Title: Efficient protein expression system
Abstract: Nucleic acid expression control sequence cassettes and vectors containing the same are provided for use in making abundant quantities of recombinant polypeptides of interest. The modified transcriptional control sequences, which include a T5 promoter sequence, are highly stable and can be used in a variety of vectors, such as plasmids.
Patent Number: 6,939,959 Issued on 09/06/2005 to Hu
| Inventors:
|
Hu; Mary ChaoHong (Edmonds, WA)
|
| Assignee:
|
ID Biomedical Corporation of Washington (Bothell, WA)
|
| Appl. No.:
|
284083 |
| Filed:
|
October 28, 2002 |
| Current U.S. Class: |
536/24.1; 435/320.1; 435/69.1; 435/325; 435/471; 435/348; 435/419; 530/350; 530/387.3 |
| Intern'l Class: |
C07H 021/04; C12N 015/00; C12N 005/00; C12P 021/06; C07K 001/00 |
| Field of Search: |
536/241
435/320.1,691,252.3,325,471,348,419,254.3
530/350,387.3
|
References Cited [Referenced By]
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| 5350690 | Sep., 1994 | Zukowski.
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| 5689056 | Nov., 1997 | Cramer et al.
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| 5756347 | May., 1998 | Sugimoto et al.
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| 5876962 | Mar., 1999 | Bishop et al.
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| 5985285 | Nov., 1999 | Titball et al.
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| 6063386 | May., 2000 | Dale et al.
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| 6194168 | Feb., 2001 | Gentz et al.
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| 6291245 | Sep., 2001 | Kopetzki et al.
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| 6436639 | Aug., 2002 | Kiefer et al.
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| Foreign Patent Documents |
| WO 94/0646/5 | Jun., 1994 | WO.
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| WO 99/1685/8 | Apr., 1999 | WO.
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|
Primary Examiner: Leffers; Gerry
Assistant Examiner: Marvich; Maria
Attorney, Agent or Firm: Seed IP Law Group PLLC
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Patent Application No.
60/348,434 filed Oct. 26, 2001, where this provisional application is incorporated
herein by reference in its entirety.
Claims
1. A nucleic acid expression control sequence cassette comprising SEQ ID NO:2.
2. A nucleic acid expression vector comprising a nucleic acid expression control
sequence cassette according to claim 1.
3. A nucleic acid expression vector comprising a nucleic acid expression control
sequence cassette that comprises the sequence set forth in SEQ ID NO:2, wherein
the vector is pT5 (SEQ ID NO:1).
4. A nucleic acid expression vector comprising a nucleic acid expression control
sequence cassette that comprises the sequence set forth in SEQ ID NO:2, wherein
the cassette is operably linked to at least one nucleic acid coding sequence.
5. The expression vector according to claim 4 wherein said at least one nucleic
acid coding sequence encodes a polypeptide selected from the group consisting of
a bacteriophage polypeptide, a bacterial polypeptide, a fungal polypeptide, a viral
polypeptide, an insect polypeptide, a plant polypeptide, and a mammalian polypeptide.
6. The expression vector according to claim 4 wherein said at least one nucleic
acid coding sequence encodes an immunogenic hybrid polypeptide comprising at least
one bacterial polypeptide.
7. The expression vector according to claim 6 wherein said immunogenic hybrid
polypeptide comprises a hybrid multivalent group A streptococcal M polypeptide.
8. The expression vector according to claim 6 wherein said immunogenic hybrid
polypeptide comprises a hybrid polypeptide of
Yersinia pestis polypeptides
F1 and V.
9. A method for producing one or more polypeptide(s), comprising:
a) culturing a cell containing the expression vector of claim 4 under conditions
sufficient to express one or more polypeptides; and
b) isolating said one or more polypeptides.
10. The method according to claim 9 wherein said one or more expressed polypeptides
are selected from the group consisting of a bacteriophage polypeptide, a bacterial
polypeptide, a fungal polypeptide, a viral polypeptide, an insect polypeptide,
a plant polypeptide, and a mammalian polypeptide.
11. The method according to claim 9 wherein said cell is selected from the group
consisting of a bacterium, a fungus, an insect cell, a plant cell, and a mammalian cell.
12. The method according to claim 9 wherein said cell is a bacterium.
13. The method according to claim 9 wherein said one or more expressed polypeptides
are in soluble form.
14. The method according to claim 9 wherein said one or more expressed polypeptides
comprise a hybrid multivalent group A streptococcal M polypeptide or a hybrid polypeptide
of
Yersinia pestis polypeptides F1 and V.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the nucleic acid expression systems,
and more specifically, to nucleic acid expression control sequence cassettes comprising
a stable bacteriophage T5 promoter and nucleic acid regulatory sequences useful
for generating efficient and stable expression vectors for high-level protein expression.
2. Description of the Related Art
A demand for the efficient production of biologics for therapeutic use is steadily
increasing as more products, such as recombinant proteins, are approved or are
nearing approval for use in humans. Bacterial fermentation processes have long
been, and still are, the major tool for production of these types of molecules.
The key objective of process optimization is to attain a high yield of product
having the required quality at the lowest possible cost, which is often determined
by the properties of a specific expression construct or system. For example, high-level
recombinant protein expression may overwhelm the metabolic capacity of a host cell,
which often impairs efficient protein production.
Hence, a need exists for identifying and developing additional nucleic acid
expression systems useful for the efficient and stable production of therapeutically
effective agents. The present invention meets such needs, and further provides
other related advantages.
BRIEF SUMMARY OF THE INVENTION
The present invention provides the discovery of a stable nucleic acid expression
control sequence for high-level expression of recombinant proteins.
In one aspect, the invention provides a nucleic acid expression control sequence
cassette, comprising (a) a transcription initiation sequence capable of remaining
hybridized under stringent conditions to a T5 promoter sequence, wherein said transcription
initiation sequence has at least basal T5 promoter transcriptional activity; (b)
at least one regulatory sequence operably linked to said transcription sequence
of (a) and capable of remaining hybridized under stringent conditions to a lac
operator sequence, wherein said at least one regulatory sequence specifically binds
a lacI repressor protein and thereby alters transcriptional activity; (c) at least
one mutated regulatory sequence of (b) wherein said at least one mutated regulatory
sequence does not specifically bind a lacI repressor protein and thereby does not
alter transcriptional activity; and (d) a translation initiation sequence. In another
embodiment, (c) is a cis-acting nucleotide sequence or transcriptional spacer comprising
up to about 30 nucleotides. In another embodiment, the aforementioned cassettes
further comprise at least one restriction enzyme recognition site at about the
3′-end and at least one restriction enzyme recognition site at about the
5′-end. In a related embodiment, the at least one restriction enzyme recognition
site at about the 5′-end is BglII and said at least one restriction enzyme
recognition site at about the 3′-end is NdeI. In a further embodiment, any
of the aforementioned cassettes comprise SEQ ID NO:2 or 3.
In another aspect, the present invention provides a nucleic acid expression vector
comprising any of the aforementioned nucleic acid expression control sequence cassette.
In certain embodiments, the expression vector may be a plasmid, a cosmid, a shuttle
vector, a viral vector, an insect vector, and a YAC, preferably a plasmid. In a
particular embodiment, the expression vector is pT5 (SEQ ID NO:1). In other embodiments,
the expression vector has the any of the aforementioned cassettes operably linked
to at least one nucleic acid coding sequence. In related embodiments, the nucleic
acid coding sequences encode a polypeptide selected from a bacteriophage polypeptide,
a bacterial polypeptide, a fungal polypeptide, a viral polypeptide, an insect polypeptide,
a plant polypeptide, or a mammalian polypeptide. In still other embodiments, there
is provided any of the aforementioned expression vectors wherein said at least
one nucleic acid coding sequence encodes an immunogenic hybrid polypeptide comprising
at least one bacterial polypeptide, preferably said immunogenic hybrid polypeptide
comprises a hybrid multivalent group A streptococcal M polypeptide or a hybrid
polypeptide of
Yersinia pestis polypeptides F1 and V.
In a further aspect, the invention provides a method for producing one or more
polypeptide(s), comprising (a) culturing a cell containing the expression vector
of claim 9 under conditions sufficient to express one or more polypeptide(s);
and (b) isolating said polypeptide(s). In one embodiment, the aforementioned method
wherein said expressed polypeptide is selected from a bacteriophage polypeptide,
a bacterial polypeptide, a fungal polypeptide, a viral polypeptide, an insect polypeptide,
a plant polypeptide, or a mammalian polypeptide. In other embodiments, said cell
is selected from the group consisting of a bacterium, a fungus, an insect cell,
a plant cell, and a mammalian cell, preferably a bacterium. In certain embodiments,
the aforementioned methods provide expressed polypeptide(s) in soluble form. In
one embodiment, any of the aforementioned methods provide expressed polypeptides
comprising a hybrid multivalent group A streptococcal M polypeptide or a hybrid
polypeptide of
Yersinia pestis polypeptides F1 and V. In another related
embodiment, any of the aforementioned methods wherein the expression vector is
pT5 (SEQ ID NO:1).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic diagram of the process for making one embodiment of
a modified T5 promoter and lac operator using PCR. This series of reactions results
in a T5 promoter operably linked to at least one functional lac operator followed
by a mutated lac operator (that can no longer function as an operator). Primers
BglQE-F (SEQ ID NO:7) and T5PRO1R (SEQ ID NO:10) were used in the first PCR reaction
(wherein T5PRO1R primes at operator I), primers NdeQE-R (SEQ ID NO:8) and T5PRO1F
(SEQ ID NO:9) were used in the second PCR reaction, and finally primers BglQE-F
and NdeQE-R were used to generate SEQ ID NO:3.
FIG. 2 shows a schematic diagram of the process for making one embodiment of
a modified T5 promoter and lac operator using PCR. This series of reactions results
in a T5 promoter operably linked to at least two functional lac operators followed
by a mutated lac operator (that can no longer function as an operator). Primers
BglQE-F (SEQ ID NO:7) and T5PRO1R (SEQ ID NO:10) were used in the first PCR reaction
(wherein T5PRO1R primes at operator II), primers NdeQE-R (SEQ ID NO:8) and T5PRO1F
(SEQ ID NO:9) were used in the second PCR reaction, and finally primers BglQE-F
and NdeQE-R were used to generate SEQ ID NO:2.
FIGS. 3A to 3D show the nucleic acid sequence of various expression
control sequences. FIG. 3A shows the T5 promoter/lac operator expression control
sequence (SEQ ID NO:4) found in the pQE-40 plasmid (Qiagen, Valencia, Calif.).
FIG. 3B shows the portion of the T5 promoter/lac operator in pQE-40 that appears
to be unstable and is often deleted (boxed sequence) when cloned (SEQ ID NO:11).
