Directed evolution of proteins for increased stability and expression using yeast display

ArticleinArchives of Biochemistry and Biophysics 526(2):174-80 · May 2012with61 Reads
DOI: 10.1016/j.abb.2012.04.022 · Source: PubMed
Abstract
The expression of recombinant proteins incorporated into the cell wall of Saccharomyces cerevisiae (yeast surface display) is an important tool for protein engineering and library screening applications. In this review, we discuss the state-of-the-art yeast display techniques used for stability engineering of proteins including antibody fragments and immunoglobulin-like molecules. The paper discusses assets and drawbacks of stability engineering using the correlation between expression density on the yeast surface and thermal stability with respect to the quality control system in yeast. Additionally, strategies based on heat incubation of surface displayed protein libraries for selection of stabilized variants are reported including a recently developed method that allows stabilization of proteins of already high intrinsic thermal stability like IgG1-Fc.
    • "However, not only the ligand binding affinity has been improved by directed evolution strategies using yeast display screening. Protocols to solely measure [121] and strategies to improve the thermal stability of antibodies applying yeast display have also been developed (reviewed by Traxlmayr and Obinger [173]). Besides the engineering of antibodies, novel antibodies against a wide panel of antigens including viral proteins from HIV [12, 178] and HCV [83] , the tuberculosis biomarker Ag85 [32] and cancer associated ligands [24] have been selected. "
    [Show abstract] [Hide abstract] ABSTRACT: Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage. In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies.
    Full-text · Article · Jan 2016
    • "Non-immunoglobulin scaffolds successfully engineered by yeast surface display comprise cystein knot peptides (knottins)2324252627, the human tenth domain of fibronectin type III (Fn3)28293031 , the lamprey variable lymphocyte receptor (VLR) [32, 33] , the hyperthermophilic DNAbinding protein Sso7d [34, 35], human serum albumin (HSA) [36], green fluorescent protein (GFP) [37, 38] , major histocompatibility complex (MHC) [39, 40], cytokines (e.g., interleukin-2, IL-2414243; interleukin-4, IL-4 [44]), growth factors (e.g., epidermal growth factor, EGF [45, 46]; vascular endothelial growth factor, VEGF [47]; hepatocyte growth factor, HGF [48]), the kringle domain [49], and hormones (e.g., leptins [50]). In addition to improving the affinity of protein binders, yeast surface display technology has also been used successfully to develop cross-reactive protein binders [51], generate conformation specific binders [52, 53], improve the stability and biophysical properties of a protein [22,545556, and engineer the function of several enzymes such as horseradish peroxidase (HRP) [57], biotin ligase BirA [58], sortase A [59], and several lipases606162, among many others. Another powerful application of yeast surface display is the identification of binding epitopes on a target protein. "
    [Show abstract] [Hide abstract] ABSTRACT: Yeast surface display is a powerful technology for engineering a broad range of protein scaffolds. This protocol describes the process for de novo isolation of protein binders from large combinatorial libraries displayed on yeast by using magnetic bead separation followed by flow cytometry-based selection. The biophysical properties of isolated single clones are subsequently characterized, and desired properties are further enhanced through successive rounds of mutagenesis and flow cytometry selections, resulting in protein binders with increased stability, affinity, and specificity for target proteins of interest.
    Full-text · Article · Jun 2015
    • "Two strategies, incorporating stabilizing/compensatory mutations and buffering the effect of destabilizing mutations by chaperones, have been demonstrated as effective solutions (for a recent overview, see Socha and). Various methods have been developed to predict and experimentally screen stabilizing mutations and can be used to maintain protein stability and evolvability (Lehmann et al., 2000; Roodveldt et al., 2005; Bommarius et al., 2006; Mayer et al., 2007; Papp et al., 2011; Traxlmayr and Obinger, 2012; Socha and). Moreover, combining stabilizing/compensatory mutations with chaperone buffering can further enhance the evolvability of enzymes and enable sustainable enzyme engineering: We recently reported the experimental evolution of a phosphotriesterase (PTE) towards increased arylesterase activity. "
    [Show abstract] [Hide abstract] ABSTRACT: The wealth of distinct enzymatic functions found in nature is impressive and the on-going evolutionary divergence of enzymatic functions continues to generate new and efficient catalysts, which can be seen through the recent emergence of enzymes able to degrade xenobiotics. However, recreating such processes in the laboratory has been met with only moderate success. What are the factors that lead to suboptimal research outputs? In this review, we discuss constraints on enzyme evolution, which can restrict evolutionary trajectories and lead to evolutionary dead-ends. We highlight recent studies that have used experimental evolution to mimic different aspects of enzymatic adaptation under simple, controlled settings to shed light on evolutionary dynamics and constraints. A better understanding of these constraints will lead to the development of more efficient strategies for directed evolution and enzyme engineering. J. Exp. Zool. (Mol. Dev. Evol.) 9999B: 1-20, 2014. © 2014 Wiley Periodicals, Inc.
    Full-text · Article · Nov 2014
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