A tightly regulated system for overproduction of bacteriophage T4 lysozyme in Escherichia coli.
Bacteriophage T4 lysozyme has been purified using the Ni-chelate affinity chromatography technique from overexpressing Escherichia coli cells by fusion to an N-terminal 6x His tail. Regulation of the lysozyme gene expression has been found to be critical during growth phase of the bacteria by comparing different plasmid constructions. Whereas a tac-promoter fusion construct alone did not lead to efficient production of T4 lysozyme because of early cell lysis, an improved repressor sequence and co-overproduction of the tac repressor resulted in high-level synthesis of the foreign protein after IPTG induction. Purification of the fusion protein from autolyzed crude cell extracts is possible in a simple one-step procedure.
Düring
K
K
University of Hamburg, Center for Applied Plant Molecular Biology, Germany.
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002846:Chromatography, Affinity; D004790:Enzyme Induction / Q000187:drug effects; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D007544:Isopropyl Thiogalactoside; D009113:Muramidase