Bacterial expression of G-protein-coupled receptors: Prediction of expression levels from sequence

Citation
H. Kiefer et al., Bacterial expression of G-protein-coupled receptors: Prediction of expression levels from sequence, RECEPT CHAN, 7(2), 2000, pp. 109-119
Citations number
14
Categorie Soggetti
Cell & Developmental Biology
Journal title
RECEPTORS & CHANNELS
ISSN journal
10606823 → ACNP
Volume
7
Issue
2
Year of publication
2000
Pages
109 - 119
Database
ISI
SICI code
1060-6823(2000)7:2<109:BEOGRP>2.0.ZU;2-C
Abstract
Eleven G-protein-coupled receptors were expressed in Escherichia coli as fu sion proteins with N-terminal glutathione-S-transferase and a C-terminal (H is)(6) tag. Expression levels varied between 0.1% and 10% of the total cell ular protein. Low expression levels, as quantified by analytical nickel che lating chromatography, coincided with a toxic effect of protein expression. Multiple linear regression analysis was used to establish a correlation be tween the occurrence of positively charged amino acid residues in the loop regions and the expression level. Indeed, 44% of the variation in expressio n levels could be attributed to the positive charge content. Consequently, this sequence feature is the major determinant of expression level. Our res ults were supported by two mutations where positive charges were introduced into loop regions of two low-expressing receptors: As predicted, these mut ations led to a considerably higher expression. A similar mutation of an ol factory receptor described previously increased expression approximately 10 0-fold and further supports our model, The data are discussed in the contes t of the "positive inside rule".