Open AccessAn inducible expression system of histidine‐tagged proteins in Streptomyces lividans for one‐step purification by Ni 2+ affinity chromatography
Author(s) -
Enguita Francisco J.,
La Fuente Juan Luis,
Martín Juan F.,
Liras Paloma
Publication year - 1996
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1996.tb08095.x
Subject(s) - multiple cloning site , affinity chromatography , biology , microbiology and biotechnology , expression vector , expression cassette , escherichia coli , thiostrepton , streptomyces , histidine , gene , coding region , selectable marker , plasmid , biochemistry , recombinant dna , vector (molecular biology) , genetics , bacteria , rna , ribosome , amino acid , enzyme
An expression and purification cassette containing the aminoglycoside phosphotransferase gene ( aph ) as selective marker has been constructed in the Escherichia coli vector pULHis2. DNA fragments inserted in the cassette can be easily subcloned in pIJ699 to give vectors for overexpression of genes in Streptomyces and purification of proteins by a one‐step procedure. The expression system uses the thiostrepton‐inducible promoter tipA for expression and a six histidine coding nucleotide sequence that is fused in frame to the foreign gene inserted in the polylinker. The pULHis2‐derived expression vector has been used satisfactorily to express and to purify the P 7 and P 8 proteins of Nocardia lactamdurans which carry out the methoxylation of cephalosporin C to 7‐methoxycephalosporin C.