into E.
coli DH10B. Viable clones only could be established in E. coli host
DH10B with the Syn 2-controlled vectors. It demonstrated that the
Syn 2-controlled vector was able to reduce the basal expression of
0.7 gene to levels that would permit isolation of stable
recombinant plasmids.
[0085] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
29 1 101 DNA Artificial Sequence Syn 1 promoter 1 agctgttgac
attgtgagcg gataacaata taatgtgtgg aattgtgagc ggataacaat 60
ttcacacaga attcattaaa gaggagaaat taactcccgg g 101 2 141 DNA
Artificial Sequence Syn 2 promoter 2 gcgccgacat cataacggtt
ctggaaaatt attttaaatt tcctcttgac attgtgagcg 60 gataacaata
taatgtgtgg aattgtgagc ggataacaat ttcacacaga attcattaaa 120
gaggagaaat taactcccgg g 141 3 50 DNA Artificial Sequence
oligonucleotide primer zc29,740 3 ttgacaatta atcatcggct cgtataatgt
gtggaattgt gagcggataa 50 4 42 DNA Artificial Sequence
oligonucleotide primer zc29,741 4 tctgatttaa tctgtatcag gctgaaaatc
ttatctcatc cg 42 5 62 DNA Artificial Sequence oligonucleotide
primer zc29,736 5 gtggaattgt gagcggataa caatttcaca cagaattcat
taaagaggag aaattaactc 60 cc 62 6 63 DNA Artificial Sequence
oligonucleotide primer zc29,738 6 gctgaaaatc ttatctcatc cgccaaaaca
cccgggagtt aatttctcct ctttaatgaa 60 ttc 63 7 50 DNA Artificial
Sequence oligonucleotide primer zc42,180 7 gcgccgacat cataacggtt
ctggcaaata ttctgaaatg agctgttgac 50 8 43 DNA Artificial Sequence
oligonucleotide primer zc42,167 8 cgccaaaaca cccgggagtt aatttctcct
ctttaatgaa ttc 43 9 63 DNA Artificial Sequence oligonucleotide
primer zc42,181 9 ttctcctctt taatgaattc tgtgtgaaat tgttatccgc
tcacaattcc acacattata 60 ttg 63 10 60 DNA Artificial Sequence
oligonucleotide primer zc42,179 10 ttctgaaatg agctgttgac attgtgagcg
gataacaata taatgtgtgg aattgtgagc 60 11 44 DNA Artificial Sequence
oligonucleotide primer zc42,734 11 cgttctggat aatgtttttt gcgccgacat
cataacggtt ctgg 44 12 51 DNA Artificial Sequence oligonucleotide
primer zc42,733 12 cgccaaaaca cccgggagtt aatttctcct ctttaatgaa
ttctgtgtga a 51 13 66 DNA Artificial Sequence oligonucleotide
primer zc42,721 13 cgacatcata acggttctgg aaaattattt taaatttcct
cttgacattg tgagcggata 60 acaata 66 14 73 DNA Artificial Sequence
oligonucleotide primer zc42,718 14 tttaatgaat tctgtgtgaa attgttatcc
gctcacaatt ccacacatta tattgttatc 60 cgctcacaat gtc 73 15 66 DNA
Artificial Sequence oligonucleotide primer zc42,719 15 cgacatcata
acggttctgg aaaaattttt taaaaaaata cttgacattg tgagcggata 60 acaata 66
16 66 DNA Artificial Sequence oligonucleotide primer zc42,720 16
cgacatcata acggttctgg aaaaaaagat caaaaaaata cttgacattg tgagcggata
60 acaata 66 17 66 DNA Artificial Sequence oligonucleotide primer
zc42,727 17 cgacatcata acggttctgg agagaaagca aaaataaatg cttgacattg
tgagcggata 60 acaata 66 18 462 DNA Homo sapiens 18 atgctgaaaa
ccctgaacct gggtagctgt gtgatcgcca ccaacctgca ggaaatccgt 60
aacggtttct ctgagatccg tggcagcgtg caggccaaag atggtaacat tgacatccgt
120 atcctgcgtc gtaccgagtc tctgcaggac accaaaccgg cgaaccgttg
ctgcctgctg 180 cgccacctgc tgcgtctgta tctggaccgt gttttcaaaa
actaccagac cccggaccac 240 tataccctgc gtaaaatcag cagcctggcc
aactccttcc tgaccatcaa aaaagacctg 300 cgtctgtgtc acgcccacat
gacctgccac tgtggtgagg aagcaatgaa aaaatacagc 360 cagattctga
