High-level expression of the mycobacterial porin MspA in Escherichia coli and purification of the recombinant protein.
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.
Heinz
Christian
C
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058, Erlangen, Germany.
Karosi
Stefanie
S
Niederweis
Michael
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D002942:Circular Dichroism; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D018272:Porins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D018272:Porins; D011994:Recombinant Proteins; C121838:mspA protein, Mycobacterium smegmatis