High-level expression of the mycobacterial porin MspA in Escherichia coli and purification of the recombinant protein
| Autor: | Stefanie Karosi, Christian Heinz, Michael Niederweis |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
DNA
Bacterial Circular dichroism Clinical Biochemistry Molecular Sequence Data Porins medicine.disease_cause Biochemistry Analytical Chemistry law.invention Mycobacterial porin law medicine Escherichia coli Amino Acid Sequence Peptide sequence Chromatography biology Base Sequence Chemistry Mycobacterium smegmatis Circular Dichroism Cell Biology General Medicine biology.organism_classification Recombinant Proteins Porin Recombinant DNA Electrophoresis Polyacrylamide Gel Bacterial outer membrane Chromatography Liquid |
| Zdroj: | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. 790(1-2) |
| ISSN: | 1570-0232 |
| Popis: | MspA is the prototype of a new family of tetrameric porins and provides the main general diffusion pathway for hydrophilic compounds through the outer membrane of Mycobacterium smegmatis. Structural analysis was hampered by the scarce amount of pure protein. After replacement of the GC-rich codons of the mspA gene by codons optimal for high-level expression in Escherichia coli, the mature MspA protein was overproduced in E. coli. The recombinant MspA (rMspA) monomer (M(r) 20000) was purified by anion exchange and hydrophobic interaction chromatography yielding 2.6 mg pure protein per liter of culture. This exceeded the yield of the native protein 10-fold. Circular dichroism revealed that rMspA is folded in a native-like structure. rMspA assembled partially to the channel-forming tetramer both during expression in E. coli and after purification in vitro. Thus, overexpression in E. coli and chromatographic purification are key steps towards a high resolution structure of MspA. |
| Databáze: | OpenAIRE |
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