TY - JOUR
T1 - Generation of a large peptide phage display library by self-ligation of whole-plasmid PCR product
AU - Kong, Xu Dong
AU - Carle, Vanessa
AU - Díaz-Perlas, Cristina
AU - Butler, Kaycie
AU - Heinis, Christian
N1 - Publisher Copyright:
© 2020 American Chemical Society
PY - 2020/11/20
Y1 - 2020/11/20
N2 - The success of phage display, used for developing target-specific binders based on peptides and proteins, depends on the size and diversity of the library screened, but generating large libraries of phage-encoded polypeptides remains challenging. New peptide phage display libraries developed in recent years rarely contained more than 1 billion clones, which appears to have become the upper size limit for libraries generated with reasonable effort. Here, we established a strategy based on whole-plasmid PCR and self-ligation to clone a library with more than 2 × 1010 members. The enormous library size could be obtained through amplifying the entire vector DNA by PCR, which omitted the step of vector isolation from bacterial cells, and through appending DNA coding for the peptide library via a PCR primer, which enabled efficient DNA circularization by end-ligation to facilitate the difficult step of vector-insertion of DNA fragments. Panning the peptide repertoires against a target yielded high-affinity ligands and validated the quality