# Patent

## Title
Vectors and methods for high throughput co-expression

## Abstract

The present invention includes vectors and methods for high throughput co-expression.

## Background
CONTINUING APPLICATION DATA
This application claims the benefit of U.S. Provisional Application Ser. No. 60/642,309, filed Jan. 7, 2005, U.S. Provisional Application Ser. No. 60/642,310, filed Jan. 7, 2005, and U.S. Provisional Application Ser. No. 60/756,028, titled “Vectors,” filed Jan. 4, 2006, each of which is incorporated by reference herein.


GOVERNMENT FUNDING
The present invention was made with government support under Grant No. NIH GM062407, awarded by the National Institutes of Health. The Government has certain rights in this invention.


BACKGROUND
With the completion of the sequencing of the human genomes and genomes of other organisms including, for example, the genomes of a wide and rapidly expanding number of prokaryotes, yeast, rice, rat, and dog, increasing attention has focused on the characterization and function of proteins, the products of genes. See, for example, Celestino et al., Gen Mol. Res. 3:421-431, 2004; Nature 436:793-800, 2005; Toh et al., Nature 438:803-819, 2005; Collins et al., Nature 422:835-847, 2003; and Cherry et al., Nature 387(6632 Suppl):67-73, 1997. The availability of sequence data and the growing impact of structural biology on biomedical research have prompted international efforts to determine protein structures on a large scale. Structural genomics (also referred to as “SG”) is a worldwide initiative aimed at determining a large number of protein structures in a high throughput mode (see, for example, Rost, Structure 6:259-63, 1998; and Stevens et al., Science 294:89-92, 2001). One such effort is the National Institutes of Health's Protein Structure Initiative, a large-scale, high-throughput (also referred to as “HTP”) effort to determine the three-dimensional atomic-level structures of a broad range of protein. These structures will be made widely available for clinical and basic studies that will expand the knowledge of the role of proteins both in normal biological processes and in disease. Initiatives, such as the Protein Structure Initiative, focus on an important aspect of proteins: the three-dimensional structures of proteins. While gene sequencing projects identify and arrange all the nucleotide bases in an organism's genetic material, efforts such as the Protein Structure Initiative will harness this genetic information to help identify and group into “families” all the natural shapes that proteins can form. To examine a protein's role in health and disease, and to explore ways to control its action, researchers seek to decipher the protein's shape, or structure. This structure reveals the physical, chemical and electrical properties of the protein and provides clues about its role in the body. See, for example, Norvell and Machalek, Nat Struct Biol 7 Suppl:931, 2000; the worldwide web at nigms.nih.gov/psi/ and rcsb.org/pdb/strucgen.html#Worldwide; and “From Genes to Proteins: NIGMS Catalogs the Shapes of Life,” NIH Record, February 2001.
In structural genomics-type high-throughput projects, thousands of genes must be inserted into expression vectors and it has become clear that protein expression and protein purification are limiting steps and a major expense. Traditional technologies of manipulating genes are too cumbersome and inefficient when one is dealing with more than a few genes at a time. See, for example, Rual et al., Curr Opin Chem Biol. 8(1):20-5, 2004.
While success rates for gene cloning are close to one hundred percent, only about twenty percent of targeted genes are successfully expressed and purified and an accurate crystal structure is obtained for only a fraction of those polypeptides that are expressed and purified. See, for example, Adams et al., Acc Chem Res 36:191-8, 2003; Brenner, Nat Struct Biol 7 Suppl:967-9, 2000; Brenner and Levitt, Protein Sci 9:197-200, 2000; Burley, Nat Struct Biol 7 Suppl:932-4, 2000; Chance et al., Biophysical Journal 82:454a-454a, 2002; Chayen, J Struct Funct Genomics 4:115-20, 2003; Lesley et al., Proc Natl Acad Sci USA 99:11664-9, 2002; and Christendat et al., Nat Struct Biol 7:903-9, 2000. Traditional technologies of manipulating genes are too cumbersome and inefficient when one is dealing with more than a few genes at a time. See, for example, Rual et al., Curr Opin Chem Biol. 8(1):20-5, 2004.
Current methodologies for determining protein structures are difficult and time-consuming. Thus, there is a need for products and methods that allow for the determination of protein structures in a low-cost and high-throughput manner.
SUMMARY OF THE INVENTION
The present invention includes expression vectors having a nucleotide sequence operably encoding a ccdB polypeptide flanked by att recombination recognition sequences, wherein the selectable marker for selection of the expression vector within a host cell is not ampicillin resistance.
In another aspect, the invention includes host cells having one or more expression vectors having a nucleotide sequence operably encoding a ccdB polypeptide flanked by att recombination recognition sequences, wherein the selectable marker for selection of the expression vector within a host cell is not ampicillin resistance.
In another aspect, the invention include methods for the expression of one or more polypeptides, the method including expressing at least one polypeptide by an expression vector having a nucleotide sequence operably encoding a ccdB polypeptide flanked by att recombination recognition sequences, wherein the selectable marker for selection of the expression vector within a host cell is not ampicillin resistance.
In another aspect, the invention includes methods for the co-expression of four or more polypeptides, the method including expressing at least one polypeptide by an expression vector having a nucleotide sequence operably encoding a ccdB polypeptide flanked by att recombination recognition sequences, wherein the selectable marker for selection of the expression vector within a host cell is not ampicillin resistance.
In another aspect, the invention includes methods of improving the solubility of one or more expressed polypeptides, the method including expressing at least one polypeptide by an expression vector having a nucleotide sequence operably encoding a ccdB polypeptide flanked by att recombination recognition sequences, wherein the selectable marker for selection of the expression vector within a host cell is not ampicillin resistance.
In another aspect, the invention includes the expression vector pDEST-C1, pDEST-C2, pDEST-C3, pDEST-CM1, pDEST-CM2, pDEST-CM3, pDEST-CM4, pDEST-CS, pDEST-CS1, pDEST-CS2, pDEST-CS3, pDEST-CS4, pDEST-CMZ1, or pDEST-CMZc1.
In another aspect, the invention includes host cells having one of more expression vectors selected from pDEST-C1, pDEST-C2, pDEST-C3, pDEST-CM1, pDEST-CM2, pDEST-CM3, pDEST-CM4, pDEST-CS, pDEST-CS1, pDEST-CS2, pDEST-CS3, pDEST-CS4, pDEST-CMZ1, and pDEST-CMZc1.
In another aspect, the invention includes polynucleotides having a nucleotide sequence operably encoding zeomycin resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR1 and attR2 sites. In some embodiments, the polynucleotide includes the G144704 cassette. In some embodiments, the G144704 cassette includes SEQ ID NO: 4, as shown in FIG. 9.
In another aspect, the invention includes expression vectors having a polynucleotide having a nucleotide sequence operably encoding zeomycin resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR1 and attR2 sites. In some embodiments, the expression vector includes a polynucleotide including a G144704 cassette. In some embodiments, the G144704 cassette includes SEQ ID NO: 4, as shown in FIG. 9.
In another aspect, the invention includes host cells having an expression vector having a polynucleotide having a nucleotide sequence operably encoding zeomycin resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR1 and attR2 sites. In some embodiments, the expression vector includes a polynucleotide including a G144704 cassette. In some embodiments, the G144704 cassette includes SEQ ID NO: 4, as shown in FIG. 9.
In another aspect, the invention includes methods for the expression of one or more polypeptides, the method including expressing at least one polypeptide by an expression vector having a polynucleotide having a nucleotide sequence operably encoding zeomycin resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR1 and attR2 sites. In some embodiments, the expression vector includes a polynucleotide including a G144704 cassette. In some embodiments, the G144704 cassette includes SEQ ID NO: 4, as shown in FIG. 9.
In another aspect, the invention includes methods for the co-expression of four or more polypeptides, the method including expressing at least one polypeptide by an expression vector having a polynucleotide having a nucleotide sequence operably encoding zeomycin resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR1 and attR2 sites. In some embodiments, the expression vector includes a polynucleotide including a G144704 cassette. In some embodiments, the G144704 cassette includes SEQ ID NO: 4, as shown in FIG. 9.
In another aspect, the invention includes methods of improving the solubility of one or more expressed polypeptides, the method including expressing at least one polypeptide by an expression vector having a polynucleotide having a nucleotide sequence operably encoding zeomycin resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR1 and attR2 sites. In some embodiments, the expression vector includes a polynucleotide including a G144704 cassette. In some embodiments, the G144704 cassette includes SEQ ID NO: 4, as shown in FIG. 9.
In another aspect, the invention includes polynucleotides having a nucleotide sequence operably encoding tetracycline resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding tetracycline zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR3 and attR4 sites. In some embodiments, the polynucleotide includes a tet Multisite. In some embodiments, the tet Multisite includes SEQ ID NO: 7 shown in FIG. 21.
In another aspect, the invention includes expression vectors including a polynucleotide having a nucleotide sequence operably encoding tetracycline resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding tetracycline zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR3 and attR4 sites. In some embodiments, the polynucleotide includes a tet Multisite. In some embodiments, the tet Multi site includes SEQ ID NO: 7 shown in FIG. 21.
In another aspect, the invention includes a host cell including an expression vector including a polynucleotide having a nucleotide sequence operably encoding tetracycline resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding tetracycline zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR3 and attR4 sites. In some embodiments, the polynucleotide includes a tet Multisite. In some embodiments, the tet Multisite includes SEQ ID NO: 7 shown in FIG. 21.
In another aspect, the invention includes methods for the expression of one or more polypeptides, the method including expressing at least one polypeptide by an expression vector including a polynucleotide having a nucleotide sequence operably encoding tetracycline resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding tetracycline zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR3 and attR4 sites. In some embodiments, the polynucleotide includes a tet Multisite. In some embodiments, the tet Multisite includes SEQ ID NO: 7 shown in FIG. 21.
In another aspect, the invention includes methods for the co-expression of four or more polypeptides, the method including expressing at least one polypeptide by an expression vector including a polynucleotide having a nucleotide sequence operably encoding tetracycline resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding tetracycline zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR3 and attR4 sites. In some embodiments, the polynucleotide includes a tet Multisite. In some embodiments, the tet Multisite includes SEQ ID NO: 7 shown in FIG. 21.
In another aspect, the invention includes methods of improving the solubility of one or more expressed polypeptides, the method including expressing at least one polypeptide by an expression vector including a polynucleotide having a nucleotide sequence operably encoding tetracycline resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding tetracycline zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR3 and attR4 sites. In some embodiments, the polynucleotide includes a tet Multisite. In some embodiments, the tet Multisite includes SEQ ID NO: 7 shown in FIG. 21.
In another aspect, the invention includes RNA interference (RNAi) vectors including a polynucleotide having a nucleotide sequence operably encoding zeomycin resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR1 and attR2 sites. In some embodiments, the polynucleotide includes a G144704 cassette. In some embodiments, the G144704 cassette includes SEQ ID NO: 4, as shown in FIG. 9.
In another aspect, the invention includes methods of producing one or more interfering RNA products, the method including expressing at least one interfering RNA product by an expression vector having a polynucleotide having a nucleotide sequence operably encoding zeomycin resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR1 and attR2 sites. In some embodiments, the expression vector includes a polynucleotide including a G144704 cassette. In some embodiments, the G144704 cassette includes SEQ ID NO: 4, as shown in FIG. 9.
In another aspect, the invention includes RNA interference (RNAi) vectors including a polynucleotide having a nucleotide sequence operably encoding tetracycline resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding tetracycline zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR3 and attR4 sites. In some embodiments, the polynucleotide includes a tet Multisite. In some embodiments, the tet Multisite includes SEQ ID NO: 7 shown in FIG. 21.
In another aspect, the invention includes methods of producing one or more interfering RNA products, the method including expressing at least one interfering RNA product by an expression vector including a polynucleotide having a nucleotide sequence operably encoding tetracycline resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding tetracycline zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide is flanked by attR3 and attR4 sites. In some embodiments, the polynucleotide includes a tet Multisite. In some embodiments, the tet Multisite includes SEQ ID NO: 7 shown in FIG. 21.
In another aspect, the invention includes the RNAi vectors pRIPPER-1, pRIPPER-2, pRIPPER-3, pRIPPER-4, pRIPPER-II, pRIPPER-III, and pRIPPER-IV.
In another aspect, the invention includes methods of producing one or more interfering RNA products, the method including expressing at least one interfering RNA product by an expression vector selected form pRIPPER-1, pRIPPER-2, pRIPPER-3, pRIPPER-4, pRIPPER-II, pRIPPER-III, or pRIPPER-IV.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic presenting the locations of the attR1 site, the attR2 site, the chloramphenical resistance gene, the ccdB gene and Primer 1 and Primer 2 in the Gateway Conversion Cassette for Reading Frame A, Reading Frame B and Reading Frame C.
FIG. 2 shows a map of the pDEST-C1 vector.
FIG. 3 presents the nucleotide sequence of the pDEST-C1 vector (SEQ ID NO: 1).
FIG. 4 shows a map of the pDEST-C2 vector.
FIG. 5 presents the nucleotide sequence of the pDEST-C2 vector (SEQ ID NO: 2).
FIG. 6 shows a map of the pDEST-C3 vector.
FIG. 7 presents the nucleotide sequence of the pDEST-C3 vector (SEQ ID NO: 3).
FIGS. 8A-8D show maps of the zeomycin gene, the Gateway cassette, and the G144704 cassette. FIG. 8A shows a map of the zeomycin cassette. FIG. 8B shows a map of the Gateway cassette. FIG. 8C shows a map of the G144704 cassette. FIG. 8D is a map of the G144704 cassette, indicating the location of various restriction enzyme sites.
FIG. 9 shows the nucleotide sequence of the G144704 cassette (SEQ ID NO: 4). The nucleotide sequences of the attR1 and attR2 sites are shaded (Encoded protein disclosed as SEQ ID NO: 45).
FIG. 10 show a SDS-PAGE demonstrating expression of various Shewanella oneidensis (“Son”) polypeptides. Lane 1 shows expression of Son-3961. Lane 2 shows expression of Son-0433. Lane 3 shows expression of Son-1358. Lane 4 shows parallel expression of Son-3961, Son-0433 and Son-1358. Lane 5 shows expression of Son-1350. Lane 6 shows expression of Son-2015. Lane 7 shows parallel expression of Son-1350 and Son-2015. Lane “M” is molecular weight markers.
FIGS. 11A and 11B show the co-expression of Clostrodium thermocellum JW-20 gene construct in pDEST-C1-C3 vectors. FIG. 11A is an SDS-PAGE of the total cell extract of the recombinant expression experiments for each Clostrodium thermocellum JW-20 gene construct. The black arrows point to the bands that correspond to the proteins expressed in the cells. FIG. 11B is a chart identifying each of the lanes. Lanes 1 through 12 are cells with two different, randomly selected, genes expressed. Lanes 13-18 are the expression testing of those cells with only one expression construct, to monitor expression of the individual proteins. Lanes 19 and 20 show expression of three non-interacting proteins.
FIG. 12 shows the co-expression of a stable protein complex for Son0433 and Son1284. Lanes 1-3 show consecutive fractions from gel-filtration. Lane 4 shows the elution from the NiNTA, 6×HIS (SEQ ID NO: 44) affinity column. Lanes 5-7 show total cell extracts; Lane 5 shows the co-expression of Son0433 and Son1284; Lanes 7 and 6 show the single expression experiments of Son0433 and Son1284, respectively.
FIG. 13 shows parallel expression of non-interacting proteins in the same cell. Lanes 13-18 are the expression studies of single genes per cell. Lanes 1-12 are the expression tests of two genes per cell. Lane 19 is the parallel expression of three genes.
FIGS. 14A and 14B show improved recombinant protein solubility with co-expression of the two individual proteins, calmodulin and the human Plasma Membrane Calcium ATPase C-terminal calmodulin binding domain (hPMCA4b). In FIG. 14A, lanes 1 and 8 are the molecular weight markers; lanes 2, 4, and 6 are the three soluble fractions that represent calmodulin hPMCA4b and co-expression of the two, respectively; lanes 3, 5 and 7 are the pellet fractions of the same growths, and lane 6 contains the soluble complex and this growth was further pursued to purification. FIG. 14B is the FPLC 280 nm chromatogram and SDS-PAGE of the indicated fraction in lane 9 showing a complex of the two proteins. Lane 10 is the same marker as lanes 1 and 8 in FIG. 14A.
FIG. 15 is an SDS-PAGE gel showing expression of the PF194 and PF1955 proteins. Each pair of lanes shows total cell fraction (“P”) and soluble (“S”) fraction, side by side. “M” represents molecular size markers (6×His tag disclosed as SEQ ID NO: 44).
FIG. 16 shows a map of the pDEST-CM1 vector.
FIG. 17 presents the nucleotide sequence of the pDEST-CM1 vector (SEQ ID NO: 5).
FIG. 18 shows a map of the pDESTCM-2 vector.
FIG. 19 presents the nucleotide sequence of the pDEST-CM2 vector (SEQ ID NO: 6).
FIG. 20 shows a map of the Multisite TetR cassette.
FIG. 21 is the nucleotide sequence of the Multisite TetR cassette (SEQ ID NO: 7).
FIG. 22 shows a map of the pDEST-CM3 vector.
FIG. 23 show the nucleotide sequence of the pDEST-CM3 vector (SEQ ID NO: 8).
FIG. 24 shows a map of the pDEST-CM4 vector.
FIG. 25 presents the nucleotide sequence of the pDEST-CM4 vector (SEQ ID NO: 9).
FIGS. 26A-26C show the various cassettes used in the construction of the pRIPPER vectors. FIG. 26A is the Gateway® cassette. FIG. 26B is the G144704 cassette. FIG. 26C is a Multisite® cassette.
FIG. 27 is a map of the pRIPPER-3 vector.
FIG. 28 is the nucleotide sequence of the pRIPPER-3 vector (SEQ ID NO: 10).
FIG. 29 is a map of the pRIPPER-1 vector.
FIG. 30 is the nucleotide sequence of the pRIPPER-1 vector (SEQ ID NO: 11).
FIG. 31 is the map of the pRIPPER-2 vector.
FIG. 32 is the nucleotide sequence of the pRIPPER-2 vector (SEQ ID NO: 12).
FIG. 33 is the map of the pRIPPER-4 vector.
FIG. 34 is the nucleotide sequence of the pRIPPER-4 vector (SEQ ID NO: 13).
FIG. 35 is a map of the pRIPPER-II vector.
FIG. 36 is the nucleotide sequence of the pRIPPER-II vector (SEQ ID NO: 14).
FIG. 37 is a map of the pRIPPER-III vector.
FIG. 38 is the nucleotide sequence of the pRIPPER-III vector (SEQ ID NO: 15).
FIG. 39 is a map of the pRIPPER-IV vector.
FIG. 40 is the nucleotide sequence of the pRIPPER-IV vector (SEQ ID NO: 16).
FIG. 41 is a map of the pDEST-CS.
FIG. 42 is the nucleotide sequence of the pDEST-CS vector (SEQ ID NO: 17).
FIG. 43 is a map of the pDEST-CS1 vector.
FIG. 44 is the nucleotide sequence of the pDEST-C1 vector (SEQ ID NO: 18).
FIG. 45 is a map of the pDEST-CS2 vector.
FIG. 46 is the nucleotide sequence of the pDEST-CS2 vector (SEQ ID NO: 19).
FIG. 47 is a map of the pDEST-CS3 vector.
FIG. 48 is the nucleotide sequence of the pDEST-CS3 vector (SEQ ID NO: 20).
FIG. 49 is a map of the pDEST-CS4 vector.
FIG. 50 is the nucleotide sequence of the pDEST-CS4 vector (SEQ ID NO: 21).
FIG. 51 is a map of the pDEST-CMZ1 vector.
FIG. 52 is the nucleotide sequence of the pDEST-CMZ1 vector (SEQ ID NO: 22).
FIG. 53 is a map of the pDEST-CMc1 vector.
FIG. 54 is the nucleotide sequence of the pDEST-CMZc1 vector (SEQ ID NO: 23).



DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION
The polynucleotides, vectors and methods of the present invention provide for the improved high throughput (HTP) expression of polypeptides and for the improved high throughput (HTP) expression of interfering RNAs.
The present invention includes vectors having a nucleotide sequence operably encoding a ccdB polypeptide flanked by att recombination recognition sequences, wherein the selectable marker for selection of the expression vector within a host cell is not resistance to the antibiotic ampicillin. Selectable markers for the expression vector within the host cell include, but are not limited to, kanamycin resistance, chloramphenicol resistance, streptomycin resistance, spectinomycin resistance, zeomycin resistance, carbenicillin resistance, tetracycline resistance, and rifampicin resistance. See, for example, Novagen 2004/2005 catalog and New England Biolabs 2005-06 Catalog. In some aspects, the present invention includes vectors having a nucleotide sequence operably encoding a ccdB polypeptide and operably encoding chloramphenicol resistance, wherein the nucleotide sequence is flanked by att recombination recognition sequences, and wherein the selectable marker for selection of the expression vector within a host cell is not ampicillin resistance. In some aspects of the present invention, the vector is an expression vector.
As used herein, the terms “polynucleotide” and “nucleotide sequence” refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
As used herein, a “vector” is a polynucleotide which transfers an inserted nucleic acid molecule into and/or between host cells. A vector may provide for the insertion of DNA or RNA into a cell, the replication of DNA or RNA, the transcription of the DNA or RNA, the translation of the DNA or RNA, and/or the processing of the translated polypeptide product. A vector may provide for more than one of the above functions. A vector may include an origin of replication, also referred to as an ori sequence or a replicon, which allows for replication of the polynucleotide in an appropriate host cell. See, for example, Novagen 2004/2005 catalog and New England Biolabs 2005-06 Catalog.
As used herein, an “expression vector” is a vector which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s). An “expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product. An expression vector may include an origin of replication, which allows for replication of the polynucleotide in an appropriate host cell. Origins of replication include, but are not limited to the ColE1 replicon, the P15A replicon, the CloDF13 replicon, or the the RSD1030 replicon. An expression vector may include a promoter, including, for example, the T7lac promoter, that provides for protein expression in the host cell. See, for example, Novagen 2004/2005 catalog and New England Biolabs 2005-06 Catalog.
As used herein, “expression” refers to the process by which a polynucleotide is transcribed into mRNA and/or the process by which the transcribed mRNA (also referred to as “transcript”) is subsequently being translated into peptides, polypeptides, or proteins. The transcripts and the encoded polypeptides are collectively referred to as gene product.
The polynucleotides and vectors of the present invention take advantage of Gateway® technology. The Gateway® cloning system is an vitro site-specific recombination technology that takes advantage of the att site-specific recombination properties of bacteriophage lambda (Hartley et al., Genome Res. 10: 1788-1795, 2000; (Landy, Annu Rev Biochem 58:913-49, 1989; Sasaki et al., J. Biotechnol. 107, 233-243, 2004; U.S. Pat. Nos. 5,888,732, 6,143,557, 6,171,861, 6,270,969, 6,277,608, and 6,720,140; and the Gateway Technology manual Version E, updated Sep. 22, 2003; available on the worldwide web at invitrogen.con/content/sfs/manuals/gatewayman.pdf) to provide a rapid and efficient way to move a gene of interest between multiple vector systems.
Polynucleotides and vectors of the present invention include one or more att recombination recognition sequences. As used herein, att recombination recognition sequences include, but are not limited to attR1, attR2, attR3, and attR4, the sequences of which are well known, and include, but are not limited to, those described in the examples included herewith and those described in Landy, Annu Rev Biochem 58:913-49, 1989; Sasaki et al., J. Biotechnol. 107, 233-243, 2004; U.S. Pat. Nos. 5,888,732, 6,143,557, 6,171,861, 6,270,969, 6,277,608, and 6,720,140; and the Gateway Technology manual Version E, updated Sep. 22, 2003; available on the worldwide web at invitrogen.com/content/sfs/manuals/gatewayman.pdf. The two recombination recognition sequences, attR1 and attR2 have been employed in the conventional gateway technology (Hartley et al., Genome Res. 10:1788-1795, 2000; (Landy, Annu Rev Biochem 58:913-49, 1989; Sasaki et al., J. Biotechnol. 107, 233-243, 2004; U.S. Pat. Nos. 5,888,732, 6,143,557, 6,171,861, 6,270,969, 6,277,608, and 6,720,140; and the Gateway Technology manual Version E, updated Sep. 22, 2003; available on the worldwide web at invitrogen.com/content/sfs/manuals/gatewayman.pdf). The recombination recognition sequences, attR3 and attR4, have been recently made available as MultiSiteTM Gateway® Three-Fragment Vector Construction Kit from Invitrogen Corp.
Gateway® technology makes gene cloning simpler, more specific and faster than traditional methods of gene cloning based on restriction enzyme digestion and ligation. Gateway® technology allows for the rapid site specific exchange of target DNA between an entry vector (containing the initial clone of the target gene) and multiple expression vectors, via recombination. In order to allow for this versatility, the Gateway® system is characterized by a DNA sequence called the Gateway® cassette. This DNA sequence contains two recombination sites attR1 and attR2 along with genes that encode chloramphenicol resistance and the “control of cell death” polypeptide, also referred to herein as ccdB (Bernard et al., J. Mol. Biol. 234, 534-541, 1993). The ccdB polypeptide is lethal to Escherichia coli (Bernard and Couturier, Mol. Gen. Genet. 226:297-304, 1991). Nucleotide sequences encoding the ccdB polypeptide are well known in the art, and include those described in the examples included herewith. A Gateway cassette allows for the selection of recombinants, as only the desired recombinants will form colonies when transformed into E. coli. 
In some aspects, polynucleotides and vectors of the present invention may have a Gateway® cassette, wherein a Gateway® cassette is a polynucleotide sequence containing the two recombination sites attR1 and attR2, along with a nucleotide sequence that operably encodes the gene product responsible for chloramphenicol resistance and the nucleotide sequence operably encoding a ccdB polypeptide. The two recombination sites, attR1 and attR2, may flank the nucleotide sequence encoding chloramphenicol resistance and the nucleotide sequence encoding a ccdB polypeptide. As used herein, a nucleotide sequence that “operably encodes” a polypeptide product with a given function includes all of the appropriate sequences necessary to result in the expression of the polypeptide product with the identified function, including, for example, coding sequences and regulatory sequences.
In some aspects, polynucleotides and vectors of the present invention may have a MultiSiteTM Gateway® cassette, wherein a MultiSiteTM Gateway® cassette is a polynucleotide sequence containing the two recombination sites attR3 and attR4, along with a nucleotide sequence that operably encodes the gene product responsible for chloramphenicol resistance and the nucleotide sequence operably encoding a ccdB polypeptide. The two recombination sites, attR3 and attR4, may flank the nucleotide sequence encoding chloramphenicol resistance and the nucleotide sequence encoding a ccdB polypeptide.
The present invention includes vectors having a nucleotide sequence operably encoding a ccdB polypeptide flanked by att recombination recognition sequences, wherein the selectable marker for selection of the expression vector within a host cell is not ampicillin resistance. Selectable markers for the expression vector within the host cell include, but are not limited to, kanamycin resistance, chloramphenicol resistance, streptomycin resistance, spectinomycin resistance, zeomycin resistance, carbenicillin resistance, tetracycline resistance, and rifampicin resistance. See, for example, Novagen 2004/2005 catalog and New England Biolabs 2005-06 Catalog. Of the more than fifty Gateway® expression vectors currently available, all encode ampicillin resistance, which is not desirable for an expression vector, due to high background problems when grown for over sixteen.
Vectors of the present invention include vectors in which the chloramphenicol resistance (chlR) gene in the Gateway® recombination cassette has been replaced with the Zeocin® resistance gene as a selective marker. Vectors of the present invention include vectors in which the technologies of the Gateway® site-specific recombination system is fused with the capacities of a multiple vector co-expression system, resulting is vectors that can express multiple proteins in a parallel manner in one E. coli strain.
A series of several vectors of the present invention, when compared to one another, may have differing replicons and resistance genes, allowing for the effective propagation, maintenance of the series of vectors in a single host cell.
The vectors of the present invention may be used in concert with any of the many available co-expression vectors, including, for example, the pET family of expression vectors (Novagen, Madison, Wis.). These vectors use a strong phage T7 promoter driven by the presence of lactose or a lactose analog (isopropyl-β-D-galactopyranoside, IPTG) to express the target protein. Other available co-expression vectors include pCDF, pRSF, and pACYC, which are compatible with pET (www.emdbiosciences.com). All four of these have compatible replication origins, and different antibiotic selection markers, so as many as all four can be stably maintained in the same E. coli cell. Thus, co-expression of up to four proteins in the same cell became possible. These vectors have been further modified (the pDUET series; see www.emdbiosciences.com) so that each vector contains two multiple cloning site, allowing for expression of up to eight different target genes simultaneously. However, while these vectors are well known, their use is limited due to the necessity of using classical restriction enzymes and ligation for cloning.
The vectors of the present invention combine aspects of co-expression vectors with the power of Gateway® technology. The vectors of the present invention may include one or more of the elements of such co-expression vectors, including, but not limited, one or more elements from a member of the pET family of vectors, the pDUET series of vectors, pCDF, pRSF, and pACYC.
The vectors of the present invention may include one or more additional elements, including, for example, elements encoding various fusion tags, fusion proteins, affinity tags, protease cleavage sites, expression signals, or promoters, including, for example, prokaryotic or eukaryotic promoters. Vectors may include, for example, an N-terminal 6×His tag (SEQ ID NO: 44) and/or an enterokinase cleavage site just before the attR1 recombination site. The vectors of the present invention may include any of the various nucleotide sequences that provide for the expression of native proteins, N- or C-terminally tagged proteins, secreted proteins, or proteins that are targeted to a subcellular location. The vectors of the present invention may include any of the various nucleotide sequences that provide a variety of promoters, poly-A addition signals, and/or elements for transient, stable and bicistronic expression.
The vectors of the present invention may include the appropriate promoters and/or origins of replication that allow for the expression of a polypeptide product in a range of host cells, including, but not limited to, bacterial host cells, including, for example, E. coli, yeast, insect cells, and mammalian cells.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 2. An example of such a vector is the pDEST-C1 vector, the nucleotide sequence of which is shown in FIG. 3.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 4. An example of such a vector is the pDEST-C2 vector, the nucleotide sequence of which is shown in FIG. 5.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 6. An example of such a vector is the pDEST-C3 vector, the nucleotide sequence of which is shown in FIG. 7.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 16. An example of such a vector is the pDEST-CM1 vector, the nucleotide sequence of which is shown in FIG. 17.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 18. An example of such a vector is the pDEST-CM2 vector, the nucleotide sequence of which is shown in FIG. 19.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 22. An example of such a vector is the pDEST-CM3, the nucleotide sequence of which is shown in FIG. 23.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 24. An example of such a vector is the pDEST-CM4 vector, the nucleotide sequence of which is shown in FIG. 25.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 41. An example of such a vector is the pDEST-CS vector, the nucleotide sequence of which is shown in FIG. 42.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 43. An example of such a vector is the pDEST-CS1 vector, the nucleotide sequence of which is shown in FIG. 44.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 45. An example of such a vector is the pDEST-CS2, the nucleotide sequence of which is shown in FIG. 46.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 47. An example of such a vector is the pDEST-CS3, the nucleotide sequence of which is shown in FIG. 48.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 49. An example of such a vector is the pDEST-CS4, the nucleotide sequence of which is shown in FIG. 50.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 51. An example of such a vector is the pDEST-CMZ1, the nucleotide sequence of which is shown in FIG. 52.
Vectors of the present invention include vectors having one or more of the elements shown in FIG. 53. An example of such a vector is the pDEST-CMc1, the nucleotide sequence of which is shown in FIG. 54.
The vectors of the present invention, when used together in the same cell, can express in a parallel manner multiple open reading frames (“ORFs”). As used herein, an ORF may include, for example, a polypeptide product, mRNA product, and an RNAi product. For example, up to two ORFs, two or more ORFs, up to three ORFs, three or more ORFs, up to four ORFs, four or more ORFs, up to six ORFs, six or more ORFs, up to eight ORFs, eight or more ORFs , up to ten ORFs, ten or more ORFs, up to twelve ORFs, twelve or more ORFs, up to sixteen ORFs, sixteen or more ORFs, up to twenty ORFs, twenty or more ORFs , up to twenty-two ORFs, twenty-two or more ORFs, up to twenty-four ORFs, twenty-four or more ORFs, up to twenty-eight ORFs, twenty-eight or more ORFs, up to thirty ORFs, thirty or more ORFs , up to thirty-two ORFs, thirty-two, or more ORFs may be expressed in a single host cell using a combination of the vectors of the present invention. Any combination of vectors of the present invention may be used in concert with any number of other available expression vectors to express multiple reading frames. The present invention also includes methods of expressing such ORFs using the one or more of the vectors of the present invention. The present invention also includes methods of expressing such ORFs using the one or more of the vectors of the present invention in combination with one or more additional expression vectors.
One, two, three, four, five, six, seven, eight, nine, ten, or more of the vectors described herein may be used in a single co-expression experiment. The vectors of the present invention may be used along with any number of currently available expression vectors and/or newly developed co-expression vectors. Examples of such vectors include, but are not limited to, those reported in the scientific literature and the many commercially available expression vectors, including those marketed, for example, Invitrogen, Novagen (Novagen 2004/2005 Catalog), Promega (Promega 2005 Life Sciences Catalog), Stratagene (Strategene 2005-06 Catalog)and New England Biolabs (2006 NEB catalog).
The expression vectors of the present invention may be used to for the expression of one or more polypeptides. The polypeptide may be homologous to the host cell, which includes, for example, proteins or peptides that are naturally encoded by the host cell, from a native DNA sequence, or a substitution, deletion, and/or insertion variant thereof. The polypeptide may also be heterologous to the host cell, expressed on a heterologous nucleic acid sequence, which includes, for example, proteins and peptides that are not naturally expressed by the host cell, proteins and peptides that are naturally expressed or encoded by the host cell, and substitutions, deletions, and/or insertion variants of proteins and peptides that are naturally expressed or encoded by the host cell. The protein or peptide may be a fusion protein, comprising two or more polypeptides that are synthesized from a nucleic acid molecule encoding both polypeptides under the control of a single set of translational control elements. The fusion protein may include a linker peptide situated between the polypeptides.
