A method for generation of arbitrary peptide libraries using genomic DNA.
Random peptide libraries can be constructed either by in vitro synthesis of random peptides, or through translation of DNA sequences from synthetic random oligonucleotides. Here we describe an alternative way of making arbitrary peptide libraries with high diversity that can be used in screening as random peptide libraries. Genomic DNA digested with a frequent-cutting restriction enzyme recognizing four nucleotides will theoretically consist of small DNA pieces with average length of 256 nucleotides, and on average around 107 fragments can be generated from a genome of 3 x 109 bases. A peptide library translated from these fragments will have sufficient diversity for some protein interaction screening experiments. Moreover, the same genome digested with a different four-cutter enzyme or ligated into different reading frames will result in different nonoverlapping libraries. A series of such libraries could be generated with genomic DNAs from different species. In this study, human genomic DNA was digested with four-cutter restriction enzymes DpnII and Tsp509I, respectively, and cloned into yeast expression vector pGADT7 to generate arbitrary peptide libraries. These libraries were used in yeast two-hybrid assays to screen for binding motifs of the PDZ domain containing protein synectin. Our results showed that in addition to various native carboxy-terminal tails, synectin could also bind to many artificial ones, some of which contained a consensus sequence--(S/T)XC-COOH.
Huang
Haiming
H
Department of Pathophysiology, National Key Laboratory of Medical Molecular Biology, Proteomics Research Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, PR China.
Gao
Youhe
Y
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015894:Genome, Human; D006801:Humans; D008024:Ligands; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D051381:Rats; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C492008:Gipc1 protein, rat; D008024:Ligands; D009479:Neuropeptides; D019151:Peptide Library; D004262:DNA Restriction Enzymes