Bacterial expression of G-protein-coupled receptors: prediction of expression levels from sequence.
Eleven G-protein-coupled receptors were expressed in Escherichia coli as fusion proteins with N-terminal glutathione-S-transferase and a C-terminal (His)6 tag. Expression levels varied between 0.1% and 10% of the total cellular protein. Low expression levels, as quantified by analytical nickel chelating chromatography, coincided with a toxic effect of protein expression. Multiple linear regression analysis was used to establish a correlation between the occurrence of positively charged amino acid residues in the loop regions and the expression level. Indeed, 44% of the variation in expression levels could be attributed to the positive charge content. Consequently, this sequence feature is the major determinant of expression level. Our results were supported by two mutations where positive charges were introduced into loop regions of two low-expressing receptors: As predicted, these mutations led to a considerably higher expression. A similar mutation of an olfactory receptor described previously increased expression approximately 100-fold and further supports our model. The data are discussed in the context of the "positive inside rule".
Kiefer
H
H
Institut für Mikrobiologie, Universität Hohenheim, Stuttgart, Germany. hans.kiefer@m-phasys.com
Vogel
R
R
Maier
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002458:Cell Fractionation; D002845:Chromatography; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D016014:Linear Models; D008969:Molecular Sequence Data; D009154:Mutation; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D011506:Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D009532:Nickel; D019204:GTP-Binding Proteins