Overproduction and purification of the Tn10-specified inner membrane tetracycline resistance protein Tet using fusions to beta-galactosidase.
Tetracycline resistance in the Enterobacteriaceae is mediated by a number of genetically related, usually plasmid-borne, determinants which specify an efflux system involving an inner membrane protein, Tet. Attempts to overproduce the Tn10 (Class B)-encoded Tet in Escherichia coli by cloning the structural gene tet downstream of the lambda PL promoter under regulation by temperature-sensitive lambda repressor cI857 were unsuccessful; induction at 42 degrees C resulted in filamentous, non-viable cells containing little detectable overproduction of the protein. However, cells containing tet fused to lacZ were resistant to tetracycline at 30 degrees C and synthesized modest amounts of a large fusion protein when induced at 42 degrees C. Fusion of the N-terminal half or the first 38 amino acids of tet to lacZ did lead to increased production of fusion proteins. Fusions could be purified by size or by LacZ immunoaffinity or substrate-affinity chromatography. In the latter method, selected detergents were required to counteract nonspecific binding of Tet to the adsorbant. Amino acid sequencing of the N-terminus of Tet-LacZ fusion proteins indicated that most molecules were blocked at this terminus. The sequence of an unblocked subpopulation was consistent with that expected from the nucleotide sequence. A collagen peptide linker, genetically placed between tet and lacZ, allowed recovery of purified Tet protein after collagenase treatment of the purified fusion protein.
Hickman
R K
RK
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
McMurry
L M
LM
Levy
S B
SB
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003094:Collagen / Q000235:genetics; D004251:DNA Transposable Elements; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D007763:Lac Operon; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D013696:Temperature; D013753:Tetracycline Resistance / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D003094:Collagen; D001616:beta-Galactosidase