EXPRESSION AND FUNCTIONAL-CHARACTERIZATION OF ESCHERICHIA-COLI NUSA AND LAMBDA-Q AS GLUTATHIONE-S-TRANSFERASE FUSION PROTEINS

The Escherichia coil transcription factor NusA and the bacteriophage l ambda antiterminator Q proteins were expressed as inducible glutathion e S-transferase (GST) fusion proteins. The fusion proteins were purifi ed under nondenaturing conditions by affinity chromatography on glutat hione agarose. Thrombin cleavage of the glutathione agarose-bound fusi on proteins yielded homogeneously pure NUsA(N+15) (5 mg/g cells) and a lmost homogeneously pure Q(N+13) protein (0.7 mg/g cells), where N + x indicates the presence of x additional amino acids at the N-terminus of the protein. The purified NUsA(N+15) exhibited the same activities as wildtype NusA in enhancement of transcriptional pausing, enhancemen t of termination at Rho-independent terminators, and enhancement of Q- mediated antitermination in vitro. The Q(N+13) protein exhibited both anti-pausing and antitermination activities in Q-mediated transcriptio n antitermination. However, the antitermination activity of Q(N+13) wa s lost gradually during storage if the thrombin used for cleavage of t he GST fusion protein was not removed. This was due to cleavage by thr ombin after Arg22 within the Q protein itself, at a noncanonical throm bin cleavage site, so the truncated protein (Q(N+22)) lacked the first 22 amino acids at the N-terminus of Q. The expression vectors describ ed here can be used to rapidly produce large quantities of these prote ins, and the truncated Q protein can be used to evaluate the requireme nt for the N-terminus of Q in antitermination, anti-pausing, interacti ons with the DNA template (gut site), and interaction with RNA polymer ase itself. (C) 1995 Academic Press, Inc.