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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.


Related in: MedlinePlus

Schematic drawing of the used approach. (a) Translation products are soluble in the reaction solution forming giant micelles. Then, the detergent DDM was added to the solution for the solubilization of the hydrophobic regions. Finally, the tag was digested by a protease, and the functional membrane protein was prepared. (b) pHtrII1–114 with large soluble tags expressed in the cell-free system. The proteins have a histidine tag and a protease site which can be used for the pull-down assay or the tag digestion, respectively.
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f1-7_51: Schematic drawing of the used approach. (a) Translation products are soluble in the reaction solution forming giant micelles. Then, the detergent DDM was added to the solution for the solubilization of the hydrophobic regions. Finally, the tag was digested by a protease, and the functional membrane protein was prepared. (b) pHtrII1–114 with large soluble tags expressed in the cell-free system. The proteins have a histidine tag and a protease site which can be used for the pull-down assay or the tag digestion, respectively.

Mentions: In the present study we developed a strategy for the membrane protein expression by using a cell-free protein synthesis system (Fig. 1a). A wheat germ cell-free system was chosen, because in it the translation reaction can proceed for longer than 60 hrs, and active proteins are yielded in milligram quantities per milliliter reaction volume. The membrane region of the pharaonis halobacterial transducer protein, pHtrII, was translated with various large soluble tags (thioredoxin, glutathione S-transferase, green fluorescent protein or maltose binding protein) (Fig. 1b). pHtrII has two transmembrane helices, and belongs to the family of two-transmembrane helical methyl-accepting chemotaxis proteins (MCPs)12–14. It forms a signaling complex with pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) in the halobacterial membrane15. This complex transmits a light signal to the sensory system which is called the two-component system in the cytoplasm 13. pHtrII activates phosphorylation cascades which modulate flagellar motors14. By using these signaling systems, Natronomonas pharaonis cells avoid harmful near-UV light, a behaviour called negative phototaxis. It is known that both, pHtrII and ppR, are stable in membranes as well as in detergent micelles16–18, and both have been well characterized over the past few years using various methods15,19. Therefore, we chose to use pHtrII as a model for membrane proteins.


Functional expression of a two-transmembrane HtrII protein using cell-free synthesis
Schematic drawing of the used approach. (a) Translation products are soluble in the reaction solution forming giant micelles. Then, the detergent DDM was added to the solution for the solubilization of the hydrophobic regions. Finally, the tag was digested by a protease, and the functional membrane protein was prepared. (b) pHtrII1–114 with large soluble tags expressed in the cell-free system. The proteins have a histidine tag and a protease site which can be used for the pull-down assay or the tag digestion, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036783&req=5

f1-7_51: Schematic drawing of the used approach. (a) Translation products are soluble in the reaction solution forming giant micelles. Then, the detergent DDM was added to the solution for the solubilization of the hydrophobic regions. Finally, the tag was digested by a protease, and the functional membrane protein was prepared. (b) pHtrII1–114 with large soluble tags expressed in the cell-free system. The proteins have a histidine tag and a protease site which can be used for the pull-down assay or the tag digestion, respectively.
Mentions: In the present study we developed a strategy for the membrane protein expression by using a cell-free protein synthesis system (Fig. 1a). A wheat germ cell-free system was chosen, because in it the translation reaction can proceed for longer than 60 hrs, and active proteins are yielded in milligram quantities per milliliter reaction volume. The membrane region of the pharaonis halobacterial transducer protein, pHtrII, was translated with various large soluble tags (thioredoxin, glutathione S-transferase, green fluorescent protein or maltose binding protein) (Fig. 1b). pHtrII has two transmembrane helices, and belongs to the family of two-transmembrane helical methyl-accepting chemotaxis proteins (MCPs)12–14. It forms a signaling complex with pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) in the halobacterial membrane15. This complex transmits a light signal to the sensory system which is called the two-component system in the cytoplasm 13. pHtrII activates phosphorylation cascades which modulate flagellar motors14. By using these signaling systems, Natronomonas pharaonis cells avoid harmful near-UV light, a behaviour called negative phototaxis. It is known that both, pHtrII and ppR, are stable in membranes as well as in detergent micelles16–18, and both have been well characterized over the past few years using various methods15,19. Therefore, we chose to use pHtrII as a model for membrane proteins.

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