Abstract
The overproduction of eukaryotic membrane proteins is a major impediment in their structural and functional characterization. Here we have used the nisin-inducible expression system of Lactococcus lactis for the overproduction of 11 mitochondrial transport proteins from yeast. They were expressed at high levels in a functional state in the cytoplasmic membrane. The results also show that the level of expression is influenced by the N-terminal regions of the transporters. Expression levels were improved >10-fold either by replacing or truncating these regions or by adding lactococcal signal peptides. The observed expression levels are now compatible with a realistic exploration of crystallization conditions. The lactococcal expression system may be used for the high-throughput functional characterization of eukaryotic membrane proteins and structural genomics.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Carrier Proteins / biosynthesis
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Carrier Proteins / chemistry
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Carrier Proteins / genetics
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Carrier Proteins / metabolism
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Gene Expression*
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Lactococcus lactis / genetics*
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Membrane Proteins / biosynthesis*
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Membrane Proteins / chemistry
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Membrane Proteins / genetics*
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Membrane Proteins / metabolism
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Mitochondria / metabolism
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Mitochondrial Proteins / biosynthesis
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Mitochondrial Proteins / chemistry
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Mitochondrial Proteins / genetics
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Mitochondrial Proteins / metabolism
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Molecular Sequence Data
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Protein Sorting Signals / genetics
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Protein Sorting Signals / physiology
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae / chemistry
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / biosynthesis
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
Substances
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Carrier Proteins
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Membrane Proteins
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Mitochondrial Proteins
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Protein Sorting Signals
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins