Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins, depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.
Article activity feed
-
-
SciScore for 10.1101/2020.09.28.316307: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Thermostability assay by FSEC-Nb: ALFA peptide-tagged hP2X3 was expressed in HEK293 cells and solubilized as described above. HEK293suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)Detergent screening by FSEC-Nb: HEK293S cells expressing ALFA-tagged OlZAC were prepared as described above. HEK293Ssuggested: RRID:CVCL_A784)Software and Algorithms Sentences Resources The micrographs were processed in RELION 3.0 for particle picking, … SciScore for 10.1101/2020.09.28.316307: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Thermostability assay by FSEC-Nb: ALFA peptide-tagged hP2X3 was expressed in HEK293 cells and solubilized as described above. HEK293suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)Detergent screening by FSEC-Nb: HEK293S cells expressing ALFA-tagged OlZAC were prepared as described above. HEK293Ssuggested: RRID:CVCL_A784)Software and Algorithms Sentences Resources The micrographs were processed in RELION 3.0 for particle picking, extraction and 2D classification70. RELIONsuggested: (RELION, RRID:SCR_016274)The plasmids shown in Fig. 9 (mEGFP-NbALFA, mCherry-NbALFA, pETNb-nALFA, pETNb-cALFA, pFBNb-cALFA, pBMNb-cALFA) have been deposited into Addgene (http://www.addgene.org/) (Addgene IDs: 159986, 159987, 159988, 159989, 159990 and 159991). http://www.addgene.org/suggested: (Addgene, RRID:SCR_002037)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
-