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All measurements were done in triplicates and all experiments were reproduced at least three times independently. Data analyses were performed using Graphpad Prism. All assay-related figures are a representative of three similar experiments.
Results
Our objective in this Obatoclax study was to over-express and investigate the substrate specificity of YjdL from E. coli. The preliminary results from uptake alanyl-peptide inhibition studies indicated that YjdL displays a lower specificity towards Ala-Ala-Ala compared to Ala-Ala. The subsequent experiments were designed to investigate this, among POTs, unusual property further.
Expression
YjdL was over-expressed in E. coli using the pTTQ18 vector. This vector has been shown to be particularly effective for over-expression of bacterial secondary transporters [10]. The final protein product is expressed as a fusion with a C-terminal hexa-His tag, in light of topology informed strategies for expression of membrane proteins in E. coli [11]. This tag was included for detection by Western blotting. Immunostaining of solubilized membrane fractions using a penta-HRP conjugate clearly showed a band at 37 kDa, not present in the membrane fractions of cells harboring the empty vector, corresponding to the over-expressed protein ( Fig. 1A). Anomalous migration of secondary transporters in SDS-gels (calculated Mw of YjdL 53.9 kDa) is a common phenomenon, possibly caused by their hydrophobicity, high binding of SDS or the retention of secondary/tertiary structure facilitating their passage through the gel [12].