Expression, purification and characterization of the IcmG and IcmK proteins of the type IVB secretion system from Coxiella burnetii
| Autor: | Katerina Arvaniti, Georgios Tsiotis, Myrto Koutantou, Athina Drakonaki, Iosif Vranakis, Cornelia Muenke, Anna Psaroulaki, Eirini Mathioudaki, Hao Xie |
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| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
Circular dichroism Size-exclusion chromatography 01 natural sciences Type IV Secretion Systems 03 medical and health sciences Bacterial Proteins Affinity chromatography 010608 biotechnology Escherichia coli Animals Humans Secretion Pathogen 030304 developmental biology Antigens Bacterial 0303 health sciences biology Chemistry Membrane Proteins Coxiella burnetii biology.organism_classification Antibodies Bacterial Recombinant Proteins Membrane protein Biochemistry Protein folding Q Fever Biotechnology |
| Zdroj: | Protein Expression and Purification. 186:105905 |
| ISSN: | 1046-5928 |
| Popis: | Coxiella burnetii, the causative agent of Q fever, is an intracellular bacterial pathogen. Studies on Coxiella have shown that a type IVB secretion system (T4BSS) contributes to the establishment of the infection by transferring protein molecules. In this report, we focus on two core proteins of the Coxiella T4BSS, namely the IcmG/DotF protein (CBU_1626) and the IcmK/DotH protein (CBU_1628). Here we present a method for the recombinant expression of IcmG and IcmK in E. coli. IcmG was purified by Strep-Tactin affinity chromatography and size exclusion chromatography, while for the purification of IcmK an additional anion exchange chromatography step was introduced. The yields of the purified IcmG and IcmK proteins were 1.2 mg/L and 3 mg/L, respectively. The purified proteins showed predominant band on SDS-PAGE gel of 37 kDa for the IcmG and 40 kDa for the IcmK. Protein folding is confirmed by circular dichroism spectroscopy. The dynamic light scattering experiment indicated that IcmG and IcmK existed in a homogenous form. Further Blue native PAGE indicates the presences of a monomeric form for the IcmK and IcmG. Our work lays the basis for functional exploration and structural determination of IcmG and IcmK proteins of Coxiella's secretion system. |
| Databáze: | OpenAIRE |
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