A novel emulsion mixture for in vitro compartmentalization of transcription and translation in the rabbit reticulocyte system

Farid J. Ghadessy and Philipp Holliger1

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

1 To whom correspondence should be addressed. e-mail: ph1{at}mrc-lmb.cam.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 References
 
Emulsion formulations used for in vitro compartmentalization (IVC) methods were found to be incompatible with protein expression in the rabbit reticulocyte (RRL) system, causing rapid discoloration and translation shutdown. Here we identify possible causes and describe a novel water-in-oil emulsion which abolished discoloration and allowed high-level in-emulsion expression of active luciferase and human telomerase using the RRL. This novel emulsion greatly expands the range of potential protein targets for IVC.

Keywords: compartmentalization/directed evolution/emulsion/rabbit reticulocyte extract/telomerase


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 References
 
Repertoire selection methods have become important tools in biology. The central tenet to all repertoire selection methodologies is a tractable linkage between the genotype and phenotype. Two main strategies have been employed to effect such a linkage. One approach, the so-called display methods, entail construction of a form of ‘genetic package’, for example a cell (bacterial or yeast) or a viral capsid displaying protein or peptide on the surface. Recently, in vitro methods such as ribosome or RNA display have been developed, whereby the linkage occurs through tethering of the genes to the proteins that they encode. Display methods have proved very successful for the isolation of protein and peptide ligands (Schatz et al., 1996Go; Griffiths and Duncan, 1998Go; Amstutz et al., 2001Go; Wittrup, 2001Go; Sidhu et al., 2003Go) and have even allowed the de novo isolation of functional protein domains (Keefe and Szostak, 2001Go).

An alternative approach is based on co-segregation of genotype and encoded phenotype into discrete, non-communicating compartments using water-in-oil emulsions (Tawfik and Griffiths, 1998Go). Emulsions are formed by two immiscible liquid phases (e.g. water and mineral oil) with one of the phases dispersed in the other as droplets of microscopic size. In vitro compartmentalization (IVC) using water-in-oil emulsions has been used for the selection of peptide ligands (Doi and Yanagawa, 1999Go; Sepp et al., 2002Go; Yonezawa et al., 2003Go) and for the directed evolution of DNA methyltransferases (Cohen et al., 2004Go), bacterial phosphotriesterase (Griffiths and Tawfik, 2003Go) and Taq polymerase (Ghadessy et al., 2001Go).

So far, IVC approaches have mainly utilized bacterial expression systems. Many proteins of interest, in particular more complex, multidomain proteins are more efficiently expressed in eukaryotic expression systems (Netzer and Hartl, 1997Go). Recently, application of the eukaryotic wheat germ extract (Yonezawa et al., 2003Go) to IVC has been described, extending the spectrum of protein targets accessible to IVC.

To further extend the capabilities of IVC methods, it would also be desirable to be able to utilize the rabbit reticulocyte (RRL) extract (Pelham and Jackson, 1976Go). In general, the RRL extract is deemed to be superior to the wheat germ system owing to the high translation activity, low nuclease and protease activity, and reduced dependence on a 5'm7GpppG cap for translation initiation. Furthermore, specific cofactors necessary for folding, assembly or correct post-translational processing of vertebrate proteins may be absent in the wheat germ system. The RRL extract has found a wide range of applications, including in vitro expression cloning (Stukenberg et al., 1997Go), protein activity/mutation analysis (Li et al., 1997Go), protein–protein interactions (Yin et al., 2002Go), directed evolution by ribosome display (He and Taussig, 1997Go) or RNA display (Roberts and Szostak, 1997Go; Keefe and Szostak, 2001Go). Furthermore, supplementation with canine microsomal membranes allows expression, translocation and post-translational modification of membrane proteins (Falk et al., 1997Go) or disulphide-bonded proteins such as the T-cell receptor (TCR)/CD3 complex (Huppa and Ploegh, 1997Go).

However, we found that current IVC emulsions were incompatible with protein expression in RRL extracts, causing rapid discoloration (Figure 1) and translation shutdown. We have investigated possible causes for this incompatibility and here we describe a novel, inert emulsion formulation compatible with efficient in-emulsion expression of eukaryotic proteins in the RRL extract.



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Fig. 1. Haemin discoloration by different emulsion formulations. Emulsions formed by mixing different oil phases with rabbit reticulocyte lysate (RRL) for 1.5 min as imaged in the visual spectrum after 15 min of incubation at 30°C. The 0.5 ml Eppendorf tubes contain equal volumes of emulsion but different emulsions display different viscosities. Emulsion formulations containing Span80 surfactant cause rapid haemin discoloration, which is partially attenuated by addition of DTT (20 mM). The Abil EM90 emulsion does not cause discoloration.