FIGS. 3C and 3D show two embodiments wherein the T5 promoter/lac operator region
is modified and surprisingly rendered stable (SEQ ID NOS:5 and 6). Lower case,
bold letters in FIGS. 3C and 3D identify the mutated lacO nucleotides (8 of 19
total), and boxed in FIG. 3D is the 32 base pair insertion that includes a mutated lacO.
FIG. 4 shows a schematic diagram of plasmid pT5 (SEQ ID NO:1) having the T5
promoter/lac operator control sequence depicted in FIG. 3D operably linked to a
nucleic acid sequence that encodes a hexavalent hybrid polypeptide (i.e., hexavalent
A.1 is a polypeptide that includes portions of M proteins from different group
A streptococci serotypes).
FIG. 5 shows a schematic diagram of plasmid pT5 having the T5 promoter/lac operator
control sequence depicted in FIG. 3D operably linked to a nucleic acid sequence
that encodes a septavalent hybrid polypeptide (i.e., septavalent B.2 is a polypeptide
that includes portions of M proteins from different group A streptococci serotypes).
FIG. 6 shows a Coomassie® blue stained SDS-PAGE of whole cell lysates of
Escherichia coli JM105 containing pT5 constructs grown in the presence or
absence of IPTG. Lane 1, uninduced pT5-Hexa A.1; Lane 2, induced pT5-Hexa A.1;
Lane 3, uninduced pT5-Hexa A.3; Lane 4, induced pT5-Hexa A.3; Lane 5, standard
molecular weight markers (bands corresponding to molecular mass 55 kDa and 36 KDa
are shown on the left); Lane 6, uninduced pT5-Septa B.2; Lane 7, induced pT5-Septa
B.2; Lane 8, uninduced pT5-Septa B.3a; and Lane 9, induced pT5-Septa B.3a. Hexa
A.3 is the same protein as Hexa A.1 and Septa B.3a is the same protein as Septa
B.2, except that silent mutations were introduced into the nucleic acid sequence
of the 3 series proteins to optimize the codons for expression in
E. coli.
The arrow on the left identifies the overexpressed Hexa A proteins and the arrow
on the right identifies the overexpressed Septa B proteins.
FIG. 7 shows a Coomassie® blue stained SDS-PAGE of whole cell lysates of
Escherichia coli JM105 containing pT5 constructs grown in the presence or
absence of IPTG. Lane 1, uninduced pT5-M18(50aa)-2; Lane 2, induced pT5-M18(50aa)-2;
and Lane 3, standard molecular weight markers (bands corresponding to molecular
mass 14 kDa and 6 KDa are shown on the right). The M18(50aa)-2 indicates that a
nucleic acid sequence encoding a dimer of the first 50 amino acids from group A
streptococci M protein from serotype 18. The arrow on the left identifies the overexpressed
M18 dimer.
FIG. 8 shows a Coomassie® blue stained SDS-PAGE of different cell fractions
of
Escherichia coli JM105 containing pT5-F1-V grown in the presence of IPTG.
Lane 1, whole cell lysate; Lane 2, standard molecular weight markers (bands corresponding
to molecular mass 55 kDa and 36 KDa are shown on the right); Lane 3, soluble fraction
from the whole cell lysate; and Lane 4, insoluble fraction from the whole cell
lysate. F1-V is a fusion protein of two
Yersinia pestis virulence proteins.
The arrow on the left identifies the overexpressed F1-V fusion protein.
DETAILED DESCRIPTION OF THE INVENTION
As noted above, the present invention is generally directed to nucleic acid expression
control sequence cassettes, which can be used to generate nucleic acid expression
vectors. When introduced to the proper host cell, these expression vectors will
stably and efficiently produce a variety of recombinant polypeptides. Furthermore,
the cassettes may be introduced into a variety of different vector backbones (such
as plasmids, cosmids, viral vectors, and the like) so that recombinant protein
expression can be accomplished in a variety of different host cells (such as bacteria,
yeast, mammalian cells, and the like). The present invention is also directed to
methods of producing and isolating recombinant proteins using the nucleic acid
expression control sequence cassettes operably linked to a nucleic acid coding
sequence. For example, without limitation, the nucleic acid expression control
sequence cassettes of this invention can be used to produce immunogenic polypeptides,
such as a hybrid group A streptococcal polypeptides or plague fusion proteins.
By way of background, and not wishing to be bound by theory, the level of recombinant
protein production from a nucleic acid expression vector is influenced by a variety
of factors, including without limitation, the copy number of the vector, the strength
of the promoter, the activity and localization of the recombinant protein being
expressed, the host cell being used, alignment of the codon usage in the recombinant
protein and host cell, and how efficiently the promoter is regulated. For example,
the pQE expression plasmids (Qiagen, Valencia, Calif.) contain an inducible expression
element consisting of phage T5 promoter and two lac operator sequences (lacO).
E. coli RNA polymerase recognizes the bacteriophage T5 promoter, which is
transcribed at a very high rate. Two lacO sequences are included in the pQE plasmids
to presumably allow more Lac repressor protein (lacI) binding to ensure efficient
repression of the powerful T5 promoter. In addition, the extremely high transcription
rate initiated at the T5 promoter can only be efficiently regulated and repressed
by the presence of high levels of lacI. Hence, to provide high levels of lacI,
the pQE vectors are typically introduced into
E. coli host strains carrying
the low-copy plasmid pREP4, which constitutively expresses lacI (has the high expressing
lacI
q mutant). Any
E. coli host strain containing both the expression
plasmid (pQE) and the repressor (pREP4) plasmid can be used for the controlled
production of recombinant proteins. Recently, a cis-repressed pQE have the coding
sequence for lacI repressor contained directly on the pQE plasmid was generated
(e.g., see pQE80L; www.qiagen.com).
Although a strong, but regulated, promoter may be desirable to more easily
produce abundant amounts of a recombinant protein, some proteins may be toxic for
a host cell even when small amounts are produced due to "leakage" of the promoter
(i.e., when a negatively regulated promoter still produces some protein). Therefore,
strong suppression of recombinant protein expression may be desirable. In other
instances, a nucleic acid expression vector may be unstable and, for reasons unknown,
a host will cause the coding sequence for a recombinant protein to be recombinantly
removed from the vector. By way of example, the expression of recombinant
Thermus
thermophilus ribonuclease H that had been cloned into pQE-40 (pQE-rnhA) was
found to be very unstable in
E. coli. The rnhA was removed from the pQE-40
plasmid and cloned into the pET-24a vector (Novagen, Madison, Wis.). The resultant
plasmid, pET-24a-rnhA, proved to be highly stable and provided high-level protein
expression in the BL21(DE3)
E. coli host cells (Novagen).
Therefore, the T7/lac operator expression control sequence between the
BglII and NdeI sites was then replaced with a T5 promoter/lac operator expression
control sequence that was generated by PCR (see FIG.
3A), to create plasmid
pET-T5-rnhA. However, the new construct showed no expression of the ribonuclease
H enzyme. Upon sequencing, it was discovered that a 32 base pair fragment of the
T5 promoter/lac operator expression control sequence was deleted in pET-T5-rnhA
(see FIG. 3B, box identifies the deletion). Part of the deletion included the -10
TATA box portion of the T5 promoter, which explained why no expression of the recombinant
rnhA gene was occurring. By way of background, and not wishing to be bound by theory,
it appears that the original T5 promoter/lac operator expression control sequence
was unstable because the duplicated lac operator sequences may have been involved
in recombination events that deleted a 32 base pair fragment from pET-T5-rnhA.
Thus, to solve this problem, site-directed mutagenesis by PCR was performed to
generate a modified T5 promoter/lac operator expression control sequence cassette,
which was stable.
The invention, therefore, relates generally to the surprising discovery, as provided
in the present disclosure, that modification of the nucleotide sequence within
a T5 promoter/lac operator expression control sequence provides a stable promoter/operator
region that results in consistent and high-level expression of recombinant proteins
in host cells, and a nucleic acid expression control sequence that can be flanked
by, for example, restriction endonuclease sites for isolation and cloning into
any desired vector. Moreover, the modified nucleic acid expression control sequence
may include one or more mutations, which can include a substitution, a deletion,
an insertion, and a combination thereof. Preferably, a modified nucleic acid expression
control sequence of the present invention has a substitution mutation, more preferably
an insertion mutation, and most preferably a combination of a substitution mutation
and insertion mutation. In a preferred embodiment, the present invention provides
a nucleic acid expression control sequence cassette comprising (a) a transcription
initiation sequence capable of remaining hybridized under stringent conditions
to a T5 promoter sequence, wherein said transcription initiation sequence has at
least basal T5 promoter transcriptional activity; (b) at least one regulatory sequence
operably linked to said transcription sequence of (a) and capable of remaining
hybridized under stringent conditions to a lac operator sequence, wherein said
at least one regulatory sequence specifically binds a lacI repressor protein and
thereby alters transcriptional activity; (c) at least one mutated regulatory sequence
of (b) wherein said at least one mutated regulatory sequence does not specifically
bind a lacI repressor protein and thereby does not alter transcriptional activity;
and (d) a translation initiation sequence.
A similar expression system relates to the T7 promoter (see U.S. Pat. Nos. 4,952496,
5,693,489, and 5,869,320), except that the T7 promoter requires a specific T7 RNA
polymerase (in contrast, transcription from the T5 promoter can occur with a host
RNA polymerase). The T7 RNA polymerase must be provided in bacterial host (typically
as a bacteriophage lysogen) and, therefore, cloning of a polynucleotide coding
sequence must first take place in a bacterial strain lacking the T7 RNA polymerase,
and then expression requires transfer to a bacterial lysogen that makes the T7
RNA polymerase. One advantage of the nucleic acid expression control system of
the present invention is that a single host cell can be used for both cloning of
a polynucleotide coding sequence and for expression of the polypeptide encoded
by a polynucleotide coding sequence. For example, any bacterial host cell that
produces lacI repressor protein (preferably a lacI expressed from the lacI
q
gene) can be used to introduce a nucleic acid expression control sequence
of the present invention carried on a vector, such as a plasmid. In addition, any
nucleic acid expression control sequence of the present invention can be used,
as described herein, with a vector that also carries the lacI
q gene
and is capable of replicating in a bacterial host (e.g., pT5, SEQ ID NO:1).
Moreover, the transcription initiation sequence is preferably capable of
remaining hybridized under stringent conditions to a T5 promoter sequence, wherein
said transcription initiation sequence has at least basal T5 promoter transcriptional
activity. Thus, a variety of T5 promoter sequences may be used, including without
limitation those described in U.S. Pat. Nos. 4,495,280 and 4,868,111. As used herein,
"basal activity" means that transcription is detectable by methods known in the
art. The surprising result of the present invention is insertion of a non-coding
cis-acting nucleic acid sequence, which functions as a transcribed spacer sequence,
stabilizes the T5 promoter/lac operator portion of the nucleic acid expression
control sequence. In one preferred embodiment, an insertion downstream of the transcription
initiation sequence and at least one regulatory sequence comprises a cis-acting
nucleotide sequence or a transcribed spacer comprising up to 32 nucleotides.
