A procedure for renaturation and purification of the extracellular Serratia marcescens nuclease from genetically engineered Escherichia coli.
Overproduction of the extracellular Serratia marcescens nuclease in Escherichia coli results in aggregation and sequestration of a large amount of the protein in inclusion bodies. Only a relatively small amount is secreted into the medium from which it can be purified following established procedures. The cell-associated insoluble protein can be solubilized in 6 M urea after breaking up the cells by sonication. Renaturation is achieved by dilution or dialysis. Subsequent phosphocellulose chromatography yields a homogeneous protein preparation which is shown by a variety of biochemical and biophysical analyses to be indistinguishable from conventionally prepared material. The high yield (> 10 mg/500-ml culture) and the ease of preparation (2 to 3 days) make this an attractive alternative to previously described procedures.
Friedhoff
P
P
Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Giessen, Germany.
Gimadutdinow
O
O
Rüter
T
T
Wende
W
W
Urbanke
C
C
Thole
H
H
Pingoud
A
A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity; D004706:Endodeoxyribonucleases / Q000096:biosynthesis / Q000302:isolation & purification*; D004722:Endoribonucleases / Q000096:biosynthesis / Q000302:isolation & purification*; D004926:Escherichia coli; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics; D014508:Urea
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D014508:Urea; D002482:Cellulose; C008881:phosphocellulose; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C021835:Serratia marcescens nuclease