Poster Presentation The 42nd Lorne Conference on Protein Structure and Function 2017

Expression, purification and characterization of sialic acid binding adhesin A (SabA) from Helicobacter pylori (#120)

Dilini Chandrapala 1 , Siew Siew Pang 1 , Terry Kwok-Schuelein 1
  1. Monash University, Clayton, VIC 3800, Notting Hill, VICTORIA, Australia

Helicobacter pylori is a highly persistent gastric pathogen and is a major causative agent of chronic gastritis and gastric cancer. Adhesion of H. pylori to gastric epithelial cells is an essential step leading to colonization and subsequently chronic infection. The H. pylori outer membrane protein SabA (sialic acid binding adhesin A) is crucial for binding to inflamed gastric tissues through interaction with sialylated glyans e.g. sialyl lewis X (sLeX). SabA consists of an N-terminal ecto-domain, a linker-region and a membrane-anchored β-barrel domain. The crystal structure of SabA ecto-domain has been solved previously in our laboratory and the putative ligand binding site identified. However, the roles of the linker-region and the β-barrel domain of SabA in determining ligand binding affinity, avidity and specificity remain unclear. In this project, full-length SabA was cloned and expressed in Escherichia coli as inclusion bodies. SabA was then refolded as a soluble membrane protein in the presence of detergent and purified by immobilized-metal affinity and size-exclusion chromatography. Purified and refolded full-length SabA migrated as a 73 kDa protein band on SDS-PAGE, and was confirmed to be SabA using mass spectrometry and SabA-specific antibodies. The secondary structure contents and folding status of full-length SabA were analyzed by circular dichroism and thermal melting experiments. Moreover, the folding status of the β-barrel domain in SabA was examined using heat modification assays. Further analyses are under way to characterize the functions of full-length SabA.