Expression of IgA-specific endopeptidases from Neisseria meningitidis in E. coli
Abstract number: 1733_916
Khamiankou V., Kazeeva T., Shevelev A.
Objective: Immunoglobulin A1-specific proteinase (Igase) is a pathogenicity factor of meningococcus (Neisseria meningitidis). These enzymes belong to trypsin-like clan of serine proteinases. They exhibit a high substrate selectivity being able to discriminate between IgA1 and IgA2 and, on the other hand, to distinguish human IgA1 and IgA1 from non-primate species. Taking in account conservancy of hinge region subjected to cleavage in human and other vertebrates, these means that Igases exhibit two-level substrate-recognition control which can be denoted as a primary and non-primary specificity. Structural basis of such a high specificity remains obscure due to unavailability of native recombinant IgA1 good for crystallographic and enzymatic studies.
Methods: We carried out expression in E. coli of two separate fragments of Igase corresponding to theoretically predicted domains, refolded and purified the proteins.
Results: N-terminal domain harboured the clearly recognized triad of residues homologous to the active centre of kallikrein, a serine protease involved to haemostasis. After two-step chromatographical purification and refolding, this protein exhibited a protease activity towards human myeloma IgA1. C-terminal portion of IgAse corresponds to a putative substrate-binding domain. This protein was expressed as a peptide with N-terminal 6His-tag in form of inclusion bodies, purified by to-step anion-exchange chromatography and refolded. As expected, the refolded protein exhibited a high binding capacity towards IgA1 from both normal and myeloma patient plasma. Besides, the binding domain tightly bound human IgA2 as confirmed in both ELISA-type and affinity chromatography tests. Likely to the native IgAse, its separated catalytic domain attacked a single peptide bound in the native IgA1, as visualised by immunoblotting with anti-IgA heavy chain antibodies under non-reducing conditions. However, similar experiment accompanied with immunoblotting under non-reducing conditions revealed slight shifting of the processing site of the isolated catalytic domain versus ones of the native enzyme.
Conclusion: We first succeeded to predict occurrence of separate domains in IgA-protease, to refold and purify them as separate proteins and to demonstrate their activity.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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