Paper Accepted in Protein Expression and Purification by Naim Hage
Naim’s first paper has very recently been accepted for publication in Protein Expression and Purification. The paper is entitled “Improved expression and purification of the Helicobacter pylori adhesin BabA through the incorporation of a hexa-lysine tag“
The paper represents the fruit of a long and at times frustrating, but finally very successful struggle to get this recombinant protein expressed and purified. A big thank you to our collaborators at Astra Zeneca R&D for their unflinching support in the past three years, and the EPSRC for funding (Grant EP/I01375X/1). We would like to thank Professor Thomas Borén and Dr. Jafar Mahdavi for the fruitful discussions and their interest in the project. We are also grateful to Professor John Atherton, University of Nottingham, for his generous gift of H. pylori J99 genomic DNA.
The paper can be downloaded from http://dx.doi.org/10.1016/j.pep.2014.10.009
Helicobacter pylori is a pathogenic bacterium that has the remarkable ability to withstand the harsh conditions of the stomach for decades. This is achieved through unique evolutionary adaptations, which include binding Lewisb antigens found on the gastric epithelium using the outer membrane protein BabA. We show here the yield of a recombinant form of BabA, comprising its putative extracellular binding domain, can be significantly increased through the addition of a hexa-lysine tag to the C-terminus of the protein. The paper describes BabA expression in the periplasmic space of Escherichia coli and its purification using immobilised metal ion affinity and size exclusion chromatography – yielding approximately 1.8 mg of protein per litre of culture. The hexa-lysine tag does not inhibit the binding activity of BabA as the recombinant protein was found to possess affinity towards HSA–Lewisb glycoconjugates.
The effect of the hexa-lysine tag on recombinant BabA expression. Coomassie staining (left) and immunodetection (right) of periplasmic extracts from E. coli shows BabA recombinant expression is significantly enhanced with the addition of a hexa-lysine tag. The (-) symbol represents uninduced cell samples while (+) represents cell samples induced with IPTG (equal amounts of sample were loaded). The BabA proteins are indicated by black arrows.
There will certainly be more papers to follow - watch this space...