Phagemid encoded small molecules for high throughput screening of chemical libraries.
A new strategy for monovalently displaying small molecules on phage surfaces was developed and applied to high throughput screening for molecules with high binding affinity to the target protein. Peptidyl carrier protein (PCP) excised from nonribosomal peptide synthetase was monovalently displayed on the surface of M13 phage as pIII fusion proteins. Small molecules of diverse structures were conjugated to coenzyme A (CoA) and then covalently attached to the phage displayed PCP by Sfp phosphopantetheinyl transferase. Because Sfp is broadly promiscuous for the transfer of small molecule linked phosphopantetheinyl moieties to apo PCP domains, this approach will enable displaying libraries of small molecules on phage surfaces. Unique 20-base-pair (bp) DNA sequences were also incorporated into the phagemid DNA so that each compound displayed on the phage surface was encoded by a DNA bar code encapsulated inside the phage coat protein. Single round selection of phage displayed small molecules achieved more than 2000-fold enrichment of small molecules with nM binding affinity to the target protein. The selection process is further accelerated by the use of DNA decoding arrays for identifying the selected small molecules.
Yin
Jun
J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Liu
Fei
F
Schinke
Martina
M
Daly
Christian
C
Walsh
Christopher T
CT
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D017104:Bacteriophage M13 / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D019151:Peptide Library; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D019151:Peptide Library; C082662:phosphopantetheinyl transferase; D004247:DNA; D017855:Transferases (Other Substituted Phosphate Groups); D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D003065:Coenzyme A