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
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".