title: Engineering trypsin-sensitive sites in a membrane transport protein.
abstract: This paper describes a systematic procedure for introducing protease-sensitive sites into bacterial integral membrane proteins. Such sites should make it possible to monitor the subcellular localization of individual domains of a topologically complex protein. Escherichia coli lac permease was used as a model. Site-directed mutagenesis, targeted to a particular periplasmic domain, was used to generate insertion derivatives containing a lysine residue in different sequence contexts. Individual mutants were then screened for lactose transport activity and efficient cleavage by trypsin. To facilitate this screen, the mutagenesis was carried out using a gene fusion encoding an easily detected, bifunctional lac permease-galactosidase hybrid. Insertions were identified in the fourth and sixth periplasmic domains (P4 and P6) that were efficiently cleaved in both the hybrid protein and in unfused lac permease. One of the P6 insertion mutants exhibited lactose transport specific activity near that of the wild-type and was shown by sequence analysis to be cleaved at the expected site in the inserted sequence. As part of this analysis, we determined the range of cellular concentrations of lac permease over which lactose uptake was linear. The activity showed a plateau at a relatively low concentration corresponding to approximately five times the wild-type level.
authors.affiliation: Department of Genetics, University of Washington, Seattle 98195, USA.
authors.forename: M H
authors.initials: MH
authors.lastname: Lee
authors.forename: C
authors.initials: C
authors.lastname: Manoil
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D015202:Protein Engineering / Q000379:methods; D027981:Symporters; D014357:Trypsin / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D014357:Trypsin