Monitoring the disulfide bond formation of a cysteine-rich repeat protein from Helicobacter pylori in the periplasm of Escherichia coli.
Helicobacter pylori infection increases the risk of cardiovascular diseases besides leading to duodenal and gastric peptic ulcerations. H. pylori cysteine-rich protein B (HcpB) is a disulfide-rich repeat protein that belongs to the family of Sel1-like repeat proteins. HcpB contains four pairs of anti-parallel alpha helices that fold into four repeats with disulfide bonds bridging the helices of each repeat. Recent in vitro oxidative refolding of HcpB identified that the formation and folding of the disulfide bond in the N-terminal repeat are the rate limiting step. Here we attempted to understand the disulfide formation of HcpB in the periplasm of Escherichia coli. The protein was expressed in wild type (possessed enzymes DsbA, B, C, and D) and knock out (Dsb enzymes deleted one at a time) E. coli strains. The soluble part of the periplasm when analyzed by SDS-PAGE and Western Blot showed that the wild type and DsbC/D knock out strains contained native oxidized HcpB while the protein was absent in the DsbA/B knock out strains. Hence the recombinant expression of HcpB in E. coli requires DsbA and DsbB for disulfide bond formation and it is independent of DsbC and DsbD. Prolonged cell growth resulted in the proteolytic degradation of the N-terminal repeat of HcpB. The delayed folding of the N-terminal repeat observed during in vitro oxidative refolding could be the reason for the enhanced susceptibility to proteolytic cleavage in the periplasm. In summary, a good correlation between in vivo and in vitro disulfide bond formation of HcpB is observed.
Coleman Softlabs, Inc., 296 Bay Road, Atherton, CA 94027, USA. v_saths@yahoo.com
Venkataramani Sathya
VS
Devi
Peer R E
PR
Mittl
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D015153:Blotting, Western; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D016480:Helicobacter pylori / Q000235:genetics; D008565:Membrane Proteins / Q000172:deficiency; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019897:Periplasm / Q000737:chemistry*; D033903:Periplasmic Proteins / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000172:deficiency; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism*
D001426:Bacterial Proteins; D004220:Disulfides; C079503:DsbB protein, Bacteria; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; C410728:hcpA protein, Helicobacter pylori; D001618:beta-Lactamases; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli