Melibiose permease of Escherichia coli: large scale purification and evidence that H+, Na+, and Li+ sugar symport is catalyzed by a single polypeptide.
As much as 20-30 mg of functional recombinant melibiose permease (Mel-6His permease) of Escherichia coli, carrying a carboxy-terminal affinity tag for metallic ions (six successive histidines), can be routinely purified from 10 g of cells (dry weight) by combining nickel chelate affinity chromatography and ion exchange chromatography. Mel-6His permease was constructed by modifying the permease gene (melB) in vitro and then overproduced in cells transformed with multicopy plasmids. The tagged permease was efficiently solubilized in the presence of 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO) and high sodium salt concentration and then selectively adsorbed on a nickel nitrilotriacetic acid (Ni-NTA) affinity resin. After the replacement of LAPAO by n-dodecyl beta-D-maltoside to maintain the activity of the soluble permease in low ionic strength media, the permease-enriched fraction (> 90%) was eluted with 0.1 M imidazole and finally purified to homogeneity (> 99%) using ion exchange chromatography. Determination of the permease N-terminal sequence shows that an initiating methionine is missing and that a Ser-Ile-Ser stretch precedes the postulated primary amino acid sequence. Purified permeases, reconstituted in liposomes, display H(+)-, Na(+)-, or Li(+)-dependent sugar binding and active transport activities similar to those of the native permease in its natural environment, proving that all three modes of symport activity are mediated by one and the same polypeptide.
Pourcher
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Laboratoire Jean Maetz, Département de Biologie Cellulaire et Moléculaire du CEA, 06230, Villefranche sur mer, France.
Leclercq
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S
Brandolin
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Leblanc
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G
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D050260:Carbohydrate Metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003902:Detergents; D004926:Escherichia coli / Q000201:enzymology*; D017353:Gene Deletion; D015870:Gene Expression; D018014:Gene Transfer Techniques; D008081:Liposomes / Q000378:metabolism; D008094:Lithium / Q000494:pharmacology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011522:Protons; D012964:Sodium / Q000494:pharmacology*; D012995:Solubility; D013329:Structure-Activity Relationship; D027981:Symporters
D003902:Detergents; D008081:Liposomes; D026901:Membrane Transport Proteins; D011522:Protons; D027981:Symporters; C021548:melibiose permease; D008094:Lithium; D012964:Sodium