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

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, in order to facilitate biochemical studies using thiolspecific modifying reagents. Of ten 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 Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in S. cerevisiae cells auxotrophic for purines, several highly functional non-cysteine 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. Keywordscysteine-less; substituted cysteine accessibility method; membrane protein; site-saturation mutagenesis; genetic selection; Quikchange mutagenesis; equilibrative nucleoside transporter A detailed understanding of how a protein functions must include structural information at some level of resolution. While x-ray crystal structures of thousands of soluble proteins have been determined to date, crystals of membrane proteins that diffract to high resolution are notoriously difficult to obtain [1]. In the absence of any high-resolution structural information for many membrane protein families, biochemical methods provide a valuable means to intuit structure. Several of these methods make use of strategically placed cysteine residues because of their unique chemistry among the natural amino acids. For example, cysteines engineered into two transmembrane helices that can be cross-linked with bifunctional thiol-reactive reagents of varying lengths provide information about the distance between the two helices in the folded structure of the protein [2]. Also, the steric and chemical environment of a cysteine residue engineered at a position of interest can be probed by the substituted cysteine Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [3][...

Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae

“…Determination of [ 3 H]cellobiose transport assays and kinetic parameters. Cellobiose transport assays were performed using a modification of the oil-stop method (19). Engineered yeast strains expressing transporter genes fused to GFP were grown to the mid-exponential phase in selective medium, washed 3ϫ with assay buffer (30 mM morpholineethanesulfonic acid [MES]-NaOH [pH 5.6] and 50 mM ethanol), and resuspended to an OD 600 of 40.…”

Role of Transmembrane Domain 4 in Ligand Permeation by Crithidia fasciculata Equilibrative Nucleoside Transporter 2 (CfNT2)

“…Expression of CfNT2 conferred upon yeast the ability to grow on medium containing inosine as the sole purine source ( Fig. 2A) (17,27) but not on adenosine (Fig. 2B), which is not a ligand of the wild-type transporter (compare with robust growth of cells expressing CfNT1, a C. fasciculata adenosine transporter (17) (Fig.…”

“…The construction of the pRS426-CuCfNT2 and pRS426-Cu-CfNT1 expression vectors is described by Liu et al (17). Leishmanial expression of CfNT2 and its cfnt2 mutant variants was from the pALTneo vector (26) with an N-terminal HA tag, pALTneo-HA, as described (27). The construction of the cysteineless cfnt2 gene (cfnt2⌬Cys) is described by Arendt et al (27).…”

“…To experimentally determine the environmental context of residues within TM4, and especially Lys 155 , SCAM (42) was utilized. Using the cysteineless variant of CfNT2, cfnt2⌬Cys (27), as a starting point, a cysteine codon was introduced at each position predicted to be part of TM4 in any of the published ENT topology predictions. Genes expressing cfnt2 mutants containing a single introduced cysteine residue (cfnt2⌬Cys-H138C through cfnt2⌬Cys-K172C) were expressed in the ⌬ldnt1/⌬ldnt2 cell line, and uptake of [ 3 H]inosine by these lines in the presence and absence of 1000ϫ unlabeled inosine was assayed.…”

“…Leishmanial expression of CfNT2 and its cfnt2 mutant variants was from the pALTneo vector (26) with an N-terminal HA tag, pALTneo-HA, as described (27). The construction of the cysteineless cfnt2 gene (cfnt2⌬Cys) is described by Arendt et al (27). All point mutants were constructed by site-directed mutagenesis of pALTneo-HA-CfNT2 or pALTneo-HA-cfnt2⌬Cys performed according to the QuikChange mutagenesis protocol (Stratagene, La Jolla, CA) with primer design based on the method of Zheng et al (28).…”