Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE credits
The development of an inexpensive, portable, stable, sensitive and selective biosensor for detection of odorants is a daunting task. Here, we hypothesized the development of a detector layer composed of the protein groups; the olfactory receptors (ORs) and the odorant binding proteins (OBPs), known to bind odorants in animal sensing. We report the design of 13 OR gene-vector constructs, and their subsequent transformation into
Escherichia (E.) coli (BL21 (DE3)-STAR-pLysS) strain. Moreover, we report the expression of several ORs into an in vitro wheat germ extract using three separate detergent mixes for protein solubilization.
In addition, we describe the design of an odorant binding protein from the Aenopheles gambiae mosquito PEST strain (OBP-PEST) gene-vector construct under an IPTG (Isopropyl β-D-1-thiogalactopyranoside) inducible promoter. OBP-PEST was heterologously expressed in E.coli with an 8 amino acid long sequence (WSPQFEK) attached C-terminally, via a thrombin cleavage site and a ﬂexible linker (GSSG). The WSPQFEK sequence, commonly referred to as a Strep-tag, enabled subsequent afﬁnity chromatography puriﬁcation of the protein, via binding to an engineered Streptavidin equivalent. Surprisingly, the OBP-PEST was found to contain a signal sequence leading to its truncation and secretion when expressed in E.coli.
Biophysical analyses were established using Circular Dichroism (CD) for the analysis of two lipocalins: Beta-lactoglobulin (BLG) and OBP-PEST. We studied the solubility, refoldability and the conformational transitions of BLG, as a result of change in solvent, pH and temperature. The secondary structure of OBP-PEST and its thermal stability was investigated.
In conclusion, this thesis work has enabled biophysical analyses of OBP-PEST and future analogs of interest to the development of a stable protein detector layer. Although further experiments are needed to fully characterize the biophysical properties, and to ﬁnd odorant substrates of OBP-PEST, it was found to be a suitable alternative to ORs in a biosensor detector layer application. More importantly, an inherent OBP-PEST signal sequence was found to mediate protein secretion when expressed heterologously in E.coli. To the best of our knowledge this is the ﬁrst lipocalin discovered to be secreted upon heterologous expression in E.coli.
We hypothesize that this signal peptide could be used as a means for targeted secretion and, hence, efﬁcient protein puriﬁcation.
2011. , 67 p.
odorant binding protein, olfaction, olfactory receptor, secretion, signal sequence