Expression of porin from Rhodopseudomonas blastica in Escherichia coli inclusion bodies and folding into exact native structure.
The homotrimeric membrane channel porin from Rhodopseudomonas blastica was expressed without signal sequence in Escherichia coli. The protein assembled in inclusion bodies in the cytosol, from which it could be recovered using urea and detergents. After purification by anion-exchange chromatography, the protein crystallized under wild-type conditions. The X-ray structure was determined at 2.2 angstroms resolution, and a comparison with the known wild-type structure showed that the recombinant porin is identical at the atomic level. The method yields porin and designed mutants thereof in 100 mg amounts, allowing for detailed functional and mechanistic studies.
Schmid
B
B
Institut für Organische Chemie und Biochemie, Albert-Ludwig-Universität, Freiburg im Breisgau, Germany.
Krömer
M
M
Schulz
G E
GE
D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D002479:Inclusion Bodies / Q000378:metabolism; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D018272:Porins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D012241:Rhodopseudomonas / Q000235:genetics / Q000378:metabolism*; D014508:Urea
D017931:DNA Primers; D046911:Macromolecular Substances; D018272:Porins; D011994:Recombinant Proteins; D014508:Urea