Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins.
We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using 'MAX' randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40,000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants.
Hughes
Marcus D
MD
School of Life and Health Sciences, Aston University Aston Triangle, Birmingham B4 7ET, UK.
Zhang
Zhan-Ren
ZR
Sutherland
Andrew J
AJ
Santos
Albert F
AF
Hine
Anna V
AV
D001665:Binding Sites; D020650:Combinatorial Chemistry Techniques; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D019151:Peptide Library; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D019151:Peptide Library; D011506:Proteins
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