|مجال التميز||تميز دراسي وبحثي|
|عنوان البحث:||Introduction of a hexalysine (6K) tag can protect from N-terminal cleavage and increase yield of recombinant proteins expressed in the periplasm of E. coli|
|رابط إلى البحث:||https://link.springer.com/protocol/10.1007/978-1-0716-1859-2_9|
|موجز عن البحث:||Recombinant expression of proteins in the periplasm of E. coli is frequently used for proteins containing disulfide bonds that are essential for protein folding and activity, as the cytosol of E. coli constitutes a reducing environment. The periplasm in contrast is an oxidative environment which supports proper protein folding. However, yields can be limited compared with cytoplasmic expression and protocols must be adjusted to avoid overloading the periplasmic transportation machinery. Another less appreciated issue with periplasmic expression is the potential generation of unwanted N-terminal cleavage products, a persistent issue which we encountered when expressing the disulfide bond containing extracellular regions of several Helicobacter pylori adhesins (BabA, BabB, BabC and LabA) in the periplasm of E. coli XL10 GOLD, a strain traditionally not used for proteins expression. Here, we describe how introducing a C-terminal hexa-lysine (6K) tag enhanced solubility and protected BabA from N-terminal proteolytic degradation (BabA), enabling crystallization and subsequent X-ray structural analysis. However. the same strategy had no advantageous effect for LabA, which using this protocol could be retrieved from the periplasm in relatively high yields (20-40 mg/L).|
|عنوان المؤتمر:||World Conference on Epidemiology-Infectious Diseases and Public Health|
|مكان الإنعقاد:||London, UK|
|طبيعة المشاركة:||Poster presentation|
|عنوان المشاركة:||Functional Characterisation of the Helicobacter pylori Outer Membrane Protein BabC|
|ملخص المشاركة:||Helicobacter pylori (H. pylori) is a pathogenic bacterium that colonises the gastric mucosa of more than 50% of all humans and a leading cause of peptic ulceration and gastric cancer worldwide. Despite the harsh conditions in the stomach, the bacterial adherence to the gastric mucosa and epithelium plays a significant role in initial colonisation and in long-term persistence by using multitude of adhesins that facilitate the adhesion to the host cells. Studies are greatly needed to understand the dynamic interplay between these adhesins, some of which have not been fully characterised. Furthermore, the need to develop alternative therapeutic strategies is important with the increased rate of antibiotic resistance associated with H. pylori eradication through targeting these adhesins.
This study is aimed at characterising the functional properties of the H. pylori outer membrane protein BabC, which has not been studied well previously with unknown structure and function. The first objectives of this study have been successfully completed through amplification of babC gene from the H. pylori strain 26695, cloning it into pOPE101, followed by transformation of the pOPE101_babC construct into E. coli XL10 Gold, and periplasmic expression and purification of BabC. BabC protein did not show any binding to Lewis a (Lea), Leb, Ley, or sialyl-Lewis x (sLex) glycoconjugates which are considered as the main glycan epitopes in gastrointestinal mucins. Analysis of amino acid sequence of the crowns of BabA J99 and BabC 26695 shows that the residues D233 and S234 in BabA crown, partially responsible for BabA binding to Leb, are missing in the amino acid sequence of the predicted BabC crown in which three hydrogen bonds will not be mediated with Leb; two hydrogen bonds between D233 and Gal5/GlcNAc3 and one hydrogen bond between S234 and Gal5. Based on this analysis, binding of BabC to Leb may not happen. Future work will reveal more about BabC and will bring us closer to achieve our aim.