Overexpression in E. coli and purification of the L. pneumophila Lpp2981 protein.
The Lpp2981 gene from Legionella pneumophila, the causative agent of Legionnaire's disease, was cloned into the pMWT7 plasmid. The construct was used to express this gene in Escherichia coli. Five different bacterial strains were tested to overexpress the gene but without success. Sequence analysis revealed a cluster of four rare codons near the 5'-end of the gene. These codons were replaced with those commonly used in E. coli. The mutated Lpp2981 gene was successfully expressed in all the E. coli strains tested. The expressed protein (with an apparent molecular mass of 30 kDa) was collected in the insoluble fraction of the cell lysate, purified as inclusion bodies and functionally reconstituted into liposomes. The highest level of overexpression was obtained in E. coli C0214 after 6 h of induction with isopropyl-β-D-thiogalactopyranoside at 37 °C, yielding 74 mg of purified protein per liter of culture. We conclude that the clustering of rare codons at the 5'-end of the open-reading frame is a critical factor for the heterologous expression of Lpp2981 in E. coli.
Department of Biosciences, Biotechnologies and Biopharmaceutic, University of Bari, Via E. Orabona 4, 70125, Bari, Italy.
Giulia
G
Giannuzzi
Nunzio
N
Lobefaro
Eleonora
E
Paradies
Angelo
A
Vozza
Giuseppe
G
Punzi
Carlo M T
CM
Marobbio
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D016952:Legionella pneumophila / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D009154:Mutation; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D003062:Codon; D008081:Liposomes; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside