Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis

Cassandra S. Arendt, Keirei Ri, Phillip Yates, Buddy Ullman

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2 to facilitate biochemical studies using thiol-specific modifying reagents. Of 10 endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site saturation mutagenesis scheme based on the Stratagene Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in Saccharomyces cerevisiae cells auxotrophic for purines, several highly functional noncysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position.

Original languageEnglish (US)
Pages (from-to)185-193
Number of pages9
JournalAnalytical Biochemistry
Volume365
Issue number2
DOIs
Publication statusPublished - Jun 15 2007

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Keywords

  • Cysteine-less
  • Equilibrative nucleoside transporter
  • Genetic selection
  • Membrane protein
  • Quikchange mutagenesis
  • Site saturation mutagenesis
  • Substituted cysteine accessibility method

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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