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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.


Effect of he type of detergent on the activity of MBP-pHtrII. MBP-pHtrII was solubilized by DDM, OG, OTG, Cholic acid, SDS and CHAPS, and exchanged completely by dialysis against buffer S (for details, see Materials and Methods) containing 0.1% DDM. ppR was applied to the column without pHtrII (control) and with MBP-tagged pHtrII. After the column was extensively washed with buffer W (for details, see Materials and Methods) to remove non-specifically bound proteins, bound proteins were eluted with buffer E (see Materials and Methods). The eluted material was collected, and then the UV-vis spectrum of ppR (λmax = 498) was measured.
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f5-7_51: Effect of he type of detergent on the activity of MBP-pHtrII. MBP-pHtrII was solubilized by DDM, OG, OTG, Cholic acid, SDS and CHAPS, and exchanged completely by dialysis against buffer S (for details, see Materials and Methods) containing 0.1% DDM. ppR was applied to the column without pHtrII (control) and with MBP-tagged pHtrII. After the column was extensively washed with buffer W (for details, see Materials and Methods) to remove non-specifically bound proteins, bound proteins were eluted with buffer E (see Materials and Methods). The eluted material was collected, and then the UV-vis spectrum of ppR (λmax = 498) was measured.

Mentions: In this report, we used DDM as a detergent for the solubilization of pHtrII. However, the kind of detergent used might have an influence on the activity of the tagged protein, and therefore, we examined the detergent dependency on the activity of the MBP tagged pHtrII (Fig. 5). pHtrII solubilized in non-ionized detergents (DDM, OG and OTG) had a much higher activity than in ionic (cholic acid and SDS) and amphoteric (CHAPS) detergents. Thus, in a cell-free protein synthesis system, non-ionized detergents might be the best choice for solubilization of tags, as it is at least in the present system. These results are consistent with previous results showing that ppR and pHtrII are not stable in the presence of cholic acid, SDS and CHAPS (ref 32 and unpublished data).


Functional expression of a two-transmembrane HtrII protein using cell-free synthesis
Effect of he type of detergent on the activity of MBP-pHtrII. MBP-pHtrII was solubilized by DDM, OG, OTG, Cholic acid, SDS and CHAPS, and exchanged completely by dialysis against buffer S (for details, see Materials and Methods) containing 0.1% DDM. ppR was applied to the column without pHtrII (control) and with MBP-tagged pHtrII. After the column was extensively washed with buffer W (for details, see Materials and Methods) to remove non-specifically bound proteins, bound proteins were eluted with buffer E (see Materials and Methods). The eluted material was collected, and then the UV-vis spectrum of ppR (λmax = 498) was measured.
© Copyright Policy
Related In: Results  -  Collection

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

f5-7_51: Effect of he type of detergent on the activity of MBP-pHtrII. MBP-pHtrII was solubilized by DDM, OG, OTG, Cholic acid, SDS and CHAPS, and exchanged completely by dialysis against buffer S (for details, see Materials and Methods) containing 0.1% DDM. ppR was applied to the column without pHtrII (control) and with MBP-tagged pHtrII. After the column was extensively washed with buffer W (for details, see Materials and Methods) to remove non-specifically bound proteins, bound proteins were eluted with buffer E (see Materials and Methods). The eluted material was collected, and then the UV-vis spectrum of ppR (λmax = 498) was measured.
Mentions: In this report, we used DDM as a detergent for the solubilization of pHtrII. However, the kind of detergent used might have an influence on the activity of the tagged protein, and therefore, we examined the detergent dependency on the activity of the MBP tagged pHtrII (Fig. 5). pHtrII solubilized in non-ionized detergents (DDM, OG and OTG) had a much higher activity than in ionic (cholic acid and SDS) and amphoteric (CHAPS) detergents. Thus, in a cell-free protein synthesis system, non-ionized detergents might be the best choice for solubilization of tags, as it is at least in the present system. These results are consistent with previous results showing that ppR and pHtrII are not stable in the presence of cholic acid, SDS and CHAPS (ref 32 and unpublished data).

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.