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Functional expression of a two-transmembrane HtrII protein using cell-free synthesis

View Article: PubMed Central - PubMed

ABSTRACT

An approach of cell-free synthesis is presented for the functional expression of transmembrane proteins without the need of refolding. The transmembrane region of the pharaonis halobacterial transducer protein, pHtrII, was translated with various large soluble tags added (thioredoxin, glutathione S-transferase, green fluorescent protein and maltose binding protein). In this system, all fusion pHtrII were translated in a soluble fraction, presumably, forming giant micelle-like structures. The detergent n-dodecyl-β-d-maltoside was added for enhancing the solubilization of the hydrophobic region of pHtrII. The activity of the expressed pHtrII, having various tags, was checked using a pull-down assay, using the fact that pHtrII forms a signaling complex with pharaonis phoborhodopsin (ppR) in the membrane, as also in the presence of a detergent. All tagged pHtrII showed a binding activity with ppR. Interestingly, the binding activity with ppR was positively correlated with the molecular weight of the soluble tags. Thus, larger soluble tags lead to higher binding activities. We could show, that our approach is beneficial for the preparation of active membrane proteins, and is also potentially applicable for larger membrane proteins, such as 7-transmembrane proteins.

No MeSH data available.


Binding activity of GST-pHtrII expressed in a cell to ppR. The relative binding activities of pHtrII to ppR without pHtrII (left), the mixture containing the ppR Y199A mutant and pHtrII (center) and the mixture containing ppR and pHtrII (right) are shown. The circle display the color of the sample obtained after elution with buffer GE (for details, see Materials and Methods). The color originates from ppR which absorbs at ~500 nm. About 100 mg functional protein was produced in a 1 liter culture.
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f6-7_51: Binding activity of GST-pHtrII expressed in a cell to ppR. The relative binding activities of pHtrII to ppR without pHtrII (left), the mixture containing the ppR Y199A mutant and pHtrII (center) and the mixture containing ppR and pHtrII (right) are shown. The circle display the color of the sample obtained after elution with buffer GE (for details, see Materials and Methods). The color originates from ppR which absorbs at ~500 nm. About 100 mg functional protein was produced in a 1 liter culture.

Mentions: It can be assumed that the soluble tags may enhance the protein expression level not only in the cell free protein synthesis but also in the cell protein synthesis. To verify this, GST-tagged pHtrII, including the transmembrane region of pHtrII, was expressed in E. coli. The obtained expression levels and yields were comparably high (100mg/L culture), however, because of the high protein concentration in the cells, GST-tagged pHtrII forms aggregation (inclusion body), therefore a refolding process was required. In order to determine if the obtained pHtrII is functional, a GST pull-down assay was performed (see Materials and Methods, and refs 21–23). Figure 6 shows the relative Optical Density (OD) at 498 nm from the adsorbed fractions of ppR alone (control), the mixture containing a ppR Y199A mutant and GSTpHtrII (Y199A), and the mixture containing wild type ppR and GST-pHtrII (W.T.). The mutant was chosen, because Tyr199 of ppR is one of the most important residues for the interaction with pHtrII17,26,29, and therefore a lower binding affinity could be expected compared to WT ppR. This could be confirmed as the fraction of the binding of the Y199A ppR mutant to pHtrII was lower than that of W.T. ppR (Fig. 6). The results suggest that GST-pHtrII can interact with ppR, and that this pull-down system functions normally. Furthermore, in this system it is comparably easy to prepare large amounts of functional pHtrII. Also, the in vitro pull-down assay used here is a simpler and easier method compared to the photo-chemical21,22,25,29 or calorimetric methods23,26 used so far for analyzing the interaction between pHtrII and ppR. Therefore, it can be concluded that the use of large soluble tags can enhance the membrane protein expression level in both, the cell and cell-free systems.


Functional expression of a two-transmembrane HtrII protein using cell-free synthesis
Binding activity of GST-pHtrII expressed in a cell to ppR. The relative binding activities of pHtrII to ppR without pHtrII (left), the mixture containing the ppR Y199A mutant and pHtrII (center) and the mixture containing ppR and pHtrII (right) are shown. The circle display the color of the sample obtained after elution with buffer GE (for details, see Materials and Methods). The color originates from ppR which absorbs at ~500 nm. About 100 mg functional protein was produced in a 1 liter culture.
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Related In: Results  -  Collection

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f6-7_51: Binding activity of GST-pHtrII expressed in a cell to ppR. The relative binding activities of pHtrII to ppR without pHtrII (left), the mixture containing the ppR Y199A mutant and pHtrII (center) and the mixture containing ppR and pHtrII (right) are shown. The circle display the color of the sample obtained after elution with buffer GE (for details, see Materials and Methods). The color originates from ppR which absorbs at ~500 nm. About 100 mg functional protein was produced in a 1 liter culture.
Mentions: It can be assumed that the soluble tags may enhance the protein expression level not only in the cell free protein synthesis but also in the cell protein synthesis. To verify this, GST-tagged pHtrII, including the transmembrane region of pHtrII, was expressed in E. coli. The obtained expression levels and yields were comparably high (100mg/L culture), however, because of the high protein concentration in the cells, GST-tagged pHtrII forms aggregation (inclusion body), therefore a refolding process was required. In order to determine if the obtained pHtrII is functional, a GST pull-down assay was performed (see Materials and Methods, and refs 21–23). Figure 6 shows the relative Optical Density (OD) at 498 nm from the adsorbed fractions of ppR alone (control), the mixture containing a ppR Y199A mutant and GSTpHtrII (Y199A), and the mixture containing wild type ppR and GST-pHtrII (W.T.). The mutant was chosen, because Tyr199 of ppR is one of the most important residues for the interaction with pHtrII17,26,29, and therefore a lower binding affinity could be expected compared to WT ppR. This could be confirmed as the fraction of the binding of the Y199A ppR mutant to pHtrII was lower than that of W.T. ppR (Fig. 6). The results suggest that GST-pHtrII can interact with ppR, and that this pull-down system functions normally. Furthermore, in this system it is comparably easy to prepare large amounts of functional pHtrII. Also, the in vitro pull-down assay used here is a simpler and easier method compared to the photo-chemical21,22,25,29 or calorimetric methods23,26 used so far for analyzing the interaction between pHtrII and ppR. Therefore, it can be concluded that the use of large soluble tags can enhance the membrane protein expression level in both, the cell and cell-free systems.

View Article: PubMed Central - PubMed

ABSTRACT

An approach of cell-free synthesis is presented for the functional expression of transmembrane proteins without the need of refolding. The transmembrane region of the pharaonis halobacterial transducer protein, pHtrII, was translated with various large soluble tags added (thioredoxin, glutathione S-transferase, green fluorescent protein and maltose binding protein). In this system, all fusion pHtrII were translated in a soluble fraction, presumably, forming giant micelle-like structures. The detergent n-dodecyl-β-d-maltoside was added for enhancing the solubilization of the hydrophobic region of pHtrII. The activity of the expressed pHtrII, having various tags, was checked using a pull-down assay, using the fact that pHtrII forms a signaling complex with pharaonis phoborhodopsin (ppR) in the membrane, as also in the presence of a detergent. All tagged pHtrII showed a binding activity with ppR. Interestingly, the binding activity with ppR was positively correlated with the molecular weight of the soluble tags. Thus, larger soluble tags lead to higher binding activities. We could show, that our approach is beneficial for the preparation of active membrane proteins, and is also potentially applicable for larger membrane proteins, such as 7-transmembrane proteins.

No MeSH data available.