"Nucleic acid" or "nucleic acid molecule" refers to any of deoxyribonucleic
acid (DNA), 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. Preferably, the nucleic acids of the
present invention are produced by PCR. Nucleic acids may be composed of monomers
that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides),
analogs of naturally occurring nucleotides (e.g., α-enantiomeric forms of
naturally-occurring nucleotides), or a combination of both. Modified nucleotides
can have modifications 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 may 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" also
includes so-called "peptide nucleic acids" (PNAs), which comprise naturally occurring
or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids
can be either single stranded or double stranded.
Further, an "isolated nucleic acid molecule" refers to a polynucleotide
molecule in the form of a separate fragment or as a component of a larger nucleic
acid construct, which has been separated from its source cell (including the chromosome
it normally resides in) at least once in a substantially pure form. For example,
a DNA molecule that encodes a recombinant polypeptide, peptide, or variant thereof,
which has been separated from a cell or from the genomic DNA of a cell, is an isolated
DNA molecule. Another example of an isolated nucleic acid molecule is a bacteriophage
promoter (e.g., T5 or T7), or nucleic acid expression control sequence cassette
of the present invention, cloned into a plasmid capable of replication in a bacterial
host cell. Still another example of an isolated nucleic acid molecule is a chemically
synthesized nucleic acid molecule. Nucleic acid molecules may be comprised of a
wide variety of nucleotides, including DNA, cDNA, RNA, nucleotide analogues, or
some combination thereof. In certain preferred embodiments, an isolated nucleic
acid molecule is an expression control sequence cassette comprising a nucleic acid
sequence as set forth in SEQ ID NOS:1, 2, 3, 5, or 6. Preferably, the nucleic acid
expression control sequence cassette is double stranded DNA.
Nucleic acid expression control sequences of this invention may be designed
for inclusion within a nucleic acid sequence cassette. As used herein, a "sequence
cassette" refers to a contiguous nucleic acid molecule that can be isolated as
a single unit and cloned as a single unit. For example, a sequence cassette may
be created enzymatically (e.g., by using type I or type II restriction endonucleases,
exonucleases, etc.), by mechanical means (e.g., shearing), by chemical synthesis,
or by recombinant methods (e.g., PCR). An advantage of the present invention is
that a nucleic acid expression control sequence comprising (a) a transcription
initiation sequence capable of remaining hybridized under stringent conditions
to a T5 promoter sequence, wherein said transcription initiation sequence has at
least basal T5 promoter transcriptional activity; (b) at least one regulatory sequence
operably linked to said transcription sequence of (a) and capable of remaining
hybridized under stringent conditions to a lac operator sequence, wherein said
at least one regulatory sequence specifically binds a lacI repressor protein and
thereby alters transcriptional activity; (c) at least one mutated regulatory sequence
of (b) wherein said at least one mutated regulatory sequence does not specifically
bind a lacI repressor protein and thereby does not alter transcriptional activity;
and (d) a translation initiation sequence, may be constructed by, for example,
PCR as a sequence cassette that is flanked by restriction endonuclease sites.
Any preferred restriction endonuclease site may be incorporated (see list of
at least 215 commercially available restriction endonucleases in the New England
Biolabs 2002 catalog, which is hereby incorporated by reference). Preferably, the
nucleic acid expression control sequence cassette comprises at least one restriction
enzyme recognition site at about the 3′-end and at least one restriction
enzyme recognition site at about the 5′-end. More preferably, the restriction
enzyme recognition site of the nucleic acid expression control sequence cassette
at about the 5′-end is BglII and the restriction enzyme recognition site
at about the 3′-end is NdeI. Preferably, the nucleic acid expression control
sequence cassette with the restriction enzyme sites at the 3′- and 5′-ends
comprises SEQ ID NOS:2 or 3.
As used herein, the term "about" or "consists essentially of" refers to ±10%
within a recited position or of any indicated structure, value, or range. In addition,
any numerical ranges recited herein are to be understood to include any integer
within that range and, where applicable (e.g., concentrations), fractions thereof,
such as one tenth and one hundredth of an integer (unless otherwise indicated).
Preferred nucleic acid expression control sequences include at least one
translation initiation sequence, which may be derived from many sources, to aid
in producing a recombinant protein of interest. In one embodiment, the translation
initiation sequence is a ribosome binding site (RBS) from the bacterial gene lacZ.
Other translation initiation sequences or ribosome binding sites may be obtained
from genes derived from mammalian coding sequences, fungal coding sequences, viral
coding sequences, plant coding sequences, bacteriophage coding sequences, and the like.
In another aspect, the nucleic acid expression control sequences comprising a
transcription initiation sequence capable of remaining hybridized under stringent
conditions to a T5 promoter sequence, at least one regulatory sequence operably
linked to the transcription sequence and capable of remaining hybridized under
stringent conditions to a lac operator sequence, and a translation initiation sequence,
also comprise a at least one mutated regulatory sequence wherein the mutated regulatory
sequence no longer functions as such. For example, an exemplary lacO sequence comprised
of 19 nucleotides may be mutated by substitution of 8 nucleotides, which can no
longer specifically bind a lacI repressor protein and thereby can no longer alter
transcriptional activity when operably linked to a transcription initiation sequence.
