Directed evolution of proteins for increased stability and expression using yeast display.
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.
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ABSTRACT: Objectives Recombinant protein subunit vaccines are formulated using protein antigens that have been synthesized in heterologous host cells. Several host cells are available for this purpose, ranging from Escherichia coli to mammalian cell lines. This article highlights the benefits of using yeast as the recombinant host.Key findingsThe yeast species, Saccharomyces cerevisiae and Pichia pastoris, have been used to optimize the functional yields of potential antigens for the development of subunit vaccines against a wide range of diseases caused by bacteria and viruses. Saccharomyces cerevisiae has also been used in the manufacture of 11 approved vaccines against hepatitis B virus and one against human papillomavirus; in both cases, the recombinant protein forms highly immunogenic virus-like particles.SummaryAdvances in our understanding of how a yeast cell responds to the metabolic load of producing recombinant proteins will allow us to identify host strains that have improved yield properties and enable the synthesis of more challenging antigens that cannot be produced in other systems. Yeasts therefore have the potential to become important host organisms for the production of recombinant antigens that can be used in the manufacture of subunit vaccines or in new vaccine development.Journal of Pharmacy and Pharmacology. 01/2015;
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ABSTRACT: Directed evolution has become a well-established tool for improving proteins and biological systems. A critical aspect of directed evolution is the selection of a suitable host organism for achieving functional expression of the target gene. To date, most directed evolution studies have used either Escherichia coli or Saccharomyces cerevisiae as a host; however, other bacterial and yeast species, as well as mammalian and insect cell lines, have also been successfully used. Recent advances in synthetic biology and genomics have opened the possibility of expanding the use of directed evolution to new host organisms such as microalgae. This review focuses on the different host organisms used in directed evolution and highlights some of the recent directed evolution strategies used in these organisms.Computational and structural biotechnology journal. 09/2012; 2:e201209012.
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ABSTRACT: For most therapeutic proteins, a long serum half-life is desired. Studies have shown that decreased antigen binding at acidic pH can increase serum half-life. In this study we aimed to investigate whether pH-dependent binding sites can be introduced into antigen binding crystallizable fragments of immunoglobulin G1 (Fcab). The C-terminal structural loops of an Fcab were engineered for reduced binding to the extracellular domain of human epidermal growth factor receptor 2 (Her2-ECD) at pH 6 compared to pH 7.4. A yeast-displayed Fcab-library was alternately selected for binding at pH 7.4 and non-binding at pH 6.0. Selected Fcab variants showed clear pH-dependent binding to soluble Her2-ECD (decrease in affinity at pH 6.0 compared to pH 7.4) when displayed on yeast. Additionally, some solubly expressed variants exhibited pH-dependent interactions with Her2-positive cells whereas their conformational and thermal stability was pH-independent. Interestingly, two of the three Fcabs did not contain a single histidine mutation but all of them contained variations next to histidines that already occurred in loops of the lead Fcab. The study demonstrates that yeast surface display is a valuable tool for directed evolution of pH-dependent binding sites in proteins.Biotechnology Journal 06/2014; · 3.71 Impact Factor