gccacttcga aaaactggaa ccgcaggcag cagtggtgaa agctctgggt 420
gaactggaca ttctgctgca gtggatggag gagaccgaat ag 462 19 153 PRT Homo
sapiens 19 Met Leu Lys Thr Leu Asn Leu Gly Ser Cys Val Ile Ala Thr
Asn Leu 1 5 10 15 Gln Glu Ile Arg Asn Gly Phe Ser Glu Ile Arg Gly
Ser Val Gln Ala 20 25 30 Lys Asp Gly Asn Ile Asp Ile Arg Ile Leu
Arg Arg Thr Glu Ser Leu 35 40 45 Gln Asp Thr Lys Pro Ala Asn Arg
Cys Cys Leu Leu Arg His Leu Leu 50 55 60 Arg Leu Tyr Leu Asp Arg
Val Phe Lys Asn Tyr Gln Thr Pro Asp His 65 70 75 80 Tyr Thr Leu Arg
Lys Ile Ser Ser Leu Ala Asn Ser Phe Leu Thr Ile 85 90 95 Lys Lys
Asp Leu Arg Leu Cys His Ala His Met Thr Cys His Cys Gly 100 105 110
Glu Glu Ala Met Lys Lys Tyr Ser Gln Ile Leu Ser His Phe Glu Lys 115
120 125 Leu Glu Pro Gln Ala Ala Val Val Lys Ala Leu Gly Glu Leu Asp
Ile 130 135 140 Leu Leu Gln Trp Met Glu Glu Thr Glu 145 150 20 66
DNA Artificial Sequence oligonucleotide primer zc29,957 20
caatttcaca cagaattcat taaagaggag aaattaacta tgctgaaaac cctgaacctg
60 ggtagc 66 21 38 DNA Artificial Sequence oligonucleotide primer
zc23,993 21 gtatcaggct gaaaatctta tctcatccgc caaaacac 38 22 64 DNA
Artificial Sequence oligonucleotide primer zc47,073 22 acaatttcac
acagaattca ttaaagagga gaaattaact atgaggggca tgaagctgct 60 gggg 64
23 64 DNA Artificial Sequence oligonucleotide primer zc47,074 23
tctgtatcag gctgaaaatc ttatctcatc cgccaaaaca tcacttcagc atcacctcca
60 ctgg 64 24 17 DNA Artificial Sequence UP element consensus
sequence 24 aaaaattttt taaaaaa 17 25 17 DNA Artificial Sequence
rrnDP1 UP element 25 aaaaaaagat caaaaaa 17 26 18 DNA Artificial
Sequence rrnBP2 UP element 26 gagagaaagc aaaaataa 18 27 64 DNA
Artificial Sequence oligonucleotide primer zc48069 27 acaatttcac
acagaattca ttaaagagga gaaattaact atgaacatta ccgacatcat 60 gaac 64
28 67 DNA Artificial Sequence oligonucleotide primer zc48070 28
tctgtatcag gctgaaaatc ttatctcatc cgccaaaaca tcagcccatt aacattgcgt
60 caagttg 67 29 1080 DNA Bacteriophage T7 0.7 virus 29 atgaacatta
ccgacatcat gaacgctatc gacgcaatca aagcactgcc aatctgtgaa 60
cttgacaagc gtcaaggtat gcttatcgac ttactggtcg agatggtcaa cagcgagacg
120 tgtgatggcg agctaaccga actaaatcag gcacttgagc atcaagattg
gtggactacc 180 ttgaagtgtc tcacggctga cgcagggttc aagatgctcg
gtaatggtca cttctcggct 240 gcttatagtc acccgctgct acctaacaga
gtgattaagg tgggctttaa gaaagaggat 300 tcaggcgcag cctataccgc
attctgccgc atgtatcagg gtcgtcctgg tatccctaac 360 gtctacgatg
tacagcgcca cgctggatgc tatacggtgg tacttgacgc acttaaggat 420
tgcgagcgtt tcaacaatga tgcccattat aaatacgctg agattgcaag cgacatcatt
480 gattgcaatt cggatgagca tgatgagtta actggatggg atggtgagtt
tgttgaaact 540 tgtaaactaa tccgcaagtt ctttgagggc atcgcctcat
tcgacatgca tagcgggaac 600 atcatgttct caaatggaga cgtaccatac
atcaccgacc cggtatcatt ctcgcagaag 660 aaagacggtg gcgcattcag
catcgaccct gaggaactca tcaaggaagt cgaggaagtc 720 gcacgacaga
aagaaattga ccgcgctaag gcccgtaaag aacgtcacga ggggcgctta 780
gaggcacgca gattcaaacg tcgcaaccgc aaggcacgta aagcacacaa agctaagcgc
840 gaaagaatgc ttgctgcgtg gcgatgggct gaacgtcaag aacggcgtaa
ccatgaggta 900 gctgtagatg tactaggaag aaccaataac gctatgctct
gggtcaacat gttctctggg 960 gactttaagg cgcttgagga acgaatcgcg
ctgcactggc gtaatgctga ccggatggct 1020 atcgctaatg gtcttacgct
caacattgat aagcaacttg acgcaatgtt aatgggctga 1080
* * * * *