Preferably, the mutated regulatory sequence also no longer remains hybridized under
stringent conditions to a lac operator sequence. Alternatively, a nucleic acid
sequence up to 150 nucleotides instead of a mutated regulatory sequence may be
used, preferably inserted downstream (i.e., to the 3′-side) of the at least
one regulatory sequence operably linked to the transcription initiation sequence.
In one preferred embodiment, the nucleic acid expression control sequence of
this
invention comprises at least one functional regulatory sequence operably linked
to a transcriptional activation sequence and at least one substitution mutated
regulatory sequence that is no longer capable of altering transcription (for illustrative
purposes, see FIG.
3C). In a more preferred embodiment, the nucleic acid
expression control sequence of this invention comprises at least two functional
regulatory sequences operably linked to a transcriptional activation sequence and
at least one insertion of a substitution mutated regulatory sequence that is no
longer capable of altering transcription (for illustrative purposes, see FIG.
3D).
Therefore, a T5 promoter/lac operator expression control sequence is surprisingly
stabilized by an insertion of a nucleic acid sequence that is non-regulatory and
is up to about 150 nucleotides in length, preferably is about 10 to about 50 nucleotides,
more preferably is about 20 nucleotides to about 40 nucleotides, and most preferably
is about 25 to about 35 nucleotides in length. In one preferred embodiment, the
insertion is a cis-acting nucleotide sequence or a transcribed spacer consisting
essentially of 32 nucleotides.
In certain aspects, the invention relates to nucleic acid vectors and constructs
that include nucleic acid expression control sequence cassettes of the present
invention, and in particular to "nucleic acid expression constructs" that include
any nucleic acid expression control sequence cassette as provided herein. In addition,
the nucleic acid expression constructs may further comprise a nucleic acid expression
control sequence of the present invention operably linked to one or more polynucleotide
coding sequences. Also provided by the present invention are nucleic acid expression
constructs, and host cells containing such nucleic acids that encode recombinant
polypeptides and variants thereof. In certain embodiments, the nucleic acid coding
sequences may encode a polypeptide selected from a bacteriophage polypeptide, a
bacterial polypeptide, a fungal polypeptide, a viral polypeptide, an insect polypeptide,
a plant polypeptide, or a mammalian polypeptide.
For example, the nucleic acid expression constructs of the present invention
can be used to express recombinant polypeptides capable of eliciting an immune
response against one or more antigens, such as the group A streptococci M proteins
or plague virulence proteins F1 and V. One aspect of the invention pertains to
isolated nucleic sequences encoding a hybrid polypeptide sequence as described
herein, as well as those sequences readily derived from isolated nucleic acid molecules
such as, for example, complementary sequences, reverse sequences and complements
of reverse sequences.
Appropriate cloning and expression vectors for use with prokaryotic and
eukaryotic hosts are described, for example, by Sambrook et al.,
Molecular Cloning:
A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), and
may include plasmids, cosmids, shuttle vectors, viral vectors and vectors comprising
a chromosomal origin of replication as disclosed therein (e.g., yeast artificial
chromosome or YAC). Generally, nucleic acid expression vectors include origins
of replication and selectable markers permitting detectable transformation of the
host cell, e.g., the ampicillin resistance gene of
E. coli and
S. cerevisiae
TRP1 gene, and an expression control sequence such as a promoter. For purposes
of the present invention, the nucleic acid expression control sequence cassettes
of this invention may be used to replace an expression control sequence already
existing in a particular desired vector. In addition, a heterologous structural
sequence may be included in appropriate phase with translation initiation sequences
and termination sequences of the vector. Optionally, a heterologous sequence can
encode a fusion protein including an amino-terminal (or a carboxy-terminal) identification
peptide imparting desired characteristics, e.g., stabilization or simplified purification
of expressed recombinant product. In particularly preferred embodiments, for example,
recombinant polypeptides are fused in-frame to a carboxy-terminal tag, which tag
may be any one of alkaline phosphatase, β-galactosidase, hexahistidine (6×His),
FLAGS® epitope tag (DYKDDDDK, SEQ ID NO:12), or GST, and the like. Most preferred
are recombinant fusion proteins that facilitate affinity detection and isolation
of the hybrid polypeptides and may include, for example, poly-His or the defined
antigenic peptide epitopes described in U.S. Pat. No. 5,011,912 and in Hopp et
al., (1988
Bio/Technology 6:1204), or the XPRESS™ epitope tag (DLYDDDDK,
SEQ ID NO:13; Invitrogen, Carlsbad, Calif.). The affinity sequence may be a hexa-histidine
tag as supplied by a vector, such as, for example, pBAD/His (Invitrogen). Alternatively,
the affinity sequence may be added either synthetically or engineered into the
primers used to recombinantly generate the nucleic acid coding sequence (e.g.,
using the polymerase chain reaction). Preferably, a recombinant polypeptide is
fused to a polyhistidine and is encoded by a recombinant nucleic acid sequence
encoding such a fusion protein.
Expression constructs for bacterial use may be constructed by inserting
into an expression vector a structural DNA sequence encoding a desired protein
together with a nucleic acid expression control sequence as described herein. The
construct may comprise one or more phenotypic selectable markers and an origin
of replication to ensure maintenance of the vector construct and, if desirable,
to provide amplification within the host. Suitable prokaryotic hosts for transformation
include
E. coli, Bacillus subtilis, Salmonella typhimurium and various species
within the genera
Pseudomonas, Streptomyces, and
Staphylococcus,
although others may also be employed as a matter of choice. Any other plasmid or
vector may be used as long as they are replicable and viable in the host.
As a representative but non-limiting example, expression vectors for bacterial
use can comprise a selectable marker and bacterial origin of replication derived
from commercially available plasmids comprising genetic elements of the well known
cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example,
pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Corp., Madison,
Wis., USA), and the T7 pET vectors (Novagen, Madison, Wis., USA). These pBR322
"backbone" sections may be combined with an appropriate nucleic acid expression
control sequence of this invention and the structural sequence to be expressed.
The pBR322 replication origin is considered medium copy, as is the replication
origin of pACYC-based vectors, in that bacteria produce about 20-80 copies of the
plasmid per cell. Low-copy vectors (less than 10 copies per cell), such as those
based on pSC101, may also be used. High copy vectors, such those based on the pUC
plasmids, may also be used. Preferably, the nucleic acid expression control sequence
of the present invention is contained in low copy vector, a medium copy vector,
or a high copy vector, and most preferably in a high copy vector.
Other vectors and constructs include chromosomal, non-chromosomal and synthetic
DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus;
yeast plasmids; yeast artificial chromosomes (YACs); vectors derived from combinations
of plasmids and phage DNA; shuttle vectors derived from combinations of plasmids
and viral DNA; viral DNA, such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used for preparation of a nucleic acid expression
construct as long as it is replicable and viable in the host cell of interest.
Further, in some preferred embodiments, nucleic acid expression constructs containing
the nucleic acid expression control sequence operably linked to polynucleotide
coding sequence(s) for polypeptide(s) and fusion protein(s) may remain extrachromosomal,
and in another preferred embodiments the expression constructs may integrate into
at least one host cell chromosome.
In another preferred embodiment, the nucleic acid expression construct has a
second
expression control sequence such as a promoter, which may be lac, lacUV5, tac,
trc, ara, trp, λ phage, T3 phage promoter, and T7 phage promoter, and more
preferably is a T7 phage promoter. The "expression control sequence" refers to
any sequences sufficient to allow expression of a protein of interest in a host
cell, including one or more promoter sequences, enhancer sequences, operator sequences
(e.g., lacO), and the like. In a preferred embodiment, the nucleic acid expression
control sequence cassette is in a plasmid and the host cell is a bacterium. More
preferably the plasmid is pT5 (SEQ ID NO:1) and the host cell is
Escherichia
coli. In certain preferred embodiments the second expression control sequence
is an "externally regulated promoter," which includes functional promoter sequences
having activity that may be altered (e.g., increased or decreased) by an additional
element, agent, molecule, component, co-factor or the like. An externally regulated
promoter may comprise, for example, a repressor binding site, an activator binding
site or any other regulatory sequence that controls expression of a polynucleotide
sequence as provided herein. In certain particularly preferred embodiments, the
externally regulated promoter is a tightly regulated promoter that is specifically
inducible and that permits little or no transcription of polynucleotide sequences
under its control in the absence of an induction signal, as is known to those familiar
with the art and described, for example, in Guzman et al. (
J. Bacteriol.,
1995, 177:4121), Carra et al. (
EMBO J., 1993, 12:35), Mayer (
Gene,
1995, 163:41), Haldimann et al. (
J. Bacteriol., 1998, 180:1277), Lutz et
al. (
Nuc. Ac. Res., 1997, 25:1203), Allgood et al. (
Curr. Opin. Biotechnol.,
1997, 8:474) and Makrides (
Microbiol. Rev., 1996, 60:512). In other preferred
embodiments of the invention, a second externally regulated promoter is present
that is inducible but that may not be tightly regulated. In certain other preferred
embodiments a second promoter is present in the expression construct of the invention
that is not a regulated promoter; such a promoter may include, for example, a constitutive
promoter such as an insect polyhedrin promoter or a yeast phosphoglycerate kinase
promoter (see, e.g., Giraud et al., 1998
J. Mol. Biol. 281:409). A nucleic
acid expression construct may also contain a transcription terminator. A vector
may also include appropriate sequences for amplifying expression.
Transcription of a DNA sequence encoding a polypeptide by higher eukaryotes
may be increased by inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter
to increase its transcription. Examples including the SV40 enhancer on the late
side of the replication origin bp 100 to 270, a cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication origin, and
adenovirus enhancers.
As noted above, in certain embodiments the vector may be a viral vector such
as
a retroviral vector. For example, retroviruses from which a retroviral plasmid
vector may be derived include, but are not limited to, Moloney Murine Leukemia
Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma
virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency
virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
While particular embodiments of nucleic acid expression control sequences are
depicted in SEQ ID NOS:1, 2, 3, 5, and 6, within the context of the present invention,
reference to one or more isolated nucleic acids includes variants of these sequences
that are substantially similar in that they are structurally similar and remain
capable of functioning as expression control sequences by being specific for one
or more regulatory proteins. As used herein, the nucleotide sequence is deemed
to be "substantially similar" if: (a) the nucleotide sequence is derived from a
transcription initiation sequence or a regulatory sequence and retain the ability
to initiate transcription or alter the level of transcription, respectively; (b)
the nucleotide sequence is capable of hybridization to the nucleotide sequences
of the present invention under stringent conditions; or (c) is a complement of
any of the sequences described in (a) and (b).
