Purification of Acyl Carrier Protein
Quorum sensing is a chemical communication tool used by bacteria to count other bacteria in the immediate vicinity. Bacteria generate signal molecules called auto-inducers to estimate local population densities. This census count allows bacteria to work together to overcome threats such as antibiotics. Our laboratory is focused on developing inhibitors for N-acylhomoserine lactone synthase—or AHL Synthase—enzymes that make signaling molecules in gram-negative bacteria, which tend to be resistant to antibiotics. AHL synthase enzymes use acyl-ACP and S-Adenosyl-L-methionine substrates to synthesize N-acylhomoserine lactone (AHL) auto-inducers.
My project is focused on purifying acyl carrier protein (ACP) that is used to make acyl-ACP substrates for AHL Synthase. The process of growing the protein starts with a small growth of E. coli, which is then transferred to a large growth. IPTG was added at the mid-log growth phase to start gene transcription. A centrifuge was used to stop protein expression and bacterial cells were concentrated to a pellet. A chemical lysing agent called BPER reagent, was used to split the bacteria open. Since ACP is an acidic protein carrying a net negative charge, this protein was purified using ion-exchange chromatography. The purity of fractions collected were checked using SDS-PAGE and fractions that contain pure protein were desalted using PD10 column and further concentrated using an Amicon 3 kD molecular weight cut off column. ACP will be enzymatically coupled to an acyl chain using a Sfp phosphopantetheinyl transferase enzyme and the activity of acyl-ACP substrates with AHL synthase enzymes will be checked using a spectrophotometric based assay.
Gibson, Jessie; Johnson, Aubrey; Frane, Nicole; and Richard, Angie, "Purification of Acyl Carrier Protein" (2014). College of Arts and Sciences Presentations. 42.