High-Level Dual Mammalian And Bacterial Protein Expression Vector

Express your protein in mammalian and bacterial cells with the pDual^® GC expression vector

High-Level Dual Mammalian and Bacterial Protein Expression Vector

Heidi A. Davis • David T. Wong • Kerstien A. Padgett • Joseph A. Sorge • Rebecca L. Mullinax

Stratagene

Stratagene has created the pDual ^® GC expression vector,* which permits high-level expression of heterologous genes in both eukaryotic and prokaryotic systems. The vector can express a fusion protein consisting of the protein of interest, three copies of the human c-myc epitope and a single copy of the HIS6 purification tag. The c-myc epitope allows for detection of the fusion protein in mammalian cell lysates and the HIS6 tag allows for purification of the fusion protein from bacterial cell lysates. Use of a dual-expression vector eliminates the need to subclone and recharacterize genes of interest when moving from one vector system to another.

In the near future, the nucleotide sequence of the genomes from many organisms will be available. 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 created a dual-expression vector for expression of proteins encoded by these genes in mammalian and bacterial cells. For expression in mammalian cells, eukaryotic genes are typically cloned first into a bacterial vector and then subcloned into a vector suitable for eukaryotic expression. Each step involves isolation and characterization of clones containing the gene of interest, requiring a significant investment of time and biological reagents. Stratagene has now eliminated the need to subclone from one vector system to another by combining the essential features of both in a dual expression vector (Figure 1).

Fig.1

seamless® Cloning Technique

The pDual GC expression vector is used in conjunction with the seamless® cloning technique.^3,4 PCR amplification of the target gene, using primers that contain the Eam1104 I restriction sites and a minimal flanking sequence, permits rapid and efficient cloning. The use of the type IIS Eam1104 I restriction enzyme eliminates any primer-related residual nucleotides that are generally present when regular restriction enzyme recognition sites are included in the PCR primer sequences. Thus, use of the Seamless cloning technique can result in expression of a protein that includes only those amino acids encoded by the target gene.

Epitope and Purification Tags

Taking advantage of the versatility of PCR cloning, the pDual GC vector offers two options for expressing a target gene. The reverse primer can be designed to contain a stop codon, which results in the target gene product being expressed in its native form. Alternatively, by designing a reverse primer that eliminates the gene’s natural termination codon, researchers can fuse their target gene to three copies of the human c-myc epitope tag (one copy is EQKLISEEDL)⁵ and a single copy of the HIS6 purification tag.⁶  The c-myc epitope and HIS6 purification tags, expressed on the C-terminus of the protein, can then be used for easy detection and purification of the fusion protein from mammalian and bacterial cell lysates, respectively. Use of the c-myc epitope and HIS6 purification tags eliminates the need to generate an antibody and optimized protein purification protocol for every target gene product, respectively.

Constitutive High-Level Mammalian Expression

The pDual GC vector contains features designed for constitutive high-level protein expression in mammalian cells³ (Figure 1). To demonstrate expression, we expressed a fusion protein consisting of firefly luciferase, and the c-myc and HIS6 epitopes in Chinese hamster ovary (CHO) mammalian cells. In enzymatic luciferase assays, we demonstrated that the luciferase fusion protein was biologically active (data not shown). Biological activity was not detected in control cells transformed with the pDual GC vector without an insert. In Western blot analyses, the luciferase fusion protein was easily detected in mammalian cell lysates with antiluciferase (Figure 2, Panel A) and anti-c-myc (Figure 2, Panel B) antibodies. To date, Stratagene has demonstrated mammalian expression of over 200 different full-length human cDNAs using the pDual GC vector.⁷

Fig.2

Inducible High-Level Bacterial Expression

The pDual GC vector contains features designed for inducible high-level protein expression in bacterial cells³ (Figure 1). To demonstrate bacterial expression, we expressed a fusion protein consisting of wild-type green fluorescent protein (GFP),⁸ plus c-myc and HIS6 epitopes in BL21-Gold cells. Results of the enzymatic GFP assays demonstrate that GFP fusion protein was biologically active; therefore, the presence of the tags did not affect its activity (data not shown). Biological activity was not detected in control cells transformed with the pDual GC vector without an insert.

HIS6-Tagged Fusion Protein in Bacterial Cell Lysates

HIS6-X tagged fusion proteins expressed in bacteria are quickly and easily purified from bacterial cell lysates. To demonstrate this, we purified the GFP fusion protein using Ni-NTA resin (Qiagen) under native conditions. The GFP 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 (results not shown).

Unique Sites for Nucleotide Sequence Insertion and Fast and Easy Subcloning

The unique Not I site between the cDNA insert and  sequences encoding the thrombin cleavage site in the pDual GC vector allows any desired nucleotide sequence to be inserted (Figure 1). For example, inserting nucleotide sequences that encode Renilla hrGFP⁹ permits visualization of fusion protein in mammalian cells. In addition, unique Pme I sites flanking the cDNA insert can be used to subclone the RBS/Kozak and cDNA sequences into other vectors (Figure 1). Digestion with Pme I creates blunt ends that can either be directly ligated to other blunt ends or to adaptors containing the desired ends.

Conclusions

Use of the pDual GC expression vector eliminates the need to obtain and validate separate expression vectors. The vector allows for high-level protein expression in mammalian and bacterial cells. The human c-myc epitope and HIS6 purification tags allow for simple detection and purification of a protein of interest, respectively. The epitope and purification tags can be removed from the fusion protein via the thrombin cleavage site that immediately precedes the tags.

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* Patent pending