"Specific for" refers to the ability of a protein (e.g., repressor, inducer)
to selectively bind a nucleic acid regulatory sequence and/or a expression regulatory
protein. Association or "binding" of a regulator protein to a specific nucleic
acid or protein generally involve electrostatic interactions, hydrogen bonding,
Van der Waals interactions, and hydrophobic interactions. Any one of these or any
combination thereof can play a role in the binding between a regulatory protein
and its ligand. Such a regulatory protein (e.g., lacI) generally associates with
a specific nucleic acid sequence (e.g., lacO) with an dissociation constant (K
d)
of at least 10
-8 M, preferably at least 10
-9 M, more preferably
at least 10
-10 M, still more preferably at least 10
-11 M
and most preferably at least 10
-12 M. Affinity and dissociation constants
may be determined by one of ordinary skill in the art using well-known techniques
(see Scatchard,
Ann. N.Y. Acad. Sci. 51:660-672, 1949).
As used herein, two nucleotide sequences are said to "hybridize" or "remain hybridized"
under conditions of a specified stringency when stable hybrids are formed between
substantially complementary nucleic acid sequences. Stringency of hybridization
refers to a description of the environment under which hybrids are annealed and
washed, which typically includes ionic strength and temperature. Other factors
that might affect hybridization include the probe size and the length of time the
hybrids are allowed to form. For example, "high," "medium" and "low" stringency
encompass the following conditions or equivalent conditions thereto: high stringency
is 0.1×SSPE or SSC, 0.1% SDS, 65° C.; medium stringency is 0.2×SSPE
or SSC, 0.1% SDS, 50° C.; and low stringency is 1.0×SSPE or SSC, 0.1%
SDS, 50° C. As used herein, the term "high stringency conditions" means that
one or more sequences will remain hybridized only if there is at least 95%, and
preferably at least 97%, identity between the sequences. In preferred embodiments,
the nucleic acid expression control sequences of this invention comprise a transcription
initiation sequence capable of remaining hybridized under stringent conditions
to a T5 promoter sequence, which includes transcription initiation sequences that
have at least basal T5 promoter transcriptional activity. In another preferred
embodiment, the nucleic acid expression control sequence of this invention comprise
a regulatory sequence capable of remaining hybridized under stringent conditions
to a lac operator sequence, which includes regulatory sequences that specifically
bind a lacI repressor protein and thereby can alter transcriptional activity when
operably linked to a transcription initiation sequence.
It should be further understood that recombinant polypeptide-encoding nucleic
acids could include variants of the natural sequence due to, for example, the degeneracy
of the genetic code (including alleles). Briefly, such "variants" may result from
natural polymorphisms or may be synthesized by recombinant methodology (e.g., to
obtain codon optimization for expression in a particular host) or chemical synthesis,
and may differ from wild-type polypeptides by one or more amino acid substitutions,
insertions, deletions, or the like. Variants encompassing conservative amino acid
substitutions include, for example, substitutions of one aliphatic amino acid for
another, such as Ile, Val, Leu, or Ala or substitutions of one polar residue for
another, such as between Lys and Arg, Glu and Asp, or Gln and Asn. Such substitutions
are well known in the art to provide variants having similar physical properties
and functional activities, such as for example, the ability to elicit and cross-react
with similar antibodies. Other variants include nucleic acids sequences that encode
a hybrid polypeptide having at least 50%, 60%, 70%, 80%, 90% or 95% amino acid
identity to polynucleotide encoded recombinant proteins. Preferred embodiments
are those having greater than 90% or 95% identity with the amino acid sequence
to the polynucleotide encoded recombinant proteins.
As will be appreciated by those of ordinary skill in the art, a nucleotide sequence
encoding a recombinant polypeptide or variant thereof may differ from the native
sequence due to codon degeneracy, nucleotide polymorphism, or nucleotide substitution,
deletion or insertion. Thus, in certain aspects the present invention includes
all degenerate nucleic acid molecules that encode peptides, polypeptides, and proteins
expressed using the nucleic acid expression control sequence of the present invention.
In another aspect, included are nucleic acid molecules that encode recombinant
polypeptide variants having conservative amino acid substitutions or deletions
or substitutions such that the recombinant polypeptide variant retains at least
one epitope capable of eliciting antibodies specific for the native protein.
In certain aspects, a nucleic acid sequence may be modified to encode a recombinant
polypeptide variant wherein specific codons of the nucleic acid sequence have been
changed to codons that are favored by a particular host and can result in enhanced
levels of expression (see, e.g., Haas et al.,
Curr. Biol. 6:315, 1996; Yang
et al.,
Nucleic Acids Res. 24:4592, 1996). For example, certain codons of
the immunogenic peptides obtained from streptococcal M proteins (and expressed
using pT5, SEQ ID NO:1) were optimized, without changing the primary sequence of
the peptides, for improved expression in
Escherichia coli (see FIG.
6).
By way of illustration and not limitation, eleven of thirteen arginine (Arg) codons
of AGG/AGA in the hexavalent A.1 hybrid polypeptide coding sequence were changed
to the Arg codons of CGT/CGC in hexavalent A.3 coding sequence. As is known in
the art, codons may be optimized for whichever host the hybrid polypeptide is to
be expressed in, including without limitation bacteria, fungi, insect cells, plant
cells, and mammalian cells. Additionally, codons encoding different amino acids
may be changed as well, wherein one or more codons encoding different amino acids
may be altered simultaneously as would best suit a particular host (e.g., codons
for arginine, glycine, leucine, and serine may all be optimized or any combination
thereof). Alternatively, codon optimization may result in one or more changes in
the primary amino acid sequence, such as a conservative amino acid substitution,
addition, deletion, or combination thereof.
Following transformation of a suitable host strain and growth
