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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.

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In vitro pull-down assay using a Ni-NTA resin. ppR was applied to the column without pHtrII (lane 1) and with MBP digested pHtrII (lane 2) and with MBP digested pHtrII in the absence of ppR (lane 3). After the column was extensively washed with buffer W (for details, see Materials and Methods) to remove nonspecifically bound proteins, bound proteins were eluted with buffer E (see Materials and Methods). The eluted material was collected, and the SDS-PAGE was performed. pHtrII was only detected in the presence of both, ppR and pHtrII, indicating that MBP digested pHtrII is functional.
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f4-7_51: In vitro pull-down assay using a Ni-NTA resin. ppR was applied to the column without pHtrII (lane 1) and with MBP digested pHtrII (lane 2) and with MBP digested pHtrII in the absence of ppR (lane 3). After the column was extensively washed with buffer W (for details, see Materials and Methods) to remove nonspecifically bound proteins, bound proteins were eluted with buffer E (see Materials and Methods). The eluted material was collected, and the SDS-PAGE was performed. pHtrII was only detected in the presence of both, ppR and pHtrII, indicating that MBP digested pHtrII is functional.

Mentions: For the preparation of un-tagged membrane proteins, it is necessary to digest the fusion soluble tag. Then, the binding activity to ppR was checked using a pull-down assay. For this, pHtrII was adsorbed onto a Ni-NTA resin containing immobilized histidine tagged fusion ppR. Figure 4 shows the adsorbed fraction of pHtrII, in the presence of histidine tagged ppR without pHtrII (lane 1), in the presence of both, ppR and MBP-digested pHtrII (lane 2), and for MBP-digested pHtrII in the absence of ppR (lane 3). As expected, specifically adsorbed pHtrII was only detected in the presence of both, ppR and pHtrII. The results show that also tagdigested pHtrII is functional.


Functional expression of a two-transmembrane HtrII protein using cell-free synthesis
In vitro pull-down assay using a Ni-NTA resin. ppR was applied to the column without pHtrII (lane 1) and with MBP digested pHtrII (lane 2) and with MBP digested pHtrII in the absence of ppR (lane 3). After the column was extensively washed with buffer W (for details, see Materials and Methods) to remove nonspecifically bound proteins, bound proteins were eluted with buffer E (see Materials and Methods). The eluted material was collected, and the SDS-PAGE was performed. pHtrII was only detected in the presence of both, ppR and pHtrII, indicating that MBP digested pHtrII is functional.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036783&req=5

f4-7_51: In vitro pull-down assay using a Ni-NTA resin. ppR was applied to the column without pHtrII (lane 1) and with MBP digested pHtrII (lane 2) and with MBP digested pHtrII in the absence of ppR (lane 3). After the column was extensively washed with buffer W (for details, see Materials and Methods) to remove nonspecifically bound proteins, bound proteins were eluted with buffer E (see Materials and Methods). The eluted material was collected, and the SDS-PAGE was performed. pHtrII was only detected in the presence of both, ppR and pHtrII, indicating that MBP digested pHtrII is functional.
Mentions: For the preparation of un-tagged membrane proteins, it is necessary to digest the fusion soluble tag. Then, the binding activity to ppR was checked using a pull-down assay. For this, pHtrII was adsorbed onto a Ni-NTA resin containing immobilized histidine tagged fusion ppR. Figure 4 shows the adsorbed fraction of pHtrII, in the presence of histidine tagged ppR without pHtrII (lane 1), in the presence of both, ppR and MBP-digested pHtrII (lane 2), and for MBP-digested pHtrII in the absence of ppR (lane 3). As expected, specifically adsorbed pHtrII was only detected in the presence of both, ppR and pHtrII. The results show that also tagdigested pHtrII is functional.

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.


Related in: MedlinePlus