A DNA molecule encoding the polypeptide may be prepared using well known recombinant DNA technology methods such as those set forth in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001) and/or Ausubel et al., eds, (Current Protocols in Molecular Biology, Green Publishing Assoc., Inc. John Wiley & Sons, Inc., N.Y., 1994).
Insertion (also referred to as “transformation” or “transfection”) of a vector of the present invention into the selected host cell may be accomplished using any of a variety of methods. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan. Transformation of a bacterial host with the vector of the present invention may be accomplished using any of a variety of methods. For example, any of the methods described in the examples herein, and any of the well known methods such as those set forth, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, may be used. Methods that may be used include, for example, such methods as calcium phosphate precipitation or electroporation. Likewise, transfection of the vectors of the present invention into mammalian cells may be accomplished using any of the many known methods. For example, transfection into mammalian cells may be accomplished using LipofectamineTM (Invitrogen Corp) according to the supplier's instructions.
Any of a variety of host-expression vector systems may be used to express the protein or peptide. These include, but are not limited to, microorganisms such as bacteria, yeast, insect cell systems, plant cell systems, or animal cell systems, including, for example, any of a variety of murine and human cells systems. The co-expression systems of the present invention may be used to express therapeutic proteins, including protein complexes, including, for example antibodies.
The vectors of the present invention provide many innovations, including, but not limited to the following. Cconstructs may be created in the same reaction and plated on different antibiotic supplemented plates. Each of these vectors can be used by itself, which allows for their use as a conventional expression vector. The throughput of expression screening is increased by testing up to four non-interacting ORFs' expression/solubility in the same cells. The vectors of the present invention can be used in methods of solubilizing proteins through soluble complex expression. The vectors of the present invention may be used in methods of high throughput (HTP) expression of already known protein complexes. And, the vectors of the present invention are compatible with the existing Gateway cloning system via the well-known L/R reaction.
In some aspects, polynucleotides and vectors of the of the present invention may include a cassette, wherein the cassette is a polynucleotide sequence containing the two recombination sites, for example attR1 and attR2 or attR3 and attR4, along with a nucleotide sequence that operably encodes the ccdB polypeptide. The two recombination sites flank the nucleotide sequence encoding encoding a ccdB polypeptide. This cassette may also include a nucleotide sequence that operably encodes a selectable marker other than chloramphenicol resistance, including for example, zeocin resistance. ZeocinTM, a registered trademark of Cayla, is the commercial name of a formulation containing Phleomycin D1, an antibiotic of the bleomycin family (also called the phleomycin family). The ble gene encodes zeocin antibiotic resistance, conferring resistance to an antibiotic of the phleomycin family (U.S. Pat. Nos. 5,021,344 and 5,118,620). In the nucleotides and vectors of the present invention, the efficiency of the Gateway® recombination reaction is not affected by the insertion of the Zeocin gene between the att recombination recognition sites.
The present invention includes a polynucleotide, also referred to herein as a “cassette,” wherein the cassette has a nucleotide sequence operably encoding zeomycin resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the nucleotide sequence operably encoding zeomycin resistance and the nucleotide sequence operably encoding a ccdB polypeptide are flanked by attR sites, including, for example, attR1 and attR2 or attR3 and attR4 recombinant recognition sites. An example of such a cassette is a cassette including one or more of the elements shown in FIGS. 8C and 8D. An example of such a cassette is the G144704 cassette, the nucleotide sequence of which is shown in FIG. 9. Another example of such a cassette is a cassette having one or more of the elements shown in FIG. 20. An example of such a cassette is the Multisite TetR cassette, the nucleotide sequence of which is shown in FIG. 21.
Vectors of the present invention allow for the tandem expression of more than one polypeptide product from a single co-expression vector. See, also, Sone et al. (Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: Modular construction of multiple cDNA expression elements using recombinant cloning, “J Biotechnol. 2005 Jun. 24 (doi: 10,1016/jbiotec.2005.02.02 1)).
The present invention also includes methods of improving the solubility of expressed polypeptides by co-expressing more than one polypeptide using one or more of the vectors described herein. The vectors described herein may be used in concert with additional, available expression vectors in such methods of improving the solubility of one or more expressed polypeptides. The solubility of an expressed polypeptide can be determined using standard methods known in the art, including any of the methods described in the examples included herewith. For example, host cells may be collected three to twenty hours after induction and the cells are lysed. Cell lysis may be accomplished using physical methods such as homogenization, sonication, French press, microfluidizer, or the like, or by using chemical methods such as treatment of the cells with EDTA and a detergent (see Falconer et al., Biotechnol. Bioengin. 53:453-458 [1997]) or by taking advantage of the lytic activities of some bacteriophage proteins (Crabtree, S. & Cronan, J. E., J. Bact., 1984, 158:354-356). In some cases, it may be advantageous to combine more than one technique.
Expression of and mRNA or polypeptide product by a vector of the present invention may be assayed by any of a wide variety of methods, including any of those described herein.
Additionally, for example, fluorescent proteins with different excitation and emission wavelengths can be used to label a target product of a cDNA or to be expressed solely. Examples of such fluorescent proteins include, for example, EGFP (BD Biosciences Clontech Inc.; GenBank accession no.: U55763) (Cormack et al., Gene 173:33-38, 1996; Zhang et al., Biochem. Biophys. Res. Commun. 227:707-711, 1996), Venus (EYFP-F46L/F64L/M153T/V163A/S175G) (Nagai et al., Nat. Biotechnol. 20:87-90, 2002), SECFP (ECFP-K27R/N165H) (Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 98:14997-15002, 2001), DsRed2 (BD Biosciences Clontech Inc.) (Matz et al., Nat. Biotechnol. 17:969-973, 1999; Terskikh et al., J. Biol. Chem. 277:7633-7636.2002) and mRFP1 (GenBank accession no.: AF506027) (Campbell et al., Proc. Natl. Acad. Sci. U.S.A. 99:7877-7882, 2002).
The present invention includes nucleotides and vectors for use in producing interfering RNA molecules for use in RNA interference (RNAi) studies. RNA is a biological process that involves sequence-specific mRNA degradation that is mediated by short interfering RNA (siRNA) molecules generated from the cleavage of dsRNA homologous to the gene targeted for silencing. The mechanism of RNAi-mediated specific gene silencing was first discovered in C. elegans and has also been found in other organisms, including Drosophila, hydra, zebrafish, and trypanasomes.
While the exact mechanism behind RNA interference is still not entirely understood, it appears that a dsRNA is processed into 20-25 nucleotide short interfering RNAs (siRNAs) by an Rnase III-like enzyme called Dicer. The siRNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs). The siRNA strands are then unwound to form activated RISCs, and the siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (discussed in Bass, B., Nature 411:428-429 (2001) and Sharp, P. A., Genes Dev. 15:485-490 (2001)). Although the phenomenon of RNAi was first characterized in C. elegans and Drosophila, RNAi has also been demonstrated to work in mammalian cells (Wianny, F. and Zernica-Goetz, M., (2000), Natrue Cell Biology Vol 2., 70-75.
Accordingly, the invention includes both novel methods and compositions for reducing nonspecific suppression and novel methods and compositions for performing RNAi to reduce expression of target genes.
Nucleotides and vectors of the present invention for use in RNAi may include any of the various nucleotides, vectors, cassettes and elements described herein.
The RNAi vectors of the present invention may be used in any of the many available RNAi systems. For example, the RNAi vectors of the present invention may used to produce an interfering RNA product in the worm Cerenohabditis elegans (C. elegans). For example, worms may be fed with bacteria transformed with one or more vectors of the present invention. Currently available methods of RNAi allow for only gene to be studied at a time. The vectors of the present invention provide HTP RNAi vectors and provide for the large scale RNAi investigation of multiple genes at a time, for example, up to four, up to eight, up to ten, up to twelve, up to sixteen, up to twenty, up to twenty-four, up to twenty-eight, or up to thirty-two genes at one time.
RNAi vectors of the present invention include vectors having one or more of the elements shown in FIG. 27. An example of such a vector is the pRIPPER-3 vector, the nucleotide sequence of which is shown in FIG. 28.
RNAi vectors of the present invention include vectors having one or more of the elements shown in FIG. 29. An example of such a vector is the pRIPPER-1 vector, the nucleotide sequence of which is shown in FIG. 30.
RNAi vectors of the present invention include vectors having one or more of the elements shown in FIG. 31. An example of such a vector is the pRIPPER-2 vector, the nucleotide sequence of which is shown in FIG. 32.
RNAi vectors of the present invention include vectors having one or more of the elements shown in FIG. 33. An example of such a vector is the pRIPPER-4 vector, the nucleotide sequence of which is shown in FIG. 34.
RNAi vectors of the present invention include vectors having one or more of the elements shown in FIG. 35. An example of such a vector is the pRIPPER-II vector, the nucleotide sequence of which is shown in FIG. 36.
RNAi vectors of the present invention include vectors having one or more of the elements shown in FIG. 37. An example of such a vector is the pRIPPER-IV vector, the nucleotide sequence of which is shown in FIG. 38.
RNAi vectors of the present invention include vectors having one or more of the elements shown in FIG. 39. An example of such a vector is the pRIPPER-IV vector, the nucleotide sequence of which is shown in FIG. 40.
The present invention also includes methods of producing one or more interfering RNAs using one or more of the vectors described herein. The present invention also includes methods of inhibiting the expression of a gene, thereby inhibiting gene function, in a host cell or animal model system, including, for example, C. elegans and transgeneic animals, such as transgenic mice, by expressing one or more interfering RNAs in the host cell or animal model system using the vectors described herein. Methods for producing and assaying the effect of interfering RNAs produced by the vectors described herein may be by any of the many available methods. See, for example, materials available on the world wide web at ambion.com/techlib/resources/RNAi/; “RNAi” A “How To” for New Users” TechNotes 11(5), Ambion, 2006; or “RNA Interference and Gene Silencing—History and Overview,” Ambion, May 20, 2002.
The vectors and method of the present invention may be used to lower production costs by allowing the use of parallel gene expression, the simultaneous expression of multiple proteins in the same cell. For example, multiple vector constructs, for example, four, sixteen, or thirty-two constructs, may be in a single expression experiment. For example, four constructs, each containing an affinity tag with a varying cleavage site can be used and four different trials can be carried out simultaneously in one experiment and therefore greatly increases protein expression and screening efficiency.
The vectors and methods of the present invention also allow for the use of different antibiotic resistance encoding vectors in addition to the Ampicillin resistance encoding pDEST vectors currently in use. Furthermore, the vectors and methods of the present invention allow for the co-expression of soluble multiple-protein complexes and for RNAi studies of certain organisms where many genes are turned on/off simultaneously. The vectors and method of the present invention will facilitate large scale operations in protein production. The vectors and method of the present invention provide for the co-expression of stable protein complexes. The vectors and method of the present invention may be used for the co-expression of endogenous and/or exogenous proteins. The vectors and method of the present invention may be used for the expression of secreted, intracellular and/or periplasmic polypeptides.
The vectors and methods of the present invention also allow for parallel gene expression and target salvaging at the gene level. Recombinant expression is a technique of choice for the synthesis of proteins of interest for structural genomics (“SG”) studies. However, based on the recent structural genomics initiatives' data, less than one half of attempted open reading frames are expressed solubly and a fraction of these actually become structures (Liu et al., Acta Crystallogr D Biol Crystallogr 61(Pt 6):679-84, 2005). The total cost and speed of the structural determinations can be deciding factors in efficiency of SG studies. When a protein does not express, or is expressed, but insoluble, using the standard recombinant protocols, then multiple approaches have to be attempted, including co-expression with potential partners (Shen et al., Proteome Sci, 3(1):3, 2005). Proteins in their native environment exist as part of complexes, bound by other, specific proteins with weak, non-covalent interactions (Sorensen and Mortensen, J Biotechnol 115(2):113-28, 2005). Soluble proteins have been shown to solubilize other, previously insoluble, proteins (Sorensen and Mortensen, J Biotechnol 115(2):113-28, 2005). The vectors and methods of the present invention allow for screening multiple proteins (and their interactions) in the same cell and can be used to facilitate the expression of proteins that are known essential parts of a stable protein-complex and to individual, non-interacting proteins.
Since many proteins are tightly bound subunits of multi-protein complexes in vivo (Wang and Chong, Proc Natl Acad Sci USA 100:478-483, 2003) co-expression of multiple target proteins is a tool for successful heterologous protein expression. It has been shown for genes that are in a stable complex in the native host, that co-expression can improve their solubility and expression yield (Bernard and Couturier, Mol. Gen. Genet. 226:297-304, 1991; Li et al., Proc Natl Acad Sci USA 94:2278-2283, 1997; and Henricksen et al., J Biol Chem, 269:11121-11132 1994). Using the vectors and methods of the present invention, genes may be co-expressed either on the same expression vector from the same promoter, or using multiple, compatible vectors each containing one target gene.
The polynucleotides and vectors of the present invention can be used in methods to perform rapid and convenient construction of many different types of expression clones carrying multiple cDNAs on a single vector for their simultaneous introduction into cells.
The polynucleotides and vectors of the present invention can be used in methods of co-expression that allow for the parallel expression of multiple proteins that are essential subunits of stable protein complexes. In most of these protein partners are difficult to identify, and once they are known, other questions arise, which requires the creation of new constructs and attempting new complexes. Addressing such question using conventional technologies is laborious and time consuming. The vectors and methods of the present invention allow such questions to be addressed in a single reaction. With the vectors and methods of the present invention, one can mix and match any proteins in a potential complex easily in hours not weeks.
Many, if not most proteins in living cells exist as part of complexes, bound by a large number of weak, non-covalent interactions. The expression of a protein in the absence of a partner protein required for it to fold properly, or for stabilization, will in most cases lead to an insoluble (incorrectly folded) product, or degradation of the target protein by the cell's normal recycling systems. The vectors and methods of the present invention provide for parallel expression of multiple proteins that form a soluble and stable complex.
The vectors and methods of the present invention may be used to simultaneously and stoichiometrically introduce multiple heterologous genes into a single living cell and has many applications in proteomic research. For example, the vectors and methods of the present invention may be in used in study of pathways, cascades, multi-unit functional protein complexes, receptor-ligand interactions and the like.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
EXAMPLES
Example 1
Vectors for High Throughput (HTP) Recombinant Co-expression of Genes in E. coli 
To develop vectors for use in high-throughput co-expression methods, various commercially available co-expression vectors were modified to make them compatible for use in the Gateways® system, marketed by Invitrogen. The nucleic acid sequences encompassing the att recombination sites of the Gateway® system were transferred into each of the commercially available co-expression vectors pCDF-1b (Novagen catalog no. 71330-3), pRSF-1b (Novagen catalog no. 1363-3), and pACYCDuetTM-1 (Novagen catalog no. 71147-3). The new vector constructs are called pDEST-C1, pDEST-C2, and pDEST-C3, respectively. The development of the pDEST-C3 vector is discussed in more detail in Example 2. The nucleotide sequence of vectors obtained from Novagen and Invitrogen is available online, for example at invitrogen.com and novagen.com.
Invitrogen's Gateway® technology allows one to quickly shuttle a cloned insert between various vectors systems, such as cloning vectors and expression vectors. The Gateway® system utilizes phage lambda site-specific att recombination sites to make cloning simpler, more specific, and faster in comparison to traditional methods utilizing restriction enzyme digestion and ligation. As marketed, the Gateway® system is suitable for the expression of only single proteins, not the co-expression of multiple proteins. The nucleic acid sequence encompassing the att recombination site is also referred to as the Gateway® cloning cassette and includes the two recombination recognitions sequences, a gene encoding chloramphenicol resistance, and the ccdB gene.
A commercially available Gateway® conversion kit (Invitrogen, catalog #118280) was used for the initial conversion of the pCDF-1b and pRSF-1b vectors to derive the pDEST-C1 and pDEST-C2 vectors. Development of the pDEST-C3 vector required a modification of the commercially available Gateway® cassette, replacing a chloramphenicol resistance gene with a gene encoding zeocin resistance. This modified cassette, called the G144704 cassette, is discussed in more detail in Example 2.
The Gateway® conversion kit includes three different versions of the Gateway® cassette, representing reading frame A, reading frame B, and reading Frame C.1 (see FIG. 1). Gateway® cassette reading frame B was used in the construction of the pDEST-C1, pDEST-C2, and pDEST-C3 vectors. FIG. 1 presents the locations of the attR1 site, attR2 site, chloramphenicol resistance gene, ccdB gene and Prime 1 and Primer 2 for Gateway Conversion Cassettes for Reading Frame A, Reading Frame B and Reading Frame C.1. Procedures were as described in more detail in Invitrogen Life Technologies Instruction Manual Gateway® Vector Conversion System with One Shot® ccdB SurvivalTM Competent Cells (Catalog no. 11828-029, Version A, 14 Jun. 2004, 25-0748, Invitrogen Life Technologies).
The PshAI restriction site present in both the pCDF-1b and pRSF-1b vectors was used for the insertion of the Gateway® cassette. The restriction enzyme PshA1 recognizes the nucleotide sequence GACNN|NNGTC (SEQ ID NO: 38). For the pDEST-C1 vector, the PshA1 recognition sequence in the pCDF-1b vector is 5′-GACAA|GAGTC-3′ (SEQ ID NO: 39) and the resultant sequence after the insertion of the Gateway cassette is 5′-GACAAATCAAC . . . GTTGATGAGTC-3′ (SEQ ID NOS 40 & 41). For the pDEST-C2 vector, the PshA1 recognition sequence in the pRSF-1b vector is 5′-GACAA|GAGTC-3′ (SEQ ID NO: 39) and the resultant sequence after the insertion of the Gateway cassette is 5′-GACAAGAGCTC . . . AAGCTTGAGTC-3′ (SEQ ID NOS 42 & 43).
The pDEST-C1 vector was selected for by growth of on streptomycin and chloramphenicol. The pDEST-C2 vector was selected for by growth on kanamycin and chloramphenicol. The orientation of the insert was verified by digestion with PstI. A proper insert results in a PstI fragment of about 500 basepairs on an agarose gel. Plasmids showing the correct digestion pattern on an agarose gel were sequenced to ensure that the destination vector was created in the proper reading frame.
FIG. 2 is a map of the pDEST-C1 vector. FIG. 3 presents the nucleotide sequence of the pDEST-C1 vector. FIG. 4 is a map of the pDEST-C2 vector. FIG. 5 presents the nucleotide sequence of the pDEST-C2 vector.
Example 2
A Zeocin Resistant Gateway Technology Cassette
Currently available co-expression vectors do not include a vector with resistance to the antibiotic zeocin as a selectable marker. In this example, a co-expression vector containing both the Gateway® cloning cassette and zeocin resistance was created by replacing the chloramphenicol gene within a Gateway® cassette with the gene encoding zeocin resistance. The resultant modified Gateway® cassette is called the G144704 Zeocin resistant Gateway® Cassette. The G144704 Zeocin resistant Gateway® Cassette was inserted into Novagen's pACYCDuetTM-1 expression vector. The resultant zeomycin resistant co-expression vector was named pDEST-C3. FIG. 6 is a map of the pDEST-C3 vector. FIG. 7 presents the nucleotide sequence of the pDEST-C3 vector.
The gene encoding zeocin resistance, shown in FIG. 8A, was obtained from the pDONR-Zeo vector (Invitrogen catalog no. 12535-035). The nucleotide sequence of the pDONOR-Zeo vector is available online at novagen.com.
Zeocin is an antibiotic that is an effective selection reagent in E. coli (Drocourt et al., Nucleic Acids Res. 18:4009, 1990). Analysis of the commercially available pDONOR-Zeo vector sequence with the New England Biolab's NEBcutter software (Vincze et al., Nucleic Acids Res. 31:3688-3691, 2003) identified the 510 nucleotide long zeomycin gene and upstream regulatory sequences of approximately 58 nucleotides (FIG. 8A). This sequence is cut by the restriction enzyme BglI at nucleotide 242. A set of primers was designed for amplification of this region by PCR which also contained a 5′ NotI site and a 3′ AgeI site (restriction enzymes that flank the CAT gene within a Gateway cassette (see FIG. 8B). The 5′ Zeocin gene primer used was 5′-GTT TCT TGC GGC CGC CAC GTT AAG GGA TTT TGG TCA-3′ (SEQ ID NO: 24) and the 3′ Zeocin gene primer used was 5′-GTT TCT TAC CGG TGT TGC AAC GAA CAG GTC ACT-3′ (SEQ ID NO: 25).
The expression vector pDEST160 (Invitrogen) was used as the source for the Gateway DNA sequences (FIG. 8B) for amplification by PCR. The sequence was analyzed for restriction enzyme recognition sites. The enzymes SacI and HindIII, on the 5′ and 3′ ends, respectively, where identified as restriction enzymes that do not cut within the cassette and, thus, allow the cloning of the cassette into the new expression vector pACYC (Novagen, San Diego, Calif.). The restriction enzymes NotI and AgeI were selected as enzymes that cut the cassette at specific locations, allowing for the removal of the CAT gene from the Gateway cassette and replacement with the Zeo gene.
Polymerase chain reaction (PCR) was used to amplify this template. Primers used in the amplification of the Gateway cassette were designed with SacI and HindIII sites on the 5′ and 3′ ends, respectively. The Gateway cassette 5′ primer was 5′-GTT TCT TGA GCT CGAT CAC AAG TTT GTA CAA AAA AGC-3′ (SEQ ID NO: 26) and the Gateway cassette 3′ primer was 5′-GTT TCT TAA GCT TAG CAG CCG GAT CTG ATC TTA-3′ (SEQ ID NO: 27).
After PCR amplification of both the Gateway cassette, the resultant PCR products were digested with the enzymes indicated in FIG. 8B by the enzymes SacI and HindIII. The cassette had to be further digested with the restriction enzyme EcoRI, as fragments B and C in FIG. 8B are indistinguishable on a gel with sizes 820 and 773 nucleotides, respectively. Cutting fragment B with EcoRI, into a 500 nucleotide and a 320 nucleotide fragment, facilitated the accurate identification and isolation of fragments A and C. Fragment A and Fragment C were purified from agarose gels using standard techniques (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989).
Fragment A and Fragment C (FIG. 8B) along with the digested Zeo PCR product were purified and mixed with the vector pACYC (digested with SacI and HindIII and gel-purified) and T4 DNA ligase. The ligation reaction was incubated overnight at 13° C. and transformed into a strain of E. coli called ccdB survival cells on plates containing both zeocin and chloramphenicol. Colonies were picked and grown up for plasmid DNA isolation. Each of the plasmids were digested with SacI, HindIII and BglI, since BglI cuts the Zeo gene, but it does not cut the commercially available Gateway cassette or pACYC. SacI and HindIII separate the plasmid and the newly inserted fragment. The correct digestion pattern as well, as correct molecular weight, were observed on agarose gel, confirming success of the new construct.
FIG. 8A shows a schematic of the cassette modification. Each site is labeled with the nucleotide number where the respective sites end. FIG. 8A shows the Zeocin gene as amplified for this experiment containing all regulatory regions. FIG. 8B shows the original Gateway cassette as amplified for this experiment. FIGS. 8C and 8D show the G144704 cassette. The G144704 cassette is also referred to herein “GatewayZeoPH.” The strategy discussed above will be used to create additional co-expression vectors encoding tetracycline resistance. In FIG. 8B, the capital letters A, B, and C represent the three fragments of the original Gateway cassette when cleaved by enzymes listed in black. Fragments A and C are retained throughout the experiments. R1 and R2 represent the attR1 attR2 sites respectively. Each of the genes are represented by the following abbreviations: Zeo, zeocin resistance gene; CAT, chloramphenicol acetyl transferase (chloramphenicol resistance gene); ccdB, encodes a protein that stabilizes gyrase covalent intermediates and is lethal for E. coli cells not containing the ccdA gene (Bernard and Couturier, Mol. Gen. Genet. 226:297-304, 1991; and Salmon et al., Mol. Gen. Genet. 244:530-538, 1994).
Sequencing of the three plasmid clones confirmed the construct. The sequence of the G144704 cassette is shown in FIG. 9. Both the orientation and DNA sequence of the new cassette has been confirmed by DNA sequencing. This plasmid was designated pDEST-C3. Using the new destination vector pDEST-C3, a number of genes have been tested for expression of the correct molecular weight proteins.
The plasmid pDEST-C3 was then utilized in a L/R recombination reaction to test that the recombination sites were intact and that genes could be cloned into the Gateway R1 R2 cassette. The efficiency of the reaction was not affected by the insertion of the Zeocin gene between the recombination sites. To date, six different genes have been tested for expression of the correct molecular weight proteins using this destination vector.
The pDEST-C3 vector, along with the pDESt-C1 and pDEST-C2 vectors described in Example 1, are three new Gateway® compatible expression/destination vectors that, when used in combination with one of the many currently available co-expression vectors allows for the expression in a parallel manner up to four genes. Each of the pDEST-C1, pDEST-C2, and pDEST-C3 vectors can also be used individually, as a conventional expression vector. Each of the vectors can be maintained individually. Each of these vectors can be further modified to include various fusion tags/proteins, protease cleavage sites, expression signals. The plasmids presently have an N-terminal 6×His tag (SEQ ID NO: 44) and pDEST-C1 and pDEST-C2 have an enterokinase cleavage site just before the attR1recombination site.
Example 3
Expression of Son Proteins
The vectors of the present invention were used to simultaneously express various Shewanella oneidensis (“Son”) polypeptides; Son-3961, Son-0433, Son-1358, Son-1350, and Son-2015. The Son-3961 polypeptide was expressed in the pDEST-15G vector; Son-0433 polypeptide was expressed in the pDEST-C3 vector; Son-1358 polypeptide was expressed in the pDEST-C2 vector; Son-1350 polypeptide was expressed in the pDEST-C1 vector; and Son-2015 was expressed in the pDEST-15 vector.
The respective plasmids were transformed into BL21 (DE3) cells (Stratagene, La Jolla, Calif.) and the cells were grown in LB media and induced with 1 mM IPTG at OD of 0.6. The cells were then allowed to grow for four hours. Cells were harvested by spinning at 13,000 RPM for 1 minute and the resulting pellet was then run on an SDS-PAGE gel. The results are shown in FIG. 10. Lane 1 shows the expression of Son-3961. Lane 2 shows the expression of Son-0433. Lane 3 shows the expression of Son-1358. Lane 4 shows the parallel expression of Son-396 1, Son-0433 and Son-1358. Lane 5 shows the expression of Son-1350. Lane 6 shows expression of Son-2015. Each of the four constructs created contains an affinity tag with varying cleavage sites. Thus, up to four different trials (see FIG. 10, lanes 4 and 7) can be carried out simultaneously in one experiment and therefore may greatly increase protein expression and screening efficiency.
Example 4
Expression of Clostrodium thermocellum JW-20 Polypeptides
Materials and Methods
For expression studies, genes cloned lab from the organism Clostrodium thermocellum JW-20 were expressed using the pDEST-C1, pDEST-C2, and pDEST-C3 vectors. Entry vectors were created with the use of pDONR-221 (kanamycin resistance) or pDONR-Zeo (Zeocin® resistance). The L/R recombination reaction was used to insert six genes into the R1-R2 sites of each of the vectors. These individual vectors were then transformed into Mach-1 E. coli cells (Invitrogen). After plasmid DNA was purified and the size of the DNA was confirmed by agarose gel electrophoresis, E. coli BL21 (DE3) cells were transformed with each of the vectors at the same time and plated on media supplemented with the appropriate antibiotics. These gene targets were chosen as proteins which had been expressed and purified successfully in earlier experiments. They were grown in one milliliter small scale test cultures overnight in LB medium. The cells were then induced with 2 mM IPTG for 5 five hours and the total cell and soluble fractions, where applicable were run on a gel. Soluble fraction of cells was achieved by incubating the cell pellet with five mg/ml lysozyme for fifteen minutes at room temperature.
For studies on the purification of protein complexes, cloned genes from the organism Shewanella oneidensis (Son) were provided Dr. Jizhong Zhou from Oak Ridge National Labs. The genes Son0433 (also called Regulator of Sigma Factor D) and Son1284 (also called Sigma Factor D) were cloned into pDEST 221. These clones were then inserted into each of the vectors (Son1284 into pDEST-C3 and Son 0433 into DEST C1) and the colonies were grown in defined PA 0.5 G media overnight. These two proteins (Son0433 and Son1284) were identified as a complex by Database of Interacting Proteins. These cultures were then inoculated into 5 L of PASM 5052 media that is self inducing and was labeled with Seleno-Methionine. The cultured were grown at 30 degrees celsius for sixteen hours. The cells were harvested by centrifugation and the cells were lysed by sonication in 50 mM HEPES buffer pH 7.6/500 mM NaCl. Both of the proteins were purified by their individual 6×HIS tags (SEQ ID NO: 44), in the same solution by one step elution with 400 mM imidazole. The eluted sample was then passed through a Superdex 75 column in 100 mM NaCl, 50 mM HEPES pH 7.6, 1 mM DTT. FIG. 12 shows the fractions from the purification, showing that the proteins formed a complex.
mRNA detection. E. coli BL21(DE3) cells containing the gene Pfu-89099 cloned into the entry clones pDEST-C1, pDEST-C2, pDEST-C3 and pET15G were grown overnight with the appropriate antibiotics and induced with 3 mM IPTG for six hours. Total RNA was isolated with the RNeasy kit from Qiagen (Valencia, Calif.) and was run on a 1.0% agarose gel. The amount of RNA was quantitated with the intensity of the band detected in the gel.
Results
Compatibility with existing system. The new vectors pDEST-C1, pDEST-C2, and pDEST-C3 are compatible with the existing Gateway cloning system via the well-known L/R reaction. The efficiency of the reaction was not affected by the insertion of the zeocin gene between the recombination sites. The proteins expressed are all the correct molecular weight with 2 shown in experiments below. Both the orientation and DNA sequence of the new cassette has been confirmed by DNA sequencing. The most widely used entry vectors used today encode kanamycin resistance. If these vectors are used, they can make the isolation of correct destination vectors from the L/R reaction with pDEST-C2 difficult and not HTP. The use of pDONR-Zeo is recommended for the use of these vectors, since other antibiotic resistance markers can interfere with the cloning into any of the four vectors.
Parallel screening of genes. In the 2×2×2 matrix shown in FIG. 11, all of the genes made their predicted protein product. The expression of two vectors at the same time is cost effective and convenient. With transformation of the third and fourth vectors, a reduced antibiotic concentration resulted in improved DNA quality. There is no apparent difference in growth rate when using any combination of the vectors used. The proteins are expressed in a similar manner in all cases, but the amount of the protein made is different in each case.
Each vector/protein construct was also expressed individually (FIG. 11). All individual expression experiments show expression of the proteins in a similar way as the parallel expression studies. This shows that our system is compatible with the previously established Gateway® system.
FIG. 11A is an SDS-PAGE of the total cell extract for each Clostrodium thermocellum JW-20 gene construct. The black arrows point to the bands that correspond to the proteins expressed in the cells. FIG. 11B is a chart identifying each of the lanes. Lanes 1 through 12 are cells with two different, randomly selected, genes expressed. Lanes 13-18 are the expression testing of those cells with only one expression construct, to monitor expression of the individual proteins. Lanes 19 and 20 are the two lanes, where three non-interacting proteins are expressed.
Parallel expression for purification. The experiments illustrate that the expression system can produce the desired protein. The data shows that varying combinations of ORFs can be expressed in parallel, in the same cell, without inhibiting each others' expression. Since these proteins are also soluble as observed during single expression experiments, the purification of all of the soluble proteins is possible, especially if the proteins expressed together have large difference in molecular weight, or only three vectors are used together. pDEST-C1 and pDEST-C2 contain an enterokinase cleavage site which can also be used for cleavage of the tag, while pDEST-C3 does not. All Gateway® compatible clones in our collection have a Tobacco Etch Virus (TEV) protease cleavage site between the gene and the recombination sequence for a final processing step. This ensures that the protein attempted for crystallization will have the native sequence, without any extra amino acids. All constructs are optimized for structural biological experiments, so when the protein is fully processed only 2 N-terminal glycines are extra to the native sequence.
Co-expression of a stable protein complex. Gateway® compatible HTP protein complex expression is the power of the present invention. Expressing more than one protein at the same time to form a stable protein complex is shown in this example. When the newly developed co-expression or parallel expression system is used, these experiments now can be carried out in a HTP manner, or with the use of all of the resources available with Gateway® compatible clones. The two genes used in here, Son0433 (Regulator of Sigma Factor D (rsd)) and Son1284 (Sigma Factor D (rpoD)), were co-expressed in culture and they co-purified in gel filtration, in the same fraction. The proteins expressed separately before the co-expression studies and the total yield of protein during co-expression was comparable, although the expression level of Son0433 was slightly affected (see a comparison of lanes 5, 6, and 7 in FIG. 12). The expression level for Son 1284 seemed to have decreased in this case. The interaction between these two proteins was not investigated. The separation of the two proteins was not possible in this co-expression experiment, due to their seemingly stable interaction with each other. Only the smaller molecular weight protein (Son 0433), due to its excess concentration compared to (Son1284) could be isolated from the mixture, by filtering the solution through a 50 kDa MW cutoff concentrator.
FIG. 12 shows the co-expression of a stable protein complex. Proteases were a problem with rpoD; the effect of proteolysis on rpoD can be seen in lane 3 of FIG. 12. Lanes 1-3 of FIG. 12 were the consecutive fractions from gel-filtration through a Superdex 75 (Amersham, Piscataway, N.J.). Lane 4 of FIG. 12 is the elution from the NiNTA, 6×HIS (SEQ ID NO: 44) affinity column. Lanes 5 through 7 are the total cell gels, with Lane 5 showing the co-expression of Son0433 and Son1284. Lanes 7 and 6 are the single expression experiments of Son0433 and Son 1284 respectively. These results for the Son0433 and Son1284 proteins are also shown in lanes 1-4 of FIG. 10.
This example facilitates the parallel expression of proteins that are essential parts of a stable protein complex. In most of these protein partners are difficult to identify, and once they are known, other questions arise, which requires the creation of new constructs and new complexes. These second set of experiments, or sub-cloning, used to be laborious and time consuming. The true power of this system lies here, since with these novel vectors, scientists now can mix and match any proteins in a potential complex easily in hours not weeks. The parallel expression testing or the parallel expression of non-interacting proteins is an added benefit, which was not anticipated. Beyond those benefits already listed, some of the other potentials of this system is that the use of expression vectors that encode for antibiotics other than ampicillin are preferred for protein expression, especially when the cells have to be grown for long times, for increased time of induction. With the vectors of the present invention, a drastic reduction of cell mass is observed, when compared to non-ampicillin resistance encoding vectors in defined media. The effects of defined media on the expression is important, since labeling of each protein with seleno-mnethionine allows for the HTP structure determination of each protein. Using currently available vectors in concert with the vectors of the present example, the amount of cells has increased.
Example 5
Parallel Gene Expression and Target Salvaging at Gene Level
This example demonstrates that three Gateway® compatible coexpression vectors, pDEST-C1, pDEST-C2, and pDEST-C3, when used together, along with a commercially available expression vector, in the same cell, can express in a parallel manner up to four ORFs.
Materials and Methods
Expression testing. The genes for all ORFs shown were cloned from the organism Clostrodium thermocellum JW-20. Entry vectors were created with the use of pDONR-221 (kanamycin resistance) or pDONR-Zeo (Zeocin® resistance). The L/R recombination reaction was used to insert six ORFs into the R1-R2 sites of each of the vectors. These individual vectors were then transformed into Mach-1 E. coli cells (Invitrogen). After plasmid DNA was purified the size of the DNA was confirmed by agarose gel electrophoresis and E. coli BL21 (DE3) cells were transformed with each of the vectors at the same time and plated on media supplemented with the appropriate antibiotics. These ORFs were chosen randomly from proteins that had been expressed and purified successfully in earlier experiments. They were grown in one milliliter small scale test cultures overnight in LB medium. The cells were then induced with 2 mM IPTG for five hours and the total cell and soluble fractions, where applicable were run on a gel. Soluble fraction of cells was achieved by incubating the cell pellet with five mg/ml lysozyme for fifteen minutes at room temperature.
Solubilization and Purification of a protein complex. The human Plasma Membrane Calcium ATPase C-terminal tail was cloned by Hua Yang. This ninety amino acid 10.5 kDa protein was truncated to amino acid 1055 through 1142. This construct was predicted to have secondary structure by JPRED (Cuff et al., Bioinformatics 14:892-3, 1998). The PCR product was cloned into pDONR-Zeo vector. The hPMCA construct was then inserted into pDEST-C1. The Calmodulin construct was supplied by Dr. Jeffrey Urbauer in a pET 15 plasmid. The two vectors were then co-transformed into BL21-DE3 cells. These cultures were then inoculated into five liters of PASM 5052 media, a self inducing media labeled with seleno-methionine. The cultures were grown at 30° C. for sixteen hours. The cells were harvested by centrifugation at 4000 g and were lysed by sonication in 50 mM HEPES buffer pH 7.6 500 mM NaCl. Both of the proteins were purified by their individual 6×HIS tags (SEQ ID NO: 44), in the same solution by one step elution with 400 mM imidazole. The eluted sample was then passed through a Superdex 75 column in 100 mM NaCl, 50 mM HEPES pH 7.6, 1 mM DTT.
Results
Compatibility with existing system. The new vectors pDEST-Cx described here are compatible with the existing Gateway® cloning system via the well-known L/R reaction. The efficiency of the reaction was not noticeably affected by the insertion of the Zeocin® gene between the recombination sites. The proteins expressed are all the correct molecular weight. Both the orientation and DNA sequence of the new DESTination vectors has been confirmed by DNA sequencing. The most widely used entry vectors encode kanamycin resistance which is not productive when using the pDEST-C1-C3 vectors. The use of pDONR-Zeo is recommended use as a pDONR vector, since other antibiotic resistance markers can interfere with each of the four vectors.
Parallel screening of genes. In the 2×2×2 matrix shown in FIG. 13, all of the genes made their predicted protein product. The expression of two vectors at the same time is the most cost effective and convenient method used here with cell yields similar as those cells expressing each construct individually. Transformation with the third and fourth vectors demonstrates a reduced success rate, but this can be overcome by reducing the concentration of the antibiotics. Nonetheless each non-interacting, co-expressed protein, was made in this system as they were when expressed individually FIG. 13. The same expression profile are seen for the co-expression of three proteins. The proteins were all known to be soluble prior to expression and the soluble cell fractions were loaded onto the gel in FIG. 13. FIG. 13 shows parallel expression of non-interacting proteins in the same cell. Lanes 13-18 are the expression studies of single genes per cell. Lanes 1-12 are the expression tests of 2 genes per cell. Lane 19 is the parallel expression of three genes. The results from this co-expression experiment are also presented in FIG. 11.
Improving protein solubility with co-expression of a protein complex. The protein complexes expressed are of greater use than just co-expression of already soluble proteins. Co-expression of previously insoluble protein with soluble, well expressing partner has been known to improve solubility. In this experiment, the insoluble human Plasma Membrane Calcium ATPase isoform 4b's (hPMCA4b) C-terminal tail was used as the insoluble protein. Previous work has shown that this part of the protein insoluble (see FIG. 13, lane 5) and it has been identified as the Calmodulin Binding Domain of the protein (Kessler et al., Biochemistry 31:11785-92, 1992). This insoluble protein was co-expressed, using our vectors, with Calmodulin, a soluble protein. These two proteins have been known to interact (Elshorst et al., Biochemistry 38:12320-32, 1999). Upon their co-expression, as shown in FIG. 14A, the two proteins are co-expressed and the previously insoluble C-terminal fragment of hPMCA4b is now soluble and in a complex with Calmodulin. This complex can also be purified together as shown by lane 9 of FIG. 14B.
Improving recombinant protein solubility with coexpression. The expression testing of the two individual proteins, calmodulin and the human Plasma Membrane Calcium ATPase C-terminal calmodulin binding domain (hPMCA4b) is shown in FIGS. 14A and 14B. The hPMCA protein was truncated for structural studies. The amino acids used here are from 1055 through 1145. This construct is the JPRED predicted structured part of the hPMCA (Cuff et al., Bioinformatics 14:892-3, 1998. In FIG. 14A, lanes 1 and 8 are the molecular weight markers; lanes 2, 4, and 6 are the three soluble fractions that represent calmodulin hPMCA4b and co-expression of the two, respectively; lanes 3, 5 and 7 are the pellet fractions of the same growths; lane 6 contains the soluble complex and this growth was further pursued to purification. FIG., 14B show the FPLC 280 nm chromatogram and the SDS-PAGE of the indicated fraction in lane 9 showing a complex of the two proteins. Lane 10 is the same marker as lanes 1 and 8 of FIG. 13.
The pDEST-C1, pDEST-C2, and pDEST-C3 coexpression vectors present at least five new innovations. One, all four of these constructs are created with the same reaction and plated on four different antibiotic supplemented plates. Two, each of these vectors can also be used by itself, which allows for their use as just a conventional expression vector. Three, increasing the throughput of expression screening by testing up to four non-interacting ORFs' expression/solubility in the same cells. Four, they are new tools for solubilizing proteins through soluble complex expression. And, five, HTP Expression of already known protein complexes. The use of parallel processing at the screening and expression level enables the structural genomics community to express, in an HTP manner, protein-protein complexes and cut costs by increasing expression efficiency of non-interacting soluble proteins.
Example 6
Copy Number and Increased Protein Solubility
Currently available recombinant protein expression system, based on very efficient and fast RNA polymerases, such as T7 polymerase, used in the DE3 cell lines are very powerful and widely used. However, the benefit of the increased efficiency with this expression system presents problems. The overabundance of the recombinant transcript causes the cells to translate the transcript faster than the protein can be folded. This folding inefficiency then interferes with soluble protein expression.
A solution for this problem with protein expression is to use the vectors of the present invention to manipulate the copy number of the vectors that encode the recombinant proteins. The vectors pDEST-C1, pDEST-C2, and pDEST-C3 have different replicons which results in different numbers of copies in the cell. This characteristic facilitates their use as co-expression vectors.
Previously, the protein PF1955 from the organism Pyrococcus furiosus was expressed and its structure determined. This effort took two years to accomplish, since the protein was not soluble. The vector pDEST527 was used to express this protein for refolding studies. The present example demonstrates that the PF1955 protein can be expressed in a complex or by itself, when using a vector with a lower copy number that that of pDEST527. The results are shown in FIG. 15, a SDS-PAGE gel of the expression experiment.
In FIG. 15, every pair of lanes show total cell fraction (“P”) and soluble (“S”) fraction side by side. Lanes 1 and 2 show expression of PF1955 protein in the vector pDEST-C1. Lanes 1 and 2 show that in the PF1955 protein is soluble when expressed in the pDEST-C1 vector. Lanes 3 and 4 of FIG. 15 show expression of the PF1955 and PF1954 proteins, each cloned into the pET24 vector. These two genes are structured in the same operon in the genome of Pyrococcus furiosus. Lanes 3 and 4 demonstrate expression of the complex using traditional methods of protein complex expression. Lanes 5 and 6 show expressions of the PF1954 protein in the pET24 vector. Lanes 7 and 8 show expression of the PF1955 protein in the pDEST-C1 vector and expression of the PF1954 protein in the pET24 vector. An expression pattern similar to the control was observed. Lanes 9 and 10 show expression of PF1955 protein in the pDEST-C3 vector and expression of the PF1954 protein in the pET24 vector. Little expression was observed. Lanes 11 and 12 show expression of the PF1955 protein in the pDEST42 vector and expression of the PF1954 protein in the pET24 vector. Both the plasmids have the same replicon, but different antibiotics resistances. These lanes show no expression of either protein from the complex, suggesting that plasmid copy number has an effect of recombinant protein expression. Lane “M” represents molecular size markers.
In the experiments of this example, the pDEST-527 vector has the 6HIS tag (SEQ ID NO: 44) of Met R S G S H H H H H H R S D I T S L Y K K A E R E (SEQ ID NO: 28) while the pDEST-C1 vector has 6HIS tag (SEQ ID NO: 44) of Met A H H H H H H V G T G S N D D D D K S T S L Y K K A E R E (SEQ ID NO: 29). The difference in HIS tags is five amino acids, encoding an enterokinase cleavage site. The pDEST-527 vector has a copy number of 40 copies per cell and the pDEST-C1 vector has a copy number of 20-40 copies per cell.
This example demonstrates that the use of an alternate copy number vector alone can improve solubility. This example also shows that different copy number plasmids, when used together, can efficiently form a complex of two proteins (PF1954 and PF1955) that interact on protein level.
Example 7
pDEST-CM Vectors
The additional vectors pDEST-CM1, pDEST-CM2, pDEST-CM3, and pDEST-CM4 were developed. To develop these vectors, the commercially available Multisite Gateway®. Three-Fragment cassette, pDEST R4-R3 (Invitrogen, catalog #12537-023) was introduced into the commercially available vectors pCDF-Duet1 (Novagen catalog #71340-3), pRSF-Duet1 (Novagen catalog #71341-3), pACYC-Duet1 (Novagen catalog #71147-3), and pET-Duet 1 (EMD Biosciences, catalog #71146-3). The nucleotide sequence of vectors obtained from Novagen and Invitrogen is available online, for example at invitrogen.com and novagen.com.
Modification of the pCDF-Duet1 vector resulted in the pDEST-CM1 vector. FIG. 16 shows a map of the pDEST-CM1 and FIG. 17 presents the nucleotide sequence of the pDEST-CM1 vector. pDEST-CM1 was created by amplifying the Gateway® Multisite cassette from pDESTR4-R3 and adding SacI and HindIII sites to the 5′ and 3′ ends, respectively. This construct was then ligated to similarly digested pCDF-Duet1, to create pDEST-CM1. The nucleotide sequence of the pCDF-Duet1 vector is available online, for example at novagen.com.
Modification of the pRSF-Duet1 vector resulted in the pDEST-CM2 vector. FIG. 18 shows a map of the pDESTCM-2 vector and FIG. 19 presents the nucleotide sequence of the pDEST-CM2 vector. pDEST-CM2 was created by amplifying the Gateway® Multisite cassette from pDESTR4-R3 and adding SacI and HindIII sites to the 5′ and 3′ ends, respectively. This construct was then ligated to similarly digested pRSF-Duet1, to create pDEST-CM1. The nucleotide sequence of the pRSF-Duet1 vector is available online, for example at novagen.com.
Modification of the pACYC-Duet1 vector resulted in the pDEST-CM3 vector. To construct the pDEST-CM3 vector, a tetracycline resistance multisite cassette (TetR multisite cassette) was first created by amplifying the multisite cassette from the pDEST R4-R3 vector by PCR with the same primers used for the creation of the pDEST-CM1, pDEST-CM2, and pDEST-CM4 vectors. The tetracycline resistance (TetR) gene was amplified from the plasmid pBR322 (Promega #D1511) using as a 5′ primer:

    GTTTCTTGCGGCCGCTTCTCATGTTTGACAGCTTATCAT (SEQ ID NO: 30) (creating a recognition site for the restriction enzyme NotI) and as a 3′ primer:
    GTTTCTTTCTAGAGACGCGATGGATATGTTCTG (SEQ ID NO: 31) (creating a recognition site for the restriction enzyme XbaI).


The two PCR reactions then were then cleaved with enzymes from New England Biolabs using standard protocols. The Multisite PCR product was digested with HindIII, SacI, NotI and XbaI, creating two fragments of interest; a 834 bp SacI-XbaI fragment and a 208 bp NotI-HindIII fragment. Fragments were gel purified using standard methods. The TetR gene PCR product was digested with NotI and XbaI and ligated to the two remaining fragments of the Multisite cassette, replacing the CamR gene with the TetR gene between the NotI and XbaI sites. This was simultaneously ligated to HindIII, SacI digested pACYCDuet-1to create pDEST-CM3 (See FIG. 20). Note this cannot be moved out using EcoNI and HindIII as the other Multisite cassette constructs were made due to internal EcoNI and HindIII sites in this construct.
FIG. 20 shows a map of the Multisite TetR cassette. FIG. 21 is the nucleotide sequence of the Multisite TetR cassette (SEQ ID NO: 7).
FIG. 22 shows a map of the pDEST-CM3 vector. FIG. 23 is the nucleotide sequence of the pDEST-C3 vector (SEQ ID NO: 8)
Modification of the pET-Duet 1 vector resulted in the pDEST-CM4 vector. FIG. 24 shows a map of the pDEST-CM4 and FIG. 25 presents the nucleotide sequence of the pDEST-CM4 vector. The pDEST-CM4 vector was created by amplifying the Gateway Multisite cassette from pDESTR4-R3 and adding SacI and HindIII sites to the 5′ and 3′ ends, respectively. This was then ligated to similarly digested pET-Duet1.
The pDEST-CM1, pDEST-CM2, pDEST-CM3, and pDEST-CM4 vectors were produced following the procedures described in Example 1 for the development of the pDEST-C1, pDEST-C2, and pDEST-C3 vectors. A Multisite Gateway® cassette with SacI and HindIII ends was cloned into the first multicloning site of each pDUET vector.
The pDEST-CM series vectors will allow the simultaneous expression of up to sixteen genes, twelve cloned by recombination into the Multisite, and four cloned by standard techniques into the second multiple cloning site on each vector.
Example 8
High Throughput RNAi Co-interference
Current RNA-mediated interference (RNAi) assays study one gene at a time. Current RNAi vectors rely on T7 promoters and Lac operator sequences. To allow for the analysis of multiple genes simultaneously, this example presents five new vectors for use in methods of high throughput RNAi co-interference. These five vectors are pRIPPER-1, pRIPPER-2, pRIPPER-3, pRIPPER-II, and pRIPPER-III. Each of these high throughput RNAi vectors contains two T7 promoters (“T7”) and two Lac Operator sequences (“LacO”) pointing towards the Gateway® cassette from both sides (See FIG. 26A). The vectors also have an antibiotic resistance gene and an origin of replication.
Each of the new vectors pRIPPER-1, pRIPPER-2, and pRIPPER-3 contain a Zeocin-labeled Gateway® cassette G144704 flanked by two T7 promoters and Lac Operator sequences (see FIG. 26B) pointing at the Gateway® sequences. This DNA cassette is from the pC3-DEST vector, described in more detail in Example 2. The 3′ end of this Gateway® cassette contains a HindIII restriction site that is the end of the site. This sequence is followed by a second T7 promoter and open reading frame with multiple cloning site and S-tagTM, followed by an AvrII site. All of these sequences are removed in order to reduce background by digestion with the two restriction enzymes AvrII and HindIII. Further downstream, a T7 terminator is retained.
To create the pRIPPER-3 vector, the pDEST-C3 vector (as described in more detail in Example 2) was altered to remove a multiple cloning site, an S-tagTM and an AvrII restriction site by digestion with the two restriction enzymes AvrII and HindIII. Then a second T7 promoter and a second Lac operator were added. The T7 promoter and Lac operator were added by ligating in the short double stranded synthetic oligonucleotide sequence formed by 5′ CTAGGTAATACGACTCACTATAGGAATTGTGAGCGGATAACAATTCCA 3′ (SEQ ID NO: 32) and 3′CATTATGCTGAGTGATATCCTTAACACTCGCCTATTGTTAAGGTTCGA 5′ (SEQ ID NO: 33). The sequence of the T7 promoter is shown in bold. The underlined sequence is the Lac Operator sequence. The proper sequence overhang needed for the AvrII enzyme recognition sequence to be complete and the sequence of the HindIII enzyme recognition sequence required for ligation are formed by the double stranded product. This sequence was synthetically created at Integrated DNA Technologies (Coralville, Iowa).
The G144704 cassette was modified by inserting a copy of the T7 promoter LacO operator in reverse orientation relative to the upstream promoter into the HindIII-AvrII sites of pDEST-C3, creating pRIPPER-3, and the novel G144704ri cassette (with a reversed-orientation promoter) surrounded by SacI and AvrII sites. This G144704ri cassette was then removed from pRIPPER-3 by digestion with SacI-AvrII, and ligated into similarly digested pCDFDuet-1, pRSFDuet-1, and pETDuet-1 to create pRIPPER-1, pRIPPER-2, and pRIPPER-4 respectively.
A map of the pRIPPER-3 vector is shown in FIG. 27 and the nucleotide sequence of the pRIPPER-3 vector (SEQ ID NO: 10) is shown in FIG. 28.
To create the pRIPPER-1 vector, a large fragment of the pRIPPER-3 vector was inserted into Novagen's pCDFDuetTM-1 vector (catalog no. 71340-3). Specifically, the sequence from between the SacI site (5′ site of pC3-DEST Zeo labeled Gateway® cassette) and the AvrII site in vector pRIPPER-3 were moved into the pCDF-DUET, creating pRIPPER-1. Clones with correct digestion pattern with AvrII, HindIII and SacI were sequenced to ensure correct sequence. A map of the pRIPPER-1 vector is shown in FIG. 29 and the nucleotide sequence of the pRIPPER-1 vector (SEQ ID NO: 11) is shown in FIG. 30.
To create the pRIPPER-2 vector, this same fragment of the pRIPPER-3 vector was inserted into Novagen's pRSFDuetTM-1 vector (catalog no. 71341-3). A map of the pRIPPER-2 vector is shown in FIG. 31 and the nucleotide sequence of the pRIPPER-2 vector (SEQ ID NO: 12) is shown in FIG. 32.
To create the pRIPPER-4 vector, this same fragment of the pRIPPER-3 vector was inserted into the pETDuet-1 vector (Novagen). A map of the pRIPPER-4 vector is shown in FIG. 33 and the nucleotide sequence of the pRIPPER-2 vector (SEQ ID NO: 13) is shown in FIG. 34.
To create the pRIPPER-II vector, the Multisite® Gateway® Cassette from Invitrogen's pDESTTM R4-R3 vector (catalog no. 12537-023) was amplified using polymerase chain reaction (PCR) and ligated into the pCDFDuet vector, using SacI and HindIII (yielding the pDEST-CM1 vector described in Example 4) followed by the insertion of an additional T7 promoter and Lac operator. The T7 promotor and lac operator sequences were inserted into the HindIII/AvrII sites of pDEST-CM1, creating pRIPPER-II. The pRIPPER-II vector is streptomycin Str(R)) and chloramphenicol resistant (Cm(R)). A map of the pRIPPER-II vector is shown in FIG. 35 and the nucleotide sequence of the pRIPPER-II vector (SEQ ID NO: 14) is shown in FIG. 36.
To create the pRIPPER-III and pRIPPER-IV vectors, the pRIPPER-II vector sequence between the ScaI and AvrII restriction sites was ligated into the pRSFDuet-1 and pETDuetTM-1 (Novagen catalog no. 71146-3) vectors, respectively. A map of the pRIPPER-III vector is shown in FIG. 37 and the nucleotide sequence of the pRIPPER-III vector (SEQ ID NO: 15) is shown in FIG. 38. A map of the pRIPPER-IV vector is shown in FIG. 39 and the nucleotide sequence of the pRIPPER-IV vector (SEQ ID NO: 16) is shown in FIG. 40.
Example 9
RNAi Vectors in Functional Assays
The RNAi vectors of the present invention may be utilized in any of the various methods of functional genomic analysis. For example, the RNAi vectors of the present invention may be used in assays utlizing Caenorhabditis elegans, including the assays described by Gonczy et al. (Gonczy et al., Nature 408(6810):331-6, 2000). For example, the RNAi vectors of the present invention will be used interfere with the expression of the Eri-1 and Rrf-3 genes in C. elegans. The RNAi vectors of the present invention will also be used interfere with the expression of the Lin-1 and Unc-22 proteins in C. elegans, genes which give the worms a multi-vulva phenotype and a stumpy phenotype, respectively. These phenotypes are recalcitrant to regular RNAi methods. The RNAi vectors of the present invention will also be used interfere with the expression Green Fluorecent Protein (GFP) and Red Fluorescent Protein in C. elegans. With the experiments outlined above, the RNAi vectors of the present invention will also be used interfere with the expression of at least six different proteins in a single C. elegans organism. The RNAi vectors of the present invention will also be used interfere with the expression of additional genes and phenotypes in C. elegans. These will show that the RNAi vectors of the present invention can be used to interfere with the expression of a large number of genes at the same time. Additional genes may include Lin-15A and lin-35 (which together give a synthetic multi-vulva phenotype), Dpy-10 (giving a dumpy phenotype), Zyg-11 (sterile phenotype) and an ORF called F33H2.8 in wormbase (giving an uncoordinated phenotype).
Example 10
Interference of URF3 and UNC-22 in C. elegans with pRIIPER Vector Constructs
C. elegans strains used in this example (strain CF1827 with GFP expression in the intestine and strain GR1373, a Eri-1 mutation no phenotype that enables increased RNAi response) were from the Caenorhabditis Genetics Center, University of Minnesota. Worms were incubated with E. coli cells that were purchased for feeding the worm, C. elegans. This strain of E. coli, was acquired from Open biosystems (pn RCE1182-9366364 feeding clone pL4440-DEST for ORF T07A9.5 (Eri-1) in E. coli strain HT115 (DE3)). The above strain of bacteria was than made competent and transformed with plasmids pRIPPER 1-Rrf-3, pRIPPER-2 Unc-22. These bacterial strains were then grown with appropriate antibiotics in liquid media and were induced with 2 mM IPTG for two hours and plated on LB plates supplemented with the appropriate antibiotics and 2 mM IPTG. The above listed worms were then plated onto the bacteria and allowed to grow for two days. Observation showed that the twitching phenotype that is associated with the Unc-22 gene's RNAi-mediated induction of the twitching phenotype. Results were observed for four worms in the GR1373 strain of worms and for one worm that does not lay the eggs is the CF1827 strain.
The RNAi constructs were made by PCRing the following primers together in the absence of any template. The primers contain the attB sequences (capitalized) and sequences that are complimentary to each other (underlined). These primers were then cycled in the PCR machine for five cycles to create the constructs used in the subsequent cloning reactions using pDONR Zeo as the DONR vector and pRIPPER 1 and pRIPPER 2 as destination vectors.
The RRF3 primers used were as follows:
5′ CTTACAAGTTTGTACAAAAAAGCAGGCTTA cttcaggtag tgatgatcta tcaaacaaat tatatgatca attttcagaa aaagtcagca aaagtttggt gaaggtggtg gagagctgca 3′ (SEQ ID NO: 34) and
5′ CTTACCACTTTGTACAAGAAAGCTGGGTG ggacggttga gacaaactgg agatggcata gcgtatttta ctacttcgag gtattcatct tgcagctctc caccaccttc acccaaacttt 3′ (SEQ ID NO: 35).
The UNC-22 primers used were as follows:
5′ CTTACAAGTTTGTACAAAAAAGCAGGCTTA tggttctccg gccttcacac ggaattcctt tccatccaaa tccaaatcga acttcggagc ctcatgcatt ggcttagcag tagcagccgc 3′ (SEQ ID NO: 36) and
5′ CTTACCACTTTGTACAAGAAAGCTGGGTG tatgaatacc gtgtcgttgc cgtcaacaaa gctgggccag gacaaccatc agattcgtct gcggctgcta ctgctaagcc aatgcatgag 3′ (SEQ ID NO: 37).
Example 11
pDEST-CS Vectors
The PDEST-CS series of vectors (pDEST-CS, pDEST-CS1, pDEST-CS2, pDEST-CS3, and pDEST-CS4) allows for the co-expression of secreted proteins. The pDEST-SC vector was created by removing the G114704 cassette from pDEST-C3 using SacI and HindIII and ligating it to similarly digested pET-26b(+) (Novagen catalog #70774-3). PDEST-CS contains the G144704p cassette as an EcoNI and HindIII fragment containing the G144704 cassette fused to the pelB sequence of pET-26b(+). This results in a vector which will create a fusion of the pelB sequence to the N-terminal of target proteins, which can target proteins for secretion into the periplasmic space in Escherichia coli. This vector is created to provide the G144704 cassette. The pDEST-CS1-4 family of vectors is then created by digestion of pDEST-CS with EcoNI and HindIII, and ligating the fragment containing the G144704p cassette to similarly digested DUET series plasmids. FIG. 41 is a map of the pDEST-CS. FIG. 42 is the nucleotide sequence of the pDEST-CS vector (SEQ ID NO: 17).
The pDEST-CS1 vector was created by removing the G114704p cassette from pDEST-CS using EcoNI and HindIII and ligating it to similarly digested pCDFDuet-1. FIG. 43 is a map of the pDEST-CS1 vector. FIG. 44 is the nucleotide sequence of the pDEST-C1 vector (SEQ ID NO: 18).
The pDEST-CS2 vector will be created by removing the GI14704p cassette from pDEST-CS using EcoNI and HindIII and ligating it to similarly digested pRSFDuet-1. There is a second EcoNIsite in the middle of the KanR gene. However, this simply means the construct will be made by a limiting digestion (a standard technique where limiting amounts of enzyme are used to give only partially digested plasmid) using a low concentration of EcoNI. Correct constructs will be selected for by KanR. If the EcoNI in the KanR gene is cut, then no colony will result. FIG. 45 is a map of the pDEST-CS2 vector. FIG. 46 is the expected nucleotide sequence of the pDEST-CS2 vector (SEQ ID NO: 19).
The pDEST-CS3 vector will be created by removing the G114704p cassette from pDEST-CS using EcoNI and HindIII, and ligating it to similarly digested pACYCDuet-1. FIG. 47 is a map of the pDEST-C3 vector. FIG. 48 is the expected nucleotide sequence of the pDEST-C3 vector (SEQ ID NO: 20).
The pDEST-CS4 vector will be created by removing the G114704p cassette from pDEST-CS using EcoNI and HindIII, and ligating it to similarly digested pETDuet-1. FIG. 49 is a map of the pDEST-C4 vector. FIG. 50 is the expected nucleotide sequence of the pDEST-CS4 vector (SEQ ID NO: 21).
Example 12
pDEST-CMZ (pSYZYGY) Family of Vectors Containing Both G144704 and Multisite Cassettes
Modification of the pDEST-CM family of vectors to include the G144704 Gateway ZeoR cassette in the second multiple cloning site will be done as follows. The pDEST-CM1, pDEST-CM2, and pDEST-CM4 vectors contain the Gateway Multisite cassette in the first multiple cloning site (MCS) of each of the available Novagen DUET vectors, pCDFDuet-1, pRSFDuet-1, and pETDuet-1, respectively, and the Gateway Multisite TetR cassette in pACYCDuet-1 (CM3). However, each of these vectors has a second multiple cloning site, accessible only by standard restriction enzyme/ligation cloning. The second MCS on each of these vectors will be replaced with the G144704 cassette, allowing Gateway recombination cloning at this site as well. Note that the recombination sites of the G144704 and Multisite differ, so that as long as the recombination reactions are performed separately, both are possible. This will be performed in a manner very similar to that described for the initial construction of the pDEST-C series of vectors. This will create a set of four vectors, pDEST-CMZ1, pDEST-CMZ2, pDEST-CMZ3, and pDEST-CMZ4 also referred to as the ‘pSYZYGY’ family, each with a total of four possible Gateway recombination sites, for a grand total of sixteen co-expressible proteins. This will be done by amplifying the G144704 cassette using primers that add NdeI and KpnI restriction sites to the 5′ and 3′ ends respectively. This will be digested with these two enzymes, and then ligated directly to each of the similarly digested pDEST-CM1-4 vectors, creating pDEST-CMZ1-4. An example map of the proposed pDEST-CMZ1 is shown below. The other three vectors will be constructed in precisely the same manner, insertion of the same cassette at the NdeI and KpnI sites on those vectors.
A map of the pDEST-CMZ1 vector is shown in FIG. 51. The expected nucleotide sequence of the pDEST-CMZ1 vector is shown in FIG. 52.
Example 13
pDEST-CMZc (pSYZYGYc) Family Containing Both G144704 and Multisite Cassettes Tagged with GFP and RFP
To create the pDEST-CMc series of vectors, the G144704 cassette will be modified to include a green fluorescent protein (GFP) marker and the Gateway Multisite cassette will be modified to include a red fluorescent protein (RFP) marker.
PCR will be used to amplify pDEST-C3 at the 3′ end of the ZeoR gene using primers abutting at their 3′ ends, which will amplify the entire vector to create a linear plasmid with SpeI and SphI restriction sites artificially added on (SpeI on the 3′ end of the ZeoR gene), followed by digestion with these enzymes to create sticky ends. Next, the green fluorescent protein (GFPuv) encoding gene from the commercially available plasmid pGFPuv (BD Biosciences/Clontech #632312) will be amplified with primers that SpeI and SphI restriction sites to the 5′ and 3′ ends of the PCR product respectively. After digestion with these enzymes, it will be ligated to the similarly digested pDEST-C3 to create pDEST-C3g. The primers will be designed such that the GFPuv encoding gene will be fused in frame with the ZeoR gene, creating a gene fusion. These are all standard molecular biology protocols. This vector will contain the new G144704g cassette, which will have all the same characteristics of the parent G144704 cassette (ZeoR, ccdB, Gateway R1 and R2 recombination sites), but additionally will express the GFPuv protein as a fusion with the ZeoR protein. This will result in colonies with a green fluorescent color when grown in the appropriate ccdA host strain in the presence of Zeocin.
In a similar manner, the gene (DsRed2) encoding the red fluorescent protein (RFP) will be amplified from the commercially available plasmid pDsRed2 (BD Biosciences/Clontech #632404), and cloned into the Gateway Multisite cassette as a 3′ fusion with the CamR gene, to create the MultisiteR cassette, which will result in red colonies.
A third construct will be made in a similar manner, fusing the RFP encoding gene to the 3′ end of the TetR gene in the Multisite TetR cassette, the only difference being that the restriction enzymes used will be SpeI and XbaI, due to the presence of an SphI site in the TetR gene, creating the Multisite TetRr cassette.
These constructs will allow for a fluorescent screen for the first step of recombination. The current selection for successful recombination, the loss of the ccdB gene in the cassette, will not work when there are two copies of this lethal gene. In the pDEST-CMZ1-4 family of vectors, which will contain two different Gateway cassettes (G144704 and Multisite), each with a ccdB gene, one can use either the ZeoR or CamR marker on the G144704 or Multisite cassette respectively to screen for recombination at one cassette or the other, but this is a two step process requiring growth of colonies under non-selective conditions, followed by replica plating and screening for colonies which have lost the marker of interest. The utility of this insertion of the GFPuv and RFP markers into the G144704g and MultisiteR cassettes respectively will reduce this selection process to one step. Colonies which have lost the color of interest, and thus contain plasmids which have successfully recombined at the desired position, can be screened directly, and moved on to the next recombination event. The G144704g cassette will be used to replace the G144704 cassettes in the second multiple cloning sites of the four pDEST-CMZ family of vectors, and the MultisiteR will replace the Multisite cassette (Multisite TetRr will replace the Multisite TetR in pDEST-CMZ3) in the other recombination site of these four vectors. This will create the pDEST-CMZc1-4 family of vectors (or ‘pSYZYGYc family), with colored markers (‘c’) for recombination screening.
A representative map of a pDEST-CMZc1 vector is shown in FIG. 53. The expected nucleotide sequence of a pDEST-CMZc1 vector is shown in FIG. 54.
Example 14
Expression of Multiple Genes in a Single Vector
The vectors of the pDEST-C series, the pDEST-CS series, and the pRIPPER series can be further modified to allow the co-expression of up to five unique proteins for each vector, allowing the expression of up to sixteen different genes, when the vectors of the present invention are used in concert with the available vectors (pDEST, pL4440, pET). In turn, the SYZYGY vectors of the present invention can be used for the co-expression of up to thirty-two proteins or, when used in RNAi, to silence thirty-two genes, since these vectors unite the traditional Gateway® and Multisite® methods into one plasmid. See also, Sone et. al. (Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: Modular construction of multiple cDNA expression elements using recombinant cloning, ” J Biotechnol. 2005 Jun. 24 (doi:10,1016/jbiotec.2005.02.021)) which describes methods for the insertion of up to five unique DNA fragments into the B1 B2 sites of a single Gateway® vector.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

## Claims

1. A polynucleotide comprising a first nucleotide sequence operably encoding zeomycin resistance and a second nucleotide sequence operably encoding a ccdB polypeptide, wherein the first nucleotide sequence and the second nucleotide sequence are flanked within the same attR1 and attR2 sites, and wherein the polynucleotide comprises a G144704 cassette having SEQ ID NO:4.

2. The polynucleotide of claim 1, wherein the polynucleotide is selected from the group consisting of pDEST-C1 (SEQ ID NO:1), pDEST-C2 (SEQ ID NO:2), pDEST-C3 (SEQ ID NO:3), pDEST-CS (SEQ ID NO:17), pDEST-CS1 (SEQ ID NO:18), pDEST-CS2 (SEQ ID NO:19), pDEST-CS3 (SEQ ID NO:20), pDEST-CS4 (SEQ ID NO:21), pDEST-CMZ1 (SEQ ID NO:22), and pDEST-CMZc1 (SEQ ID NO:23).

3. An expression vector comprising the polynucleotide of claim 1.

4. A host cell comprising the expression vector of claim 3.

5. A polynucleotide comprising a first nucleotide sequence operably encoding tetracycline resistance and a second nucleotide sequence operably encoding a ccdB polypeptide, wherein the first nucleotide sequence and the second nucleotide sequence are flanked within the same attR3 and attR4 sites, and wherein the polynucleotide comprises a tet Multisite having SEQ ID NO: 7.

6. The polynucleotide of claim 5 wherein the polynucleotide is selected from the group consisting of pDEST-CM1 (SEQ ID NO:5), pDEST-CM2 (SEQ ID NO:6), pDEST-CM3 (SEQ ID NO:8), pDEST-CM4 (SEQ ID NO:9), pDEST-CMZ1 (SEQ ID NO:22), and pDEST-CMZc1 (SEQ ID NO:23).

7. An expression vector comprising the polynucleotide of claim 5.

8. A host cell comprising the expression vector of claim 7.

9. An RNA interference (RNAi) vector comprising the polynucleotide of claim 1.

10. An RNA interference (RNAi) vector comprising the polynucleotide of claim 5.

11. An RNAi vector selected from the group consisting of pRIPPER-1 (SEQ ID NO:11), pRIPPER-2 (SEQ ID NO:12), pRIPPER-3 (SEQ ID NO:10), pRIPPER-4 (SEQ ID NO:13), pRIPPER-II (SEQ ID NO:14), pRIPPER-III (SEQ ID NO:15), and pRIPPER-IV (SEQ ID NO:16).

12. A method of producing one or more interfering RNA products, the method comprising expressing at least one interfering RNA product by an RNAi vector of claim 11.

13. A host cell comprising the RNAi vector of claim 11.

14. The RNA interference (RNAi) vector of claim 9, wherein the RNAi vector is selected from the group consisting of pRIPPER-1 (SEQ ID NO:11), pRIPPER-2 (SEQ ID NO:12), pRIPPER-3 (SEQ ID NO:10), and pRIPPER-4 (SEQ ID NO:13).

15. The RNA interference (RNAi) vector of claim 10, wherein the RNAi vector is selected from the group consisting of pRIPPER-II (SEQ ID NO:14), pRIPPER-III (SEQ ID NO: 15), and pRIPPER-IV (SEQ ID NO:16).

16. An RNA interference (RNAi) vector comprising a polynucleotide cassette, the polynucleotide cassette comprising a nucleotide sequence operably encoding zeomycin resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the polynucleotide cassette is flanked by attR1 and attR2 sites, and wherein the polynucleotide cassette comprises a G144704 cassette having SEQ ID NO: 4.

17. An RNA interference (RNAi) vector of claim 16, wherein the RNAi vector is selected from the group consisting of pRIPPER-1 (SEQ ID NO:11), pRIPPER-2 (SEQ ID NO:12), pRIPPER-3 (SEQ ID NO:10), and pRIPPER-4 (SEQ ID NO:13).

18. The RNA interference (RNAi) vector of claim 16, wherein the G144704 cassette having SEQ ID NO: 4 is flanked on either end by a T7 promoter and a Lac Operator sequence.

19. An RNA interference (RNAi) vector comprising a polynucleotide cassette, the polynucleotide cassette comprising a nucleotide sequence operably encoding tetracycline resistance and a nucleotide sequence operably encoding a ccdB polypeptide, wherein the polynucleotide cassette is flanked by attR3 and attR4 sites, and wherein the polynucleotide cassette flanked by attR3 and attR4 sites comprises a tet Multisite having SEQ ID NO: 7.

20. An RNA interference (RNAi) vector of claim 19, wherein the RNAi vector is selected from the group consisting of pRIPPER-II (SEQ ID NO:14), pRIPPER-III (SEQ ID NO: 15), and pRIPPER-IV (SEQ ID NO:16).

21. The RNA interference (RNAi) vector of claim 19, wherein the tet Multisite having SEQ ID NO: 7 is flanked on either end by a T7 promoter and a Lac Operator (LacO) sequence.