Isolation of novel single-chain Cro proteins targeted for binding to the bcl-2 transcription initiation site by repertoire selection and subunit combinatorics.
New designed DNA-binding proteins may be recruited to act as transcriptional regulators and could provide new therapeutic agents in the treatment of genetic disorders such as cancer. We have isolated tailored DNA-binding proteins selected for affinity to a region spanning the transcription initiation site of the human bcl-2 gene. The proteins were derived from a single-chain derivative of the lambda Cro protein (scCro), randomly mutated in its recognition helices to construct libraries of protein variants of distinct DNA-binding properties. By phage display-afforded affinity selections combined with recombination of shuffled subunits, protein variants were isolated, which displayed high affinity for the target bcl-2 sequence, as determined by electrophoretic mobility shift and biosensor assays. The proteins analyzed were moderately sequence-specific but provide a starting point for further maturation of desired function.
Jonas
Kristina
K
Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden.
Van Der Vries
Erhard
E
Nilsson
Mikael T I
MT
Widersten
Mikael
M
D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D019254:Genes, bcl-2 / Q000235:genetics*; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D024363:Transcription Initiation Site; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D019151:Peptide Library; D021122:Protein Subunits; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins