Mutation of High-Affinity Methionine Permease Contributes to Selenomethionyl Protein Production in Saccharomyces cerevisiae.
The production of selenomethionine derivatives of recombinant proteins allows phase determination by single- or multi-wavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic ... proteins that require posttranslational modification, the toxic effects of selenomethionine often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both selenomethionine and selenate, and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to selenomethionine by SMR-94 strain remains unclear. Here, we report the characterization of selenomethionine-resistant mutants of Saccharomyces cerevisiae, and the identification of a mutant allele of the MUP1 gene, encoding high-affinity methionine permease, which confers selenomethionine resistance. Although the total Met uptake by the mup1 mutant (SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture condition, the mup1 mutant showed higher productivity of SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.
Mesh Terms:
5-Methylcytosine, Acetylation, Animals, Blastocyst, Cricetinae, DNA (Cytosine-5-)-Methyltransferase, DNA Methylation, DNA Modification Methylases, Embryonic Development, Epigenesis, Genetic, Female, Fluorescent Antibody Technique, Gene Expression, Genitalia, Male, Histones, In Situ Hybridization, Insulin-Like Growth Factor II, Male, Membrane Proteins, Mesocricetus, Pregnancy, Reverse Transcriptase Polymerase Chain Reaction, Spermatozoa
5-Methylcytosine, Acetylation, Animals, Blastocyst, Cricetinae, DNA (Cytosine-5-)-Methyltransferase, DNA Methylation, DNA Modification Methylases, Embryonic Development, Epigenesis, Genetic, Female, Fluorescent Antibody Technique, Gene Expression, Genitalia, Male, Histones, In Situ Hybridization, Insulin-Like Growth Factor II, Male, Membrane Proteins, Mesocricetus, Pregnancy, Reverse Transcriptase Polymerase Chain Reaction, Spermatozoa
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Date: Aug. 06, 2010
PubMed ID: 20693451
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