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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.


In vitro pull-down assay using a Ni-NTA resin. ppR was applied to the column without pHtrII (control) and with Trx-tagged pHtrII (Trx), GST-tagged pHtrII (GST), GFP-tagged pHtrII (GFP), MBP-tagged pHtrII (MBP) and pHtrII1–159His (His(from E. coli), as a positive control). 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 the UV-vis spectrum of ppR (λmax = 498) was then measured. The circles display the color and absorbance of ppR bound to pHtrII for each column. All fusion pHtrII represent functional proteins because the fractions of ppR bound to pHtrII are much higher than that of the control.
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f3-7_51: In vitro pull-down assay using a Ni-NTA resin. ppR was applied to the column without pHtrII (control) and with Trx-tagged pHtrII (Trx), GST-tagged pHtrII (GST), GFP-tagged pHtrII (GFP), MBP-tagged pHtrII (MBP) and pHtrII1–159His (His(from E. coli), as a positive control). 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 the UV-vis spectrum of ppR (λmax = 498) was then measured. The circles display the color and absorbance of ppR bound to pHtrII for each column. All fusion pHtrII represent functional proteins because the fractions of ppR bound to pHtrII are much higher than that of the control.

Mentions: In an effort to determine if the soluble tagged pHtrII is functional, we performed an in vitro pull-down assay (see Materials and Methods, and refs 21–23). ppR was adsorbed onto a Ni-NTA resin containing immobilized histidine tagged fusion pHtrII proteins. Figure 3 shows the adsorbed fraction of ppR in the absence (lane 1) and presence of TrxpHtrII (lane 2), GST-pHtrII (lane 3), GFP-pHtrII (lane 4), MBP-pHtrII (lane 5) and pHtrII1–159 (lane 6). pHtrII1–159 was expressed in E. coli cells, purified by the column chromatography and used as a positive control. Because ppR maximally absorbs light at 498 nm, the concentration of ppR can easily be determined by the color and the absorbance (Fig. 3). Specifically adsorbed ppR was detected in the presence of all fusion pHtrII, indicating that all tagged pHtrII have a certain binding activity to ppR. As already mentioned, in these experiments, purified untagged ppR (0.8mM) and all histidine tagged pHtrII (0.08mM) were mixed in the molar ratio of 1 :10. Therefore, the bound fraction of ppR to pHtrII represents the activity related to the folded fraction of soluble tagged pHtrII.


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 (control) and with Trx-tagged pHtrII (Trx), GST-tagged pHtrII (GST), GFP-tagged pHtrII (GFP), MBP-tagged pHtrII (MBP) and pHtrII1–159His (His(from E. coli), as a positive control). 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 the UV-vis spectrum of ppR (λmax = 498) was then measured. The circles display the color and absorbance of ppR bound to pHtrII for each column. All fusion pHtrII represent functional proteins because the fractions of ppR bound to pHtrII are much higher than that of the control.
© Copyright Policy
Related In: Results  -  Collection

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

f3-7_51: In vitro pull-down assay using a Ni-NTA resin. ppR was applied to the column without pHtrII (control) and with Trx-tagged pHtrII (Trx), GST-tagged pHtrII (GST), GFP-tagged pHtrII (GFP), MBP-tagged pHtrII (MBP) and pHtrII1–159His (His(from E. coli), as a positive control). 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 the UV-vis spectrum of ppR (λmax = 498) was then measured. The circles display the color and absorbance of ppR bound to pHtrII for each column. All fusion pHtrII represent functional proteins because the fractions of ppR bound to pHtrII are much higher than that of the control.
Mentions: In an effort to determine if the soluble tagged pHtrII is functional, we performed an in vitro pull-down assay (see Materials and Methods, and refs 21–23). ppR was adsorbed onto a Ni-NTA resin containing immobilized histidine tagged fusion pHtrII proteins. Figure 3 shows the adsorbed fraction of ppR in the absence (lane 1) and presence of TrxpHtrII (lane 2), GST-pHtrII (lane 3), GFP-pHtrII (lane 4), MBP-pHtrII (lane 5) and pHtrII1–159 (lane 6). pHtrII1–159 was expressed in E. coli cells, purified by the column chromatography and used as a positive control. Because ppR maximally absorbs light at 498 nm, the concentration of ppR can easily be determined by the color and the absorbance (Fig. 3). Specifically adsorbed ppR was detected in the presence of all fusion pHtrII, indicating that all tagged pHtrII have a certain binding activity to ppR. As already mentioned, in these experiments, purified untagged ppR (0.8mM) and all histidine tagged pHtrII (0.08mM) were mixed in the molar ratio of 1 :10. Therefore, the bound fraction of ppR to pHtrII represents the activity related to the folded fraction of soluble tagged pHtrII.

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.