 

    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 References
 
Previously described water-in-oil emulsion formulations for IVC abolish translation in the rabbit reticulocyte system

We first examined in-emulsion expression of firefly luciferase in the TNT T7 coupled rabbit reticulocyte lysate (RRL) system (Promega) as a sensitive measure of transcription/translation efficiency (a detailed description of emulsification procedures is given as supplementary material, available at PEDS online). We tested the two previously described emulsions used for IVC: E1 [4.5% v/v sorbitan monooleate (Span80) (Sigma), + 0.5% v/v polyoxyethylene sorbitan monooleate (Tween80) (Sigma)] used for expression of HaeIII methylase in S30 bacterial extracts (Tawfik and Griffiths, 1998Go) and the heat-stable emulsion E2 [4.5% v/v Span80 (Fluka) + 0.4% v/v Tween80 (Sigma Ultra) + 0.05% v/v Triton-X100 (Sigma)] used in the directed evolution of Taq DNA polymerase (Ghadessy et al., 2001Go). After incubation and disruption of the emulsion, luciferase activity was measured. For both E1 and E2 we found essentially negligible luciferase activity, typically <1% of the non-emulsified reaction (Figure 2).



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Fig. 2. Expression of firefly luciferase activity. In-emulsion expression of firefly luciferase is compared with in-solution expression of firefly luciferase activity using the rabbit reticulocyte extract. Emulsions are prepared with or without the addition of DTT (20 mM) by stirring for 3 min (grey bars) or 4 min (white bars).

 
We also noted a striking colour change of the RRL from a bright red to a rusty brown. Discoloration was rapid and occurred within minutes after emulsification (Figure 1). Furthermore, we found that all of the surfactants (either alone or dissolved in mineral oil), but not the mineral oil by itself, inhibited protein expression and caused various degrees of discoloration. As transcription by T7 RNA polymerase and also transcription/translation in a bacterial S30 extract are not affected by emulsification using the E1 formulation (Tawfik and Griffiths, 1998Go), we concluded that the inhibition of protein expression was due to interference of components in the emulsion mixture with the mammalian translation machinery.

We noticed that the discoloration appeared to parallel the inhibition of translation (e.g. mineral oil alone did not cause either). The normal, bright-red colour of the RRL is due to the presence of haemin, which is essential for its activity. Haemin discoloration to a rusty brownish hue suggested an oxidative process. Oxidative stress can activate the eIF-2{alpha} kinase pathway, which is involved in the regulation of mammalian translation [reviewed by Dever (1999Go)]. Different eIF-2{alpha} kinases respond to different types of stress signals. Among these, HRI, a kinase expressed in haematopoietic cells, is activated by haemin oxidation and triggers a shutdown of translation (Dever, 1999Go).

Supplementation with antioxidants is not sufficient to restore translation

In order to prevent or reverse haemin oxidation, which we hoped would restore translation in the RRL extract, we tested different antioxidants or reducing compounds. We tried to remove oxidative species from the oil phase (the mineral oil–surfactant mixture) or from the main surfactant component Span80 by extraction with aqueous 1 M dithiothreitol (DTT) or by direct reaction with a potent reducing agent [polystyrylmethyltrimethylammonium borohydride coupled to beads (Novabiochem)] or from the water phase by addition of DTT or ascorbic acid. Although discoloration was slowed (Figure 1), none of these yielded any improvements in translation efficiencies. Furthermore, treatment of the oil phase with borohydride beads (and to a lesser extent DTT) appeared to reduce significantly the emulsion-forming properties of the surfactant–oil phase, leading to unstable emulsions (data not shown).

Only when we combined DTT (at 20 mM final concentration added to the water phase) with an altered oil phase comprising a minimal amount of just one surfactant (1.5% v/v Span 80 in mineral oil) did we observe both reduced discoloration (Figure 1) and a small but consistent improvement in in-emulsion expression levels (Figure 2). However, levels of DTT above 20 mM were found to inhibit translation in the RRL expression system (data not shown).

Supplementation with chaperones is not sufficient to restore translation

The fact that even millimolar concentrations of DTT did not restore more than residual translation activity suggested that factors other than oxidation might be contributing to translation inhibition in the emulsion. It has been known for some time that addition of bovine serum albumin (BSA) (containing traces of denatured protein produced by freeze–thawing) can induce a translation shutdown in RRL (Matts et al., 1993Go). We suspected that some protein denaturation might occur during the emulsification process, as it entails the vigorous mixing of a hydrophilic water phase with a hydrophobic oil–surfactant mixture. We used the previously described translation shutdown by BSA (Matts et al., 1993Go) to measure the effect of supplementing the RRL lysates with the chaperones HSP70 and HSP90 with and without emulsification using emulsions E1 and E2. However, again these produced negligible improvements on in-emulsion translation levels (data not shown), which were non-additive with improvements achieved by addition of DTT and could be equalled or surpassed by the addition of small amounts (5%) of glycerol (data not shown), which is known to protect proteins from denaturation.

