Protein Expression Protocol for the Catalytic Domain of an Adenylate Cyclase Membrane-Anchored by a Vibrio Quorum Sensing Receptor

Abstract

The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported ( Beltz et al., 2016 ). This protocol describes the expression and membrane preparation for these chimeric proteins.

Keywords: Adenylate cyclase (AC); CqsS; French press; Membrane protein; Protein expression; Quorum sensing (QS).

Figure 1.. Comparison of the two MCS of pETDuet-1 (A) and pETDuet-3 (B)

Figure 2.. Short French press protocol.

A. Equipment of the French Pressure Cell; B. Aminco French Pressure Cell (taken out of the fridge 4 °C); C. Attention! Before use: Grease the O-rings and back-up rings of the stamp with glycerol! Insert the stamp into the cell body so that the inner cell body surface is covered with glycerol as well! D. Place the cell body with the stamp upside down into the stand with the opening facing upwards. E. Place the flow valve into the hole. F. Fill in the cell solution (looks milky). G. Put the lid on top. H. Close the flow valve. I. Place the French Pressure Cell into the gadget and… (J) close the bracket. K. Start the French press by turning the hand gear on ‘high’ and (L) switch on the pump. M. When the French press set up 1,100 psi… (N) open carefully the flow valve such that… (O) a flow drop by drop is visible. P. Attention! Stop in time so you can easily open the bracket and the stamp does not hit the ground! Q. When you are done, flip the switch to ‘down’ and turn on the pump again. The hydraulic lift moves down and you can take out the French Pressure Cell. R. Your cells are lysed properly when you can see the scale through the tube. S. Put your sample back on ice for the next step.

Figure 3.. Western blots (A-C) and SDS-PAGE (D)