Expression in Escherichia coli of cytoplasmic portions of the cystic fibrosis transmembrane conductance regulator: apparent bacterial toxicity of peptides containing R-domain sequences.
Large peptide segments (148-479 amino acids) of the cystic fibrosis transmembrane conductance regulator, which are projected to represent cytoplasmic portions of this large membrane protein, were expressed in Escherichia coli using two T7 RNA polymerase vectors, pET11a and pRSET. Five of the nine peptides were readily expressed at high levels (15-35 mg/liter) and one at an intermediate level (10 mg/liter), but three could not be expressed at >1.5 mg/liter regardless of efforts to further optimize the system. Preinduction testing of these latter plasmids failed to demonstrate any plasmid instability, while bacterial survival was drastically curtailed upon induction, beyond that observed with the other plasmids. Peptides containing the second half of exon 13 (residues 700-830; R domain) appear to be especially toxic to the expressing bacteria. Peptides including this hydrophilic segment may be inhibiting the bacterial permeases which are known to he homologous to other portions of CFTR.
Yike
I
I
Department of Pediatrics, Willard A. Birnbaum Cystic Fibrosis Research Center, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Zhang
Y
Y
Ye
J
J
Dearborn
D G
DG
D000595:Amino Acid Sequence; D000667:Ampicillin / Q000494:pharmacology; D001483:Base Sequence; D002969:Clavulanic Acids / Q000494:pharmacology; D003001:Cloning, Molecular; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D005091:Exons; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D017421:Sequence Analysis; D013982:Ticarcillin / Q000494:pharmacology
C505032:CFTR protein, human; D002969:Clavulanic Acids; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D007544:Isopropyl Thiogalactoside; D000667:Ampicillin; C043215:ticarcillin-clavulanic acid; D013982:Ticarcillin