We also attempted a direct inhibition of the eIF-2{alpha} kinase pathway. Unfortunately, no specific inhibitors of eIF-2{alpha} kinases are currently known. We therefore used the unspecific kinase inhibitors 2-aminopurine (2-AP) and cAMP (both from Sigma). Although cAMP produced a small improvement in translation, it proved non-additive with the beneficial effects of DTT (data not shown).

Development of a novel inert oil phase for water-in-oil emulsions

Our failure to rescue translation using the previously described IVC emulsion formulations E1 and E2 with a variety of additives prompted us to search for a novel, inert emulsion formulation that would be compatible with protein expression in the RRL. We tested a range of different surfactants (see supplementary material) for their ability to form stable emulsions and support protein expression in RRL extracts. Of these only one, the silicone-based surfactant polysiloxane–polycetyl–polyethylene glycol copolymer (Abil EM90) (Goldschmidt), afforded a striking improvement over previous surfactant formulations.

Emulsions formed with Abil EM90 (4% v/v) dissolved in light mineral oil did not cause any noticeable haemin discoloration (Figure 1) even after prolonged incubation (24 h) at 37°C. Abil EM90 also yielded a stable, homogeneous emulsion with an average compartment size of 2–5 µM (Figure 3), providing ~109–1010 compartments/ml for IVC. Most importantly, in-emulsion protein expression in the RRL (as assessed by expression of luciferase activity) was improved dramatically, reaching up to 40% of a non-emulsified reaction (Figure 2). The ~2-fold reduction in expression efficiency compared with in-solution expression of luciferase is comparable to that observed using bacterial extracts (data not shown). Presumably, it reflects sequestration of reagents to compartments too small to support efficient protein expression.



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Fig. 3. Abil EM90 water-in-oil emulsion. A water-in-oil emulsion formed by Abil EM90 (4% v/v) in mineral oil (stirring for 4 min) imaged in the visual spectrum in phase-contrast mode. The distance between the bars is 10 µm. Aqueous compartments vary in size between 2 and 5 µm.

 
In-emulsion expression of active human telomerase

The TNT T7 coupled RRL system (Promega) is optimized for expression of firefly luciferase. As a more stringent test we investigated whether the Abil EM90 emulsion would support expression of a more complex eukaryotic protein such as telomerase. Human telomerase is a ribonucleoprotein comprising an RNA component (human telomeric RNA: hTR) and a protein component (hTERT). Telomerase is poorly expressed in prokaryotic systems but had been shown to be expressed in functional form in RRL extracts (Weinrich et al., 1997Go). We therefore tested in-emulsion expression of human telomerase by the RRL in the Abil EM90 emulsions.

For telomerase expression we used linear templates encoding the hTERT and hTR subunits under the control of a T7 promoter. Expression templates were co-emulsified with a telomerase substrate oligonucleotide (TS). After breaking of the emulsion, the TS oligonucleotide allows the detection of in-emulsion telomerase activity by the Telomerase Repeat Amplification Protocol (TRAP) PCR assay (Kim et al., 1994Go) (detailed descriptions of hTERT-hTR constructs, expression and TRAP assay are given in the supplementary material). The TRAP assay detects the presence of the characteristic TTAGGG telomeric repeats appended to the 3' end of the TS substrate oligonucleotide. Telomerase activity is signified by the production of a characteristic ladder (with 6 bp spacing for each repeat).

The TRAP PCR assay results showed that active telomerase was indeed expressed within the emulsion (Figure 4). Control experiments, comprising expression of either the telomerase protein subunit (hTERT) or telomeric RNA (hTR) on their own or co-expression of hTR with an inactive, truncated hTERT subunit (hTERT{Delta}), did not produce detectable telomerase activity either in emulsion or in solution (Figure 4).



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Fig. 4. In-emulsion expression of active human telomerase. Telomerase activity for (Ain-solution expression and (Bin-emulsion expression of human telomerase in rabbit reticulocyte extract. TRAP PCR products are resolved on a 20% PAGE gel. Telomerase activity results in the appearance of ladder with 6 bp spacing.

 
Conclusion

We have described a novel emulsion formulation that permits functional in-emulsion expression using the rabbit reticulocyte system. The water-in-oil emulsions that are formed by the silicone-based surfactant Abil EM90 are homogeneous and stable over prolonged periods. Aqueous compartments range between 2 and 5 µm in diameter, which is ideal for directed evolution methods based on IVC, enabling the formation of between 109 and 1010 compartments/ml of emulsion.

