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

Chien-Hsien Lee,Min-Feng Hsu,Andrew H.-J Wang,Chia-Cheng Chou

doi:10.1038/srep15086

Chien-Hsien Lee, Min-Feng Hsu + Show 2 more

Open Access

https://doi.org/10.1038/srep15086

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Abstract

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 (Nin orientation). To create a suitable linker for orientating the target TM proteins with the periplasmic N-termini (Nout 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 Nout 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 Nin or Nout by evaluating the net expression of the fusion proteins.

Highlights

We have developed a quick and easy method to obtain N-terminal topological information concerning the recombinant TM proteins inserted into the E. coli plasma membrane
Because the C-terminus of D94N is oriented in the membrane as Cin[12], fusing a TM protein with a naturally occurring N-terminus orientation (Nin) orientation downstream to D94N should potentially increase its expression
The N-terminal orientation of each TM protein was assessed by evaluating its expression after insertion into D94N-3TM and D94N fusion vectors by Western blotting (WB) analysis, where the fusion proteins were all at the anticipated molecular weight (Fig. 4B,C)

Summary

Introduction

By combining the protein tags for C-terminal orientation determination with D94N-related fusion partners, a more comprehensive topological information of a target TM protein can be obtained. The increased expression could suggest that the fused TM target protein has a Nin orientation.

Results

Conclusion