Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast

Traxlmayr, Michael W.; Faissner, Maximilian; Stadlmayr, Gerhard; Hasenhindl, Christoph; B, Antes; Rüker, Florian; Obinger, Christian

doi:10.1016/j.bbapap.2012.01.006

Cited by 51 publications

(51 citation statements)

References 18 publications

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“…13 These anomalies are contributions from the aromatic or cystinyl side-chains within the far-UV. 18 The minimum at 230 nm was also reported for an IgG1-Fc 19 and described for some V L domains, 20,21 the latter attributed to the interaction of the aromatic residues with the conserved Trp35.…”

Section: Elongation Of the C-terminal Domain Of An Anti-amyloid β Sinmentioning

confidence: 97%

Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency

Rivera-Hernández

Argany

Blasco-Moreno

et al. 2013

mAbs

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Section: Elongation Of the C-terminal Domain Of An Anti-amyloid β Sinmentioning

confidence: 97%

Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency

Rivera-Hernández

Argany

Blasco-Moreno

et al. 2013

mAbs

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“…Here we report that yeast also produces BDNF primarily in an inactive and misfolded form. Yeast surface display has been used to identify better-folded and -secreted variants of single-chain T-cell receptors (19), antibody Fc regions (20), and epidermal growth factor receptor (21), among others (22). Thus, yeast surface display approaches were employed to improve the protein-folding and -processing properties of BDNF.…”

mentioning

confidence: 99%

Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae

Burns

Malott

Metcalf

et al. 2014

Appl Environ Microbiol

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cBrain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employing Saccharomyces cerevisiae display and secretion systems, it was found that BDNF was poorly expressed and partially inactive on the yeast surface and that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. Thus, for the purpose of increasing the compatibility of yeast as an expression host for BDNF, directed-evolution approaches were employed to improve BDNF folding and expression levels. Yeast surface display was combined with two rounds of directed evolution employing random mutagenesis and shuffling to identify BDNF mutants that had 5-fold improvements in expression, 4-fold increases in specific TrkB binding activity, and restored p75 binding activity, both as displayed proteins and as secreted proteins. Secreted BDNF mutants were found largely in the form of soluble homodimers that could stimulate TrkB phosphorylation in transfected PC12 cells. Site-directed mutagenesis studies indicated that a particularly important mutational class involved the introduction of cysteines proximal to the native cysteines that participate in the BDNF cysteine knot architecture. Taken together, these findings show that yeast is now a viable alternative for both the production and the engineering of BDNF.

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“…Recently, we have described the directed evolution of stabilized IgG1-Fc scaffolds by an application of strong heat shock to IgG1-Fc libraries displayed on yeast [27]. After four rounds of selection 17 variants with intact FcRn, CD16a and Protein A binding and increased thermal stability could be selected by FACS.…”

Section: Resultsmentioning

confidence: 99%

Creating stable stem regions for loop elongation in Fcabs — Insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations

Hasenhindl

Lai

Delgado

et al. 2014

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules.

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Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast

Cited by 51 publications

References 18 publications

Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency

Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency

Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae

Creating stable stem regions for loop elongation in Fcabs — Insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations

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