This novel emulsion formulation extends the scope of directed evolution (and other) methods based on IVC to more complex, vertebrate protein targets that may be difficult to express in bacterial or wheat germ extracts or require specific cofactors, chaperones, post-translational modifications or processing for activity. Emulsions formed by Abil EM90 appear to be inert and (in contrast to previous IVC emulsions) free of oxidant contaminants. Hence they may also prove useful for the study and directed evolution of redox-sensitive proteins.


    Acknowledgements
 
We thank S.Magdassi (University of Jerusalem, Israel) and R.J.Jackson (University of Cambridge) for helpful discussions.


    References
 Top
 Abstract
 Introduction
 Results and discussion
 References
 
Amstutz,P., Forrer,P., Zahnd,C. and Pluckthun,A. (2001) Curr. Opin. Biotechnol., 12, 400–405.[CrossRef][ISI][Medline]

Cohen,H.M., Tawfik,D.S. and Griffiths,A.D. (2004) PEDS, 17, 3–11.[Medline]

Dever,T.E. (1999) Trends Biochem. Sci., 24, 398–403.[CrossRef][ISI][Medline]

Doi,N. and Yanagawa,H. (1999) FEBS Lett., 457, 227–230.[CrossRef][ISI][Medline]

Falk,M.M., Buehler,L.K., Kumar,N.M. and Gilula,N.B. (1997) EMBO J., 16, 2703–2716.[Abstract/Free Full Text]

Ghadessy,F.J., Ong,J.L. and Holliger,P. (2001) Proc. Natl Acad. Sci. USA, 98, 4552–4557.[Abstract/Free Full Text]

Griffiths,A.D. and Duncan,A.R. (1998) Curr. Opin. Biotechnol., 9, 102–108.[CrossRef][ISI][Medline]

Griffiths,A.D. and Tawfik,D.S. (2003) EMBO J., 22, 24–35.[Abstract/Free Full Text]

He,M. and Taussig,M.J. (1997) Nucleic Acids Res., 25, 5132–5134.[Abstract/Free Full Text]

Huppa,J.B. and Ploegh,H.L. (1997) J. Exp. Med., 186, 393–403.[Abstract/Free Full Text]

Keefe,A.D. and Szostak,J.W. (2001) Nature, 410, 715–718.[CrossRef][ISI][Medline]

Kim,N.W. et al. (1994) Science, 266, 2011–2015.[ISI][Medline]

Li,J. et al. (1997) Science, 275, 1943–1947.[Abstract/Free Full Text]

Matts,R.L., Hurst,R. and Xu,Z. (1993) Biochemistry, 32, 7323–7328.[ISI][Medline]

Netzer,W.J. and Hartl,F.U. (1997) Nature, 388, 343–349.[CrossRef][ISI][Medline]

Pelham,H.R. and Jackson,R.J. (1976) Eur. J. Biochem., 67, 247–256.[Abstract]

Roberts,R.W. and Szostak,J.W. (1997) Proc. Natl Acad. Sci. USA, 94, 12297–12302.[Abstract/Free Full Text]

Schatz,P.J., Cull,M.G., Martin,E.L. and Gates,C.M. (1996) Methods Enzymol., 267, 171–191.[ISI][Medline]

Sepp,A., Tawfik,D.S. and Griffiths,A.D. (2002) FEBS Lett., 532, 455–458.[CrossRef][ISI][Medline]

Sidhu,S.S., Bader,G.D. and Boone,C. (2003) Curr. Opin. Chem. Biol., 7, 97–102.[CrossRef][ISI][Medline]

Stukenberg,P.T., Lustig,K.D., McGarry,T.J., King,R.W., Kuang,J. and Kirschner,M.W. (1997) Curr. Biol., 7, 338–348.[ISI][Medline]

Tawfik,D.S. and Griffiths,A.D. (1998) Nat. Biotechnol., 16, 652–656.[ISI][Medline]

Weinrich,S.L. et al. (1997) Nat. Genet., 17, 498–502.[ISI][Medline]

Wittrup,K.D. (2001) Curr. Opin. Biotechnol., 12, 395–399.[CrossRef][ISI][Medline]

Yin,Y., Wang,Z.-H., Mora-Garcia,S., Li,J., Yoshida,S., Asami,T. and Chory,J. (2002) Cell, 109, 181–191.[ISI][Medline]

Yonezawa,M., Doi,N., Kawahashi,Y., Higashinakagawa,T. and Yanagawa,H. (2003) Nucleic Acids Res., 31, e118.[Abstract/Free Full Text]

Received January 22, 2004; accepted February 18, 2004 Edited by Peter Hudson