An improved system for ribonuclease Ba expression.
The extracellular ribonuclease from Bacillus amyloliquefaciens (barnase, RNase Ba) is a well-characterized enzyme extensively used in structure-function studies. A new system for efficient expression and purification of barnase has been developed. The strong regulated expression cassette with the Pr promoter of lambda phage and the cooperative expression of barnase and barstar under its control have been applied to expression of these proteins in Escherichia coli. The expression cassette containing the Pr promoter of E. coli lambda phage under cI repressor regulation and the nucleotide sequence coding for barnase and barstar structural genes were merged into the plasmid pTN441, which was used for large-scale barnase production. The phoA signal peptide was used to express the target protein into cell periplasm. The purification of RNase Ba was carried out in two steps: the initial sample was concentrated followed by RP-HPLC. The system provides a stable yield of homogeneous protein of about 100-150 mg per liter of culture medium.
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
A L
AL
Okorokov
R W
RW
Hartley
K I
KI
Panov
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease