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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

SDS-PAGE analysis of cell-free protein synthesis of tagged pHtrII. T, S and P represent total translational product, soluble fraction and pellet after low speed centrifugation, respectively. The bands marked by a star in the gel represent the tagged pHtrII1–114 obtained by cell-free translation.
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f2-7_51: SDS-PAGE analysis of cell-free protein synthesis of tagged pHtrII. T, S and P represent total translational product, soluble fraction and pellet after low speed centrifugation, respectively. The bands marked by a star in the gel represent the tagged pHtrII1–114 obtained by cell-free translation.

Mentions: Commercially available tag genes, Trx, GST, GFP and MBP, were used here. They are cloned into a plasmid vector and all constructed plasmids were analyzed using an automated sequencer to confirm the expected nucleotide sequences. In the presence of 16mM Mg2+, mRNAs of various soluble tagged pHtrIIs were synthesized with a SP6 RNA polymerase, using the plasmids as templates. These synthesized mRNAs were precipitated with ethanol, dissolved in a dialysis buffer, and then mixed with the wheat germ extract for protein synthesis. The purification of the wheat embryos, and the preparation of the cell-free extract were performed as described previously20. Fusion pHtrII, displayed in Figure 1b, were expressed as a soluble fraction (Fig. 2), to which 0.1% DDM was added for 1 hour for solubilization at room temperature under gentle stirring.


Functional expression of a two-transmembrane HtrII protein using cell-free synthesis
SDS-PAGE analysis of cell-free protein synthesis of tagged pHtrII. T, S and P represent total translational product, soluble fraction and pellet after low speed centrifugation, respectively. The bands marked by a star in the gel represent the tagged pHtrII1–114 obtained by cell-free translation.
© Copyright Policy
Related In: Results  -  Collection

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

f2-7_51: SDS-PAGE analysis of cell-free protein synthesis of tagged pHtrII. T, S and P represent total translational product, soluble fraction and pellet after low speed centrifugation, respectively. The bands marked by a star in the gel represent the tagged pHtrII1–114 obtained by cell-free translation.
Mentions: Commercially available tag genes, Trx, GST, GFP and MBP, were used here. They are cloned into a plasmid vector and all constructed plasmids were analyzed using an automated sequencer to confirm the expected nucleotide sequences. In the presence of 16mM Mg2+, mRNAs of various soluble tagged pHtrIIs were synthesized with a SP6 RNA polymerase, using the plasmids as templates. These synthesized mRNAs were precipitated with ethanol, dissolved in a dialysis buffer, and then mixed with the wheat germ extract for protein synthesis. The purification of the wheat embryos, and the preparation of the cell-free extract were performed as described previously20. Fusion pHtrII, displayed in Figure 1b, were expressed as a soluble fraction (Fig. 2), to which 0.1% DDM was added for 1 hour for solubilization at room temperature under gentle stirring.

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