An inducible expression system of histidine-tagged proteins in Streptomyces lividans for one-step purification by Ni2+ affinity chromatography.
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 P7 and P8 proteins of Nocardia lactamdurans which carry out the methoxylation of cephalosporin C to 7-methoxycephalosporin C.
Enguita
F J
FJ
Department of Ecology, Genetics and Microbiology, Faculty of Biology, University of León, Spain.
de la Fuente
J L
JL
Martín
J F
JF
Liras
P
P
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006639:Histidine / Q000378:metabolism; D019868:Kanamycin Kinase; D008780:Methyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D009615:Nocardia / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013302:Streptomyces / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013883:Thiostrepton / Q000494:pharmacology; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C093122:7-alpha-cephem-methoxylase; D006639:Histidine; D006899:Mixed Function Oxygenases; D008780:Methyltransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D013883:Thiostrepton