Figure 9
From: Characterisation of a natural variant of the γ-butyrolactone signalling receptor
Heterologous expression and Western analysis of ScbR. A. Heterologous expression and purification of ScbRM145 for the creation of ScbR antibodies. Crude cell extracts from E. coli JM101 harbouring pTE88 before (lane 1) and after (lane 2) induction with 0.2 % (w/v) of L-rhamnose. The MalE-ScbRM145 fusion protein present in the induced fraction was then purified with an amylose resin (lane 3). MalE-ScbRM145 was cleaved with Factor Xa to separate MalE from ScbRM145 (lane 4). ScbRM145 was further purified with a heparin column (lane 5). Arrows show the protein bands representing each protein. Theoretical molecular weight of MalE-ScbRM145, MalE and ScbRM145 are noted in brackets. All protein fractions were analysed on 12 % (w/v) SDS-PAGE followed by staining with Coomassie blue. M denotes for prestained protein molecular weight ladders (SM0431 (old version) (Fermentas).and Precision Plus Protein “All Blue” Standard (BioRad)). B. Heterologous expression of ScbRM145 and ScbRM600 for gel retardation assays and Western analysis of both forms of ScbR. ScbR-antibodies were generated using recombinant ScbRM145 shown in (a). ScbRM145 and ScbRM600 were expressed in E. coli JM101/pIJ6120 and pTE58 harbouring scbRM145 and scbRM600, respectively. In Western analysis, ScbR was detected with a sample of the recombinant ScbRM145 (“rScbRM145”) and in cell-free extracts (CE) of E. coli JM101/pIJ6120 and pTE58 (“ScbRM145” and “ScbRM600”). In CE of E. coli JM101 harbouring the empty expression vector pIJ2925 (“E. coli CE”) no ScbR was found. Comparable amounts of ScbR were detected with same amounts of total CE proteins.