Over-expression, purification, and confirmation of Bacillus anthracis transcriptional regulator NprR

• Published in: Protein expression and purification 2016-09, Vol.125, p.83-89
• Main Authors: Rice, Amy J., Woo, Jerry K., Khan, Attiya, Szypulinski, Michael Z., Johnson, Michael E., Lee, Hyunwoo, Lee, Hyun
• Format: Article
• Language: English
• Subjects: Bacillus anthracis; Bacillus anthracis - genetics; Bacillus anthracis - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Cell Line; Gene Expression; Gene Expression Regulation, Bacterial; Humans; Purification; Quorum Sensing; Surface Plasmon Resonance; Tandem Mass Spectrometry; The neutral protease regulator (NprR); Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - isolation & purification; Transcription Factors - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2015.08.030

•Cloning, expression, and purification of B. anthracis AqsR protein.•A lowered induction temperature resulted in a reasonable yield.•High purity of AqsR protein obtained by IMAC and gel filtration chromatography.•Full-length AqsR protein was confirmed with mass spectroscopy.•Peptide binding kinetics were determined using surface plasmon resonance. Quorum sensing (QS) has been recognized as an important biological phenomenon in which bacterial cells communicate and coordinate their gene expression and cellular processes with respect to population density. Bacillus anthracis is the etiological agent of fatal pulmonary anthrax infections, and the NprR/NprX QS system may be involved in its pathogenesis. NprR, renamed as aqsR for anthrax quorum sensing Regulator, is a transcriptional regulator that may control the expression of genes required for proliferation and survival. Currently, there is no protocol reported to over-express and purify B. anthracis AqsR. In this study, we describe cloning, purification, and confirmation of functional full-length B. anthracis AqsR protein. The AqsR gene was cloned into the pQE-30 vector with an HRV 3C protease recognition site between AqsR and the N-terminal His6-tag in order to yield near native AqsR after the His-tag cleavage, leaving only two additional amino acid residues at the N-terminus.