Introduction of a C-terminal hexa-lysine tag increases thermal stability of the LacDiNac binding adhesin (LabA) exodomain from Helicobacter pylori

Paraskevopoulou, Vasiliki; Artiaga, Ver�nica Garc�a; Rowlinson, Rachel; Winkler, G. Sebastiaan; Gellert, Paul; Stolnik, Snow; Overman, Ross; Falcone, Franco H.

Abstract

Helicobacter pylori is a pathogenic microorganism infecting approximately 50% of the global population, and establishes life-long colonization despite the hostile stomach environment. H. pylori employs a wide range of outer membrane proteins (adhesins) for epithelial attachment, which specifically bind to glycans or non-carbohydrate structures expressed on the gastric epithelium. A recently described adhesin from H. pylori is LabA, named after its ability to bind to a disaccharide present in gastric mucus (LacdiNAc-specific adhesin). Here, we describe the recombinant expression of LabA from H. pylori strains J99 and 26695 in E. coli. High yields of recombinant LabA were obtained using periplasmic expression. We found that the addition of a C-terminal hexalysine (6K) tag enhanced the thermal stability of LabA without affecting its secondary structure, using differential scanning fluorimetry and circular dichroism spectroscopy. In contrast to our previous report for another H. pylori adhesin (BabA), the 6K tag did not enhance recombinant protein yield or solubility. Both versions of LabA, with or without the 6K tag, were expressed and isolated from the periplasmic space of Escherichia coli, with a surprisingly high yield of at least 40?mg/L for each independent preparation, following a two-step purification protocol. The proteins were analyzed with mass spectrometry (MS). Unlike its reported effect on stability of BabA, the 6K tag did not appear to protect the N-term of recombinant LabA from partial periplasmic degradation.

Citation

Paraskevopoulou, V., Artiaga, V. G., Rowlinson, R., Winkler, G. S., Gellert, P., Stolnik, S., …Falcone, F. H. (2019). Introduction of a C-terminal hexa-lysine tag increases thermal stability of the LacDiNac binding adhesin (LabA) exodomain from Helicobacter pylori. Protein Expression and Purification, 163, 1-8. https://doi.org/10.1016/j.pep.2019.105446

• Journal Article Type: Article
• Acceptance Date: Jun 30, 2019
• Online Publication Date: Jul 1, 2019
• Publication Date: Jul 9, 2019
• Deposit Date: Sep 19, 2019
• Publicly Available Date: Sep 19, 2019
• Journal: Protein Expression and Purification
• Print ISSN: 1046-5928
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 163
• Article Number: 105446
• Pages: 1-8
• DOI: https://doi.org/10.1016/j.pep.2019.105446
• Keywords: Biotechnology; Helicobacter pylori; LabA; Periplasmic expression; Hexa-lysine tag; Thermal stability; Outer membrane porin
• Public URL: https://nottingham-repository.worktribe.com/output/2297331
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S1046592819302566?via%3Dihub