split and mix
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2020 ◽  
Vol 48 (11) ◽  
pp. e63-e63 ◽  
Author(s):  
Laurens Lindenburg ◽  
Tuomas Huovinen ◽  
Kayleigh van de Wiel ◽  
Michael Herger ◽  
Michael R Snaith ◽  
...  

Abstract Site-saturation libraries reduce protein screening effort in directed evolution campaigns by focusing on a limited number of rationally chosen residues. However, uneven library synthesis efficiency leads to amino acid bias, remedied at high cost by expensive custom synthesis of oligonucleotides, or through use of proprietary library synthesis platforms. To address these shortcomings, we have devised a method where DNA libraries are constructed on the surface of microbeads by ligating dsDNA fragments onto growing, surface-immobilised DNA, in iterative split-and-mix cycles. This method—termed SpliMLiB for Split-and-Mix Library on Beads—was applied towards the directed evolution of an anti-IgE Affibody (ZIgE), generating a 160,000-membered, 4-site, saturation library on the surface of 8 million monoclonal beads. Deep sequencing confirmed excellent library balance (5.1% ± 0.77 per amino acid) and coverage (99.3%). As SpliMLiB beads are monoclonal, they were amenable to direct functional screening in water-in-oil emulsion droplets with cell-free expression. A FACS-based sorting of the library beads allowed recovery of hits improved in Kd over wild-type ZIgE by up to 3.5-fold, while a consensus mutant of the best hits provided a 10-fold improvement. With SpliMLiB, directed evolution workflows are accelerated by integrating high-quality DNA library generation with an ultra-high throughput protein screening platform.


2010 ◽  
Vol 46 (13) ◽  
pp. 2209 ◽  
Author(s):  
Grégory Upert ◽  
Christoph A. Merten ◽  
Helma Wennemers
Keyword(s):  

2009 ◽  
Vol 48 (20) ◽  
pp. 3661-3664 ◽  
Author(s):  
Kirsten Belser ◽  
Tünde Vig Slenters ◽  
Conelious Pfumbidzai ◽  
Grégory Upert ◽  
Laurent Mirolo ◽  
...  

2009 ◽  
Vol 121 (20) ◽  
pp. 3715-3718 ◽  
Author(s):  
Kirsten Belser ◽  
Tünde Vig Slenters ◽  
Conelious Pfumbidzai ◽  
Grégory Upert ◽  
Laurent Mirolo ◽  
...  

2005 ◽  
Vol 24 (10) ◽  
pp. 1158-1163 ◽  
Author(s):  
Philipp Krattiger ◽  
Roman Kovàsy ◽  
Jefferson D. Revell ◽  
Helma Wennemers

2003 ◽  
Vol 42 (15) ◽  
pp. 1722-1724 ◽  
Author(s):  
Philipp Krattiger ◽  
Catherine McCarthy ◽  
Andreas Pfaltz ◽  
Helma Wennemers
Keyword(s):  

2001 ◽  
Vol 3 (1) ◽  
pp. 20-27 ◽  
Author(s):  
Sid Topiol ◽  
John Davies ◽  
Sundararajan Vijayakuma ◽  
James R. Wareing

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