The proteins encoded by the rbs operon of Escherichia coli: II. Use of chimeric protein constructs to isolate and characterize RbsC.
Chimeric genes encoding full-length copies of rbsA and rbsC connected by segments coding for short bridge peptides were constructed and expressed in Escherichia coli. Surprisingly, the chimeric genes complemented the strain in which rbsA and rbsC were deleted. The chimeric proteins were overproduced, and the products were purified by affinity chromatography. In order to obtain highly purified protein, a poly-His leader peptide was incorporated so that Ni-chelate affinity chromatography could be employed. The leader peptide and the bridge peptide were designed with factor Xa-cleavable sites to permit recovery of the individual RbsA and RbsC protein. A rbsC gene encoding a poly-His leader was also constructed and expressed. Both the chimeric RbsA-C species and the poly-HisRbsC were produced at levels that permitted isolation of the equivalent of milligram quantities of RbsC per liter of culture. This is a substantial increase in amounts from any previous RbsC production vectors. All proteins from the rbs operon have now been overproduced and substantially purified.
Zaitseva
J
J
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Zhang
H
H
Binnie
R A
RA
Hermodson
M
M
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000345:Affinity Labels / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression / Q000235:genetics; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D010957:Plasmids; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D002352:Carrier Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C102053:RbsA protein, E coli; C102054:RbsC protein, E coli; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000241:Adenosine