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Study protein function more efficiently with standardized clones
Rebecca L. Mullinax Heidi Davis David T. Wong
Kelly Wynne Vilma Nioko
Leonardo DeLeon SanDEe Soares Joseph A. Sorge Edward Marsh
Stratagene
Spencer Stevens Chris Hansen Brian Schilling
Phenogenex
Stratagene now offers sequence-validated and expression-tested human cDNA in a single dual- expression vector. These clones eliminate the time spent cloning, sequencing, and expression testing new genes and allow gene analysis experiments to begin immediately. The versatile dual-expression vector permits proteins to be expressed and detected in mammalian cells and proteins to be expressed in and purified from bacterial cells.
In the near future, the human genome will be completely sequenced. The next and more challenging step will be to characterize the biological role of each gene and the way in which the encoded protein functions in the cell. To facilitate this characterization, Stratagene has cloned the open reading frame (ORF) of selected human cDNA into a dual-expression vector. Potential uses for these expressed proteins include analyzing protein function, defining both protein-protein and protein-DNA interactions, elucidating pathways, studying protein degradation, determining the effects of over-expression, and preparing antigen.
Fig.1
The pDual GC expression vector* was designed for high-level protein expression in mammalian and bacterial cells ( Figure 1). The dominant selectable marker is the neomycin phosphotransferase gene, which is under the dual control of the b-lactamase and SV40 promoters in bacterial and mammalian cells, respectively. The tandemly arranged bacterial Shine-Dalgarno1 and mammalian Kozak2 consensus sequences permit mRNA to be translated efficiently.
To create the GeneConnection expression-tested clone collection, the gene encoding b-lactamase (ampr) between the Eam1104 I sites in the pDual GC vector is replaced with a human ORF using the seamless cloning method3.
Clone Selection and Availability
GeneConnection expression-tested clones were selected based on their biological function and potential applications. They include kinases, DNA-binding proteins, transferases, transporters, oncogenes, cytochromes, proteases, inflammatory response proteins, cellular matrix proteins, metabolic proteins, synthases, esterases, zinc-finger proteins, and ribosomal proteins. For further information regarding clone selection and availability, visit www.stratagene.com.
Validated Nucleotide Sequence
The nucleotide sequence of the ORF of all expression-tested clones has been determined by double-pass sequencing. A comparison between the clone and a published sequence is provided with each clone.
Epitope and Purification Tags
All clones express a fusion protein consisting of the protein encoded by the cDNA insert, the thrombin cleavage site, three copies of the c-myc epitope tag, and a single copy of the HIS6 purification tag ( Figure 1). The c-myc epitope is derived from the human c-myc gene and contains 10 amino acid residues (EQKLISEEDL).4 This allows for convenient and sensitive detection of expressed proteins with anti-c-myc antibody. The HIS6 purification tag consists of six histidine residues and permits quick and easy purification of the fusion protein from bacterial cells.5
Constitutive High-Level Mammalian Expression
The GeneConnection clones contain features designed for constitutive high-level protein expression in mammalian cells.3 The vector contains the promoter and enhancer region of the human cytomegalovirus (CMV)### immediate early gene for constitutive expression of the pDual GC clones in either transiently or stably transfected mammalian cells.
To determine the protein expression level of the pDual GC vector, firefly luciferase activity was measured in transiently transfected Chinese hamster ovary (CHO) cells. Luciferase was chosen because it can be assayed both enzymatically and immunologically. Results of the enzymatic luciferase assays (Figure 2) demonstrate that the fusion protein is biologically active. Control transfections, with the reagents alone or the pDual GC vector without an insert, show low background levels.
Fig.2
To demonstrate that it is easy to detect the luciferase fusion protein, we performed Western blot analyses with anti-c-myc antibody using cell lysates prepared from transfected cells. The cells were transfected with pDual GC with a cDNA insert encoding luciferase, pCMV-Script (with an insert encoding luciferase containing a carboxy terminal c-myc epitope) or the pDual GC vector without an insert. To verify that the luciferase fusion protein was being detected, we also performed Western blot analyses with an antiluciferase antibody. Results indicate that the luciferase fusion protein is easily detected in both Western blot analyses (Figure 3).
Fig.3
Prior to commercial release, all expression-tested clones are tested by Western blot analysis with the anti-c-myc antibody to verify mammalian expression of a protein in CHO cells of the predicted molecular weight (Figure 4).
Fig.4
Inducible High-Level Bacterial Expression
The GeneConnection expression-tested clones contain features designed for inducible high-level protein expression in bacterial cells. The vector contains the hybrid T7/lac O promoter and lac repressor gene (lac I) to regulate protein expression. Therefore, expression is inducible using isopropyl-1-thio-b-D-galatopyranoside (IPTG) in BL21(DE3) bacterial cells that contain the T7 RNA polymerase.
HIS6-Tagged Fusion Protein in Bacterial Cell Lysates
To demonstrate the inducible expression in bacterial cells, we expressed a fusion protein consisting of wild-type green fluorescent protein (GFP),7 c-myc, and HIS6 in BL21-Gold cells. GFP was chosen because it is easily detected under long wavelength UV in induced plate and liquid cultures and requires the formation of a homodimer for biological activity. Results of the enzymatic GFP assays demonstrate that GFP tagged with c-myc and HIS6 was biologically active; therefore, the presence of the tags did not affect its activity (data not shown). Biological activity was not detected in cells transformed with the pDual GC vector without an insert.
Fusion proteins consisting of the protein encoded by the cDNA insert, c-myc, and HIS6 expressed in bacteria are quickly and easily purified from bacterial cell lysates. To demonstrate this, we purified the wild-type GFP fusion protein using Ni-NTA resin (Qiagen) under native conditions. The fusion protein also contains a thrombin cleavage site between GFP and the c-myc epitope. Following purification, the c-myc epitope and HIS6 purification tags were separated from GFP by incubation with thrombin (Figure 5). The GFP was biologically active during protein purification and cleavage (data not shown).
Fig.5
Unique Site for Nucleotide Sequence Insertion
The unique Not I site between the cDNA insert and thrombin cleavage site in the pDual GC vector allows any desired nucleotide sequence to be inserted (Figure 1). Not I was chosen as it is estimated to occur only once in 100,000 bases in the human genome and is, therefore, unlikely to be present in most cDNA. For example, inserting nucleotide sequences that encode GFP permits visualization of cellular proteins in mammalian cells. Alternatively, sequences encoding a translation stop codon would result in translational termination at the inserted sequence.
Fast and Easy Subcloning
Unique Pme I sites flanking the cloned cDNA can be used to subclone the RBS/Kozak and cDNA sequences into other vectors. Pme I is estimated to occur only once in 70,000 bases in the human genome and, hence, is unlikely to be present in most cDNA. Digestion with Pme I creates blunt ends that can either be directly ligated to other blunt ends or to adaptors containing the desired ends. Being able to directly subclone the sequence-verified cloned cDNA enables the known nucleotide sequence to be preserved.
Conclusions
The GeneConnection expression-tested clones offer sequence-validated and mammalian expression-tested human cDNA in a dual prokaryotic and eukaryotic expression vector. Using sequence-validated clones makes it possible to draw valid conclusions based on the function of a protein. The dual expression vector eliminates the need to obtain and validate separate expression vectors. The vector contains a hybrid bacterial promoter for inducible bacterial expression, the CMV promoter and enhancer region for constitutive mammalian expression, and tandem consensus sequences for optimal translation initiation in both systems. The c-myc epitope and HIS6 purification tags at the carboxy terminus of the expressed protein allow proteins to be detected and purified, respectively. The tags can be separated from the protein encoded by the cDNA via a thrombin cleavage site.
Acknowledgments
The authors would like to thank the DNA sequencers at Phenogenex for their excellent technical assistance.
REFERENCES
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Shine, J., et al. (1974) Proc. Natl. Acad. Sci. USA 71: 1342-1436.
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Kozak, M. (1986) Cell 44: 283-292.
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Padgett, K., et al. (1996) Gene 161: 31-35.
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Evan, G.I., et al. (1985) Mol. Cell Biol. 5: 3610-3616.
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Hochuli, E., et al., (1987) J. Chromatog. 411: 177.
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Chalfie, M., et al. (1994) Science 263: 802-905.
* Patent pending
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