Bacterial expression of a eukaryotic membrane protein in fusion to various Mistic orthologs.
Mistic, a bacterial membrane-associating protein family, uniquely found in Bacillus species. It enhances expression of eukaryotic membrane proteins at the bacterial membrane. Mistic from B. subtilis (M110), expresses at the Escherichia coli membrane, however its shorter orthologs have been recently shown to be mainly cytoplasmic with varying membrane affinities. Based on that, we hypothesized that the expression level of membrane proteins fused to Mistic is correlated with the degree of membrane association of the particular Mistic protein. We compared expression levels by various Mistic proteins as fusion partners for the Aplysia californica Kv1.1 (aKv1.1) channel as a cargo membrane protein. Mistic from B. atrophaeus (M4), which has the highest membrane association among the shorter orthologs, enhanced expression of the transmembrane domain of aKv1.1 to the highest extent. In contrast, M1, which consists of the 84 C-terminal amino acids of M110 is the most soluble protein and showed the least capacity to express the channel. A chimeric Mistic, constructed with the first alpha-helix (H1) of M110 N-terminally fused to M4, did not increase the level of expression of aKv1.1 beyond those of either the M110 or the M4 fusions. The channel fused to M110, M4 or the aforementioned H1-M4 chimera, expresses in the highest quantity and quality among Mistic proteins, providing suitable sample for structural studies. Our data support the concept that expression levels of 'Misticated' membrane proteins are related to the independent chaperoning character of Mistic via direct membrane association, rather than related to specific sequence-dependent interaction with the E. coli translocon machinery.
Dvir
Hay
H
Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
Choe
Senyon
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008027:Light; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012542:Scattering, Radiation; D016415:Sequence Alignment
D001426:Bacterial Proteins; D008565:Membrane Proteins; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins