Metalloadsorption by Escherichia coli cells displaying yeast and mammalian metallothioneins anchored to the outer membrane protein LamB.
Yeast (CUP1) and mammalian (HMT-1A) metallothioneins (MTs) have been efficiently expressed in Escherichia coli as fusions to the outer membrane protein LamB. A 65-amino-acid sequence from the CUP1 protein of Saccharomyces cerevisiae (yeast [Y] MT) was genetically inserted in permissive site 153 of the LamB sequence, which faces the outer medium. A second LamB fusion at position 153 was created with 66 amino acids recruited from the form of human (H) MT that is predominant in the adipose tissue, HMT-1A. Both LamB153-YMT and LamB153-HMT hybrids were produced in vivo as full-length proteins, without any indication of instability or proteolytic degradation. Each of the two fusion proteins was functional as the port of entry of lambda phage variants, suggesting maintenance of the overall topology of the wild-type LamB. Expression of the hybrid proteins in vivo multiplied the natural ability of E. coli cells to bind Cd2+ 15- to 20-fold, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions.
Sousa
C
C
Centro Nacional de Biotecnología, CSIC, Madrid, Spain.
Kotrba
P
P
Ruml
T
T
Cebolla
A
A
De Lorenzo
V
V
D000327:Adsorption; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D002104:Cadmium / Q000378:metabolism*; D002352:Carrier Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D018272:Porins; D011991:Receptors, Virus / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; C500769:CUP1-1 protein, S cerevisiae; D002352:Carrier Proteins; D005656:Fungal Proteins; D018272:Porins; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; C007121:copper thionein; C016532:maltoporins; D002104:Cadmium; D008668:Metallothionein