Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1824, Issue 4, Pages 542-549Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2012.01.006
Keywords
Stability engineering; Antibody engineering; Crystallizable domain; Yeast display; Directed evolution
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Funding
- Christian Doppler Research Association (CDG)
- doctoral program BioToP - Biomolecular Technology of Proteins of the Austrian Science Funds [FWF-W1224]
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We have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and T-m-values up to 85 degrees C was developed. Besides library construction by error prone PCR, strong heat stress at 79 degrees C for 10 min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time. (C) 2012 Elsevier B.V. All rights reserved.
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