Recombinant Renilla Reniformis GFP Displays Low Toxicity

New Vitality™ hrGFP—new organism, new performance

Recombinant Renilla Reniformis GFP Displays Low Toxicity

Katie Felts • Brenda Rogers • Keith Chen • Henry Ji • Joe Sorge • Peter Vaillancourt
Stratagene

We have cloned and expressed the gene for the Renilla reniformis green fluorescent protein, and have fully humanized the gene using codons preferred in highly expressed human genes.*^,Y High-level expression of functional fluorescent protein in retrovirally transduced cells is substantially more consistent and less toxic over time and passage number for this new humanized R. reniformis GFP (hrGFP) than for the redshifted, humanized Aeqourea GFP (EGFP). Two reporter vectors are now available with hrGFP downstream of an internal ribosomal entry site (IRES) ^±±, allowing bicistronic expression of your gene of interest and hrGFP as well as a third retroviral reporter vector.

Green fluorescent protein (GFP) has become a versatile tool for monitoring mammalian expression and protein localization in the fields of biochemistry, molecular and cell biology, high-throughput screening and gene discovery.^1,2 GFPs have been identified in a wide range of coelenterates, and while recently the number of cloned GFPs has expanded, to date the best characterized proteins are those from the jellyfish Aequorea victoria and the anthazoan Renilla reniformis.

The purified native R. reniformis GFP is reported to have certain characteristics that implicate it as a more attractive alternative to the Aequorea protein for use as a biological marker³. Renilla GFP absorbs light with a 5-fold higher extinction coefficient than wild-type Aeqourea GFP, and 2.5-fold more efficiently than red-shifted variants of the Aequorea protein. Renilla GFP has a broader range of pH stability than Aequorea GFP, and is more resistant to organic solvents, detergents and proteases. In addition, Renilla GFP exists in solution as a non-dissociable homodimer at all concentrations, so the exposed surface area is less hydrophobic and less likely to interact with other proteins in the cell. Aequorea GFP forms weak homodimers at moderate to low concentrations, and is often cytotoxic. Due to this cytotoxicity, researchers have often been frustrated in their attempts to produce stable GFP-expressing cells lines using the Aequorea protein⁴.

While the native R. reniformis protein has been well characterized, cloning this gene has been difficult. We cloned the R. reniformis gene and chemically synthesized a fully humanized⁵ 238 amino acid open reading frame for expression. The humanized R. reniformis GFP (hrGFP) was cloned into the pFB retroviral vector for expression analysis. Figure 1 shows the results of transduction of a range of mammalian cell types with this vector.

Fig.1

Spectral Analysis

The hrGFP gene was inserted into a mammalian expression vector and expressed in CHO cells, and a spectral analysis was performed on crude CHO cell extracts. The fluorescence profile for the cloned protein is virtually identical to that reported for the native protein, with a single major excitation peak at 500 nm (compared with 498 nm for the native protein). The 500 nm peak is preceded by a vibrational shoulder at approximately 470 nm, a characteristic of the native Renilla GFP³. The emission spectra show a single peak at 506 nm for the cloned protein, compared with the reported maximum of 509 nm for the native protein (data not shown).

Low Toxicity

Fig.2

Production of stable GFP-expressing cell lines has been problematic for many researchers due to toxicity of the Aequorea GFP⁴. In order to compare the relative tolerance of cells for high-level expression of the GFPs over time and passage number, we transduced HeLa cells with retroviral vectors containing either the hrGFP gene, or that for the red-shifted, humanized Aequorea protein (EGFP)². Cells were infected at an equivalent multiplicity of infection with one or the other virus, and then passaged for several weeks in the absence of any selection. At various times following infection, the transduced populations were assayed for fluorescence intensity by FACS, and for viability by visual inspection and fluorescence microscopy. As can be seen in the Figure 2 and Figure 3, at 3 days post-infection, both hrGFP and EGFP show an equivalent, high level of fluorescence. Following trypsinization for the first passage on day 4, we observed a high degree of cell death for the EGFP expressing cells, and by 1 week post-infection, the EGFP-expressing population as a whole showed a substantially diminished fluorescence intensity, while the hrGFP population remained essentially as bright as on day 3. By two weeks post-infection, over half of the EGFP-expressing population exhibited no detectable fluorescence, with the remaining cells peaking at an intensity that is greater than 15-fold below that for hrGFP, which again remained unchanged.

Fig.3

Innovative Vectors

Three Vitality™ hrGFP reporter vectors are currently available for mammalian expression: pIRES-hrGFP-1 and pIRES-hrGFP-2, and the pFB-hrGFP retroviral vector.

The pIRES-hrGFP vectors contain an IRES between the multiple cloning site (MCS) and hrGFP, allowing both your gene of interest and hrGFP expression from the CMV promoter.  The pIRES-hrGFP-1 vector also contains a FLAG tag C-terminal to your gene of interest, and the pIRES-hrGFP-2 vector contains an Hemagglutinin (HA) tag C-terminal to your gene of interest.  These vectors also contain a LoxP site where either hygromycin, puromycin, or neomycin resistance cassettes can be quickly inserted using site-specific recombination to provide your choice of drug resistance markers (Figure 4). The pFB-hrGFP retroviral vector is ideal for mammalian expression in difficult to transfect cell lines.

Fig.4

Conclusion

The humanized R. reniformis green fluorescent protein displays a level of fluoresence comparable to that for EGFP when expressed in a range of mammalian cell types. When expressed at high levels in human cells, the hrGFP is well tolerated over several passages, in contrast to EGFP, which is generally cytotoxic after transduction of the EGFP gene. We have carried a transduced population of HeLa cells for over 2 months in the absence of selection, and have observed no significant loss of overall fluorescence, and minimal cytopathic effect for the hrGFP-expressing cells. On the other hand, substantial cell death for the EGFP-expressing population resulted in a selection for cells exhibiting low fluoresence or no detectable fluorescence.

REFERENCES

1. Chalfie, M. et. al. (1994). Science 263: 802-805.

2. Tsien, R.Y. (1998). Annu. Rev. Biochem. 67: 509-44.

3. Ward, W.W. et. al. (1980). Phytochem. and Phytobiol. 31: 611-615.

4. Hanazono et. al. (1997). Hum. Gene Ther. 8: 1313-1319.

5. Haas, J. et. al. (1996). Curr. Biol. 6(3): 315-324.

* Patent pending