Determination of the first methionine of GluQ-RS. The cloning strategy utilized is shown at the top. A fragment from the stop codon of dksA to the end of gluQ-rs gene was amplified from S. flexneri genomic DNA with the primers ATGGQRSF/ATGGQRSR and cloned into a pET15c vector using the restriction sites BamHI and XhoI. Therefore this clone represents the operon, but dksA was replaced with the plasmid encoded fragment pelB. The transcription of this plasmid, named pATGGQRS, is controlled by the T7 promoter and translation is controlled by the Shine-Dalgarno (SD) sequence of pelB, both contained within the plasmid. The GluQ-RS protein synthesized has a histidine tag (H6) at the C-terminus, facilitating protein purification. The putative ρ-independent terminator is represented by the stem loop symbol upstream of gluQ-rs gene , and the black box in the gene indicates the two possible peptides, depending of which methionine is utilized. Bottom: The SDS –polacrylamide gel electrophoresis showing the supernatant extract (S) and the partially purified protein (Pur) produced by cells carrying the recombinant plasmid. The predominant band indicated by the arrow was excised and subjected to amino terminal sequencing, yielding the following amino acid sequence: T-D-T-Q-Y-I-G-R-F-A-P. This corresponds to the sequence following the second methionine. The size of the molecular markers (M) are given in kDa.
Caballero et al. BMC Microbiology 2012 12:226 doi:10.1186/1471-2180-12-226