MELIBIOSE PERMEASE OF ESCHERICHIA-COLI - LARGE-SCALE PURIFICATION ANDEVIDENCE THAT H+, NA+, AND LI+ SUGAR SYMPORT IS CATALYZED BY A SINGLEPOLYPEPTIDE

As much as 20-30 mg of functional recombinant melibiose permease (Mel- 6His permease) of Escherichia coli, carrying a carboxy-terminal affini ty 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 perm ease was constructed by modifying the permease gene (melB) in vitro an d then overproduced in cells transformed with multicopy plasmids. The tagged permease was efficiently solubilized in the presence of 3-(laur ylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO) and high sodium s alt concentration and then selectively adsorbed on a nickel nitrilotri acetic acid (Ni-NTA) affinity resin, After the replacement of LAPAO by n-dodecyl beta-D-maltoside to maintain the activity of the soluble pe rmease in low ionic strength media, the permease-enriched fraction (> 90%) was eluted with 0.1 M imidazole and finally purified to homogenei ty (> 99%) using ion exchange chromatography. Determination of the per mease N-terminal sequence shows that an initiating methionine is missi ng and that a Ser-Ile-Ser stretch precedes the postulated primary amin o acid sequence, Purified permeases, reconstituted in liposomes, displ ay H+-, Na+-, or Li+-dependent sugar binding and active transport acti vities similar to those of the native permease in its natural environm ent, proving that all three modes of symport activity are mediated by one and the same polypeptide.