Combining Rational and Random Strategies in β-Glucosidase Zm-p60.1 Protein Library Construction

<div><p>Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins’ structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations’ effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.</p></div

High Throughput Screening Method for Identifying Potential Agonists and Antagonists of Arabidopsis thaliana Cytokinin Receptor CRE1/AHK4

The CRE1/AHK4 cytokinin receptor is an important component of plants’ hormone signaling systems, and compounds that can alter its activity have potential utility for studying the receptor’s functions and/or developing new plant growth regulators. A high throughput method was developed for screening compounds with agonist or antagonist properties toward the CRE1/AHK4 cytokinin receptor in a single experiment using the Nanodrop II liquid handling system and 384-well plates. Potential ligands are screened directly, using a reporter system in which receptor signaling activity triggers expression of β-galactosidase in Escherichia coli. This enzyme generates a fluorescent product from a non-fluorescent substrate, allowing the agonistic/antagonistic behavior of tested compounds to be assayed in relation to that of an internal standard (here the natural ligand, trans-zeatin). The method includes a robust control procedure to determine false positive or false negative effects of the tested compounds arising from their fluorescent or fluorescent-quenching properties. The presented method enables robust, automated screening of large libraries of compounds for ability to activate or inhibit the Arabidopsis thaliana cytokinin receptor CRE1/AHK4

Bioinformatics-based estimation of variability in the set of 167 β-glycosidases defined by Zhao et al. [13] at the position corresponding to W373 in β-glucosidase Zm-p60.1.

<p>The whole alignment is included as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108292#pone.0108292.s002" target="_blank">File S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108292#pone.0108292.s003" target="_blank">S3</a>.</p

Hydrolysis rates for substrates <i>p</i>NPG (A) and <i>t</i>ZOG (B) by β-glucosidase Zm-p60.1 variants produced in the course of saturation mutagenesis of the W373position.

<p>Hydrolysis rates for substrates <i>p</i>NPG (A) and <i>t</i>ZOG (B) by β-glucosidase Zm-p60.1 variants produced in the course of saturation mutagenesis of the W373position.</p

Simulation of random clone picking in randomized codon based saturation mutagenesis and calculation of the average number of clones needed to detect a new variant in libraries generated by NNK, NNN and NNM randomization.

<p>Simulation of random clone picking in randomized codon based saturation mutagenesis and calculation of the average number of clones needed to detect a new variant in libraries generated by NNK, NNN and NNM randomization.</p

Comparison of projected costs of randomized codon-based saturation mutagenesis library creation and one-by-one site-directed mutagenesis (SDM).

<p>Empirically determined results for the combined β-glucosidase Zm-p60.1 mutagenesis approach are also shown (experimental).</p