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Determining the N-terminal orientations of recombinant transmembrane proteins in the Escherichia coli plasma membrane

In silico algorithms have been the common approach for transmembrane (TM) protein topology prediction. However, computational tools may produce questionable results and experimental validation has proven difficult. Although biochemical strategies are available to determine the C-terminal orientation...

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Autores principales: Lee, Chien-Hsien, Chou, Chia-Cheng, Hsu, Min-Feng, Wang, Andrew H.-J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604451/
https://www.ncbi.nlm.nih.gov/pubmed/26462555
http://dx.doi.org/10.1038/srep15086
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author Lee, Chien-Hsien
Chou, Chia-Cheng
Hsu, Min-Feng
Wang, Andrew H.-J.
author_facet Lee, Chien-Hsien
Chou, Chia-Cheng
Hsu, Min-Feng
Wang, Andrew H.-J.
author_sort Lee, Chien-Hsien
collection PubMed
description In silico algorithms have been the common approach for transmembrane (TM) protein topology prediction. However, computational tools may produce questionable results and experimental validation has proven difficult. Although biochemical strategies are available to determine the C-terminal orientation of TM proteins, experimental strategies to determine the N-terminal orientation are still limited but needed because the N-terminal end is essential for membrane targeting. Here, we describe a new and easy method to effectively determine the N-terminal orientation of the target TM proteins in Escherichia coli plasma membrane environment. D94N, the mutant of bacteriorhodopsin from Haloarcula marismortui, can be a fusion partner to increase the production of the target TM proteins if their N-termini are in cytoplasm (N(in) orientation). To create a suitable linker for orientating the target TM proteins with the periplasmic N-termini (N(out) orientation) correctly, we designed a three-TM-helix linker fused at the C-terminus of D94N fusion partner (termed D94N-3TM) and found that D94N-3TM can specifically improve the production of the N(out) target TM proteins. In conclusion, D94N and D94N-3TM fusion partners can be applied to determine the N-terminal end of the target TM proteins oriented either N(in) or N(out) by evaluating the net expression of the fusion proteins.
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spelling pubmed-46044512015-12-07 Determining the N-terminal orientations of recombinant transmembrane proteins in the Escherichia coli plasma membrane Lee, Chien-Hsien Chou, Chia-Cheng Hsu, Min-Feng Wang, Andrew H.-J. Sci Rep Article In silico algorithms have been the common approach for transmembrane (TM) protein topology prediction. However, computational tools may produce questionable results and experimental validation has proven difficult. Although biochemical strategies are available to determine the C-terminal orientation of TM proteins, experimental strategies to determine the N-terminal orientation are still limited but needed because the N-terminal end is essential for membrane targeting. Here, we describe a new and easy method to effectively determine the N-terminal orientation of the target TM proteins in Escherichia coli plasma membrane environment. D94N, the mutant of bacteriorhodopsin from Haloarcula marismortui, can be a fusion partner to increase the production of the target TM proteins if their N-termini are in cytoplasm (N(in) orientation). To create a suitable linker for orientating the target TM proteins with the periplasmic N-termini (N(out) orientation) correctly, we designed a three-TM-helix linker fused at the C-terminus of D94N fusion partner (termed D94N-3TM) and found that D94N-3TM can specifically improve the production of the N(out) target TM proteins. In conclusion, D94N and D94N-3TM fusion partners can be applied to determine the N-terminal end of the target TM proteins oriented either N(in) or N(out) by evaluating the net expression of the fusion proteins. Nature Publishing Group 2015-10-14 /pmc/articles/PMC4604451/ /pubmed/26462555 http://dx.doi.org/10.1038/srep15086 Text en Copyright © 2015, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Lee, Chien-Hsien
Chou, Chia-Cheng
Hsu, Min-Feng
Wang, Andrew H.-J.
Determining the N-terminal orientations of recombinant transmembrane proteins in the Escherichia coli plasma membrane
title Determining the N-terminal orientations of recombinant transmembrane proteins in the Escherichia coli plasma membrane
title_full Determining the N-terminal orientations of recombinant transmembrane proteins in the Escherichia coli plasma membrane
title_fullStr Determining the N-terminal orientations of recombinant transmembrane proteins in the Escherichia coli plasma membrane
title_full_unstemmed Determining the N-terminal orientations of recombinant transmembrane proteins in the Escherichia coli plasma membrane
title_short Determining the N-terminal orientations of recombinant transmembrane proteins in the Escherichia coli plasma membrane
title_sort determining the n-terminal orientations of recombinant transmembrane proteins in the escherichia coli plasma membrane
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604451/
https://www.ncbi.nlm.nih.gov/pubmed/26462555
http://dx.doi.org/10.1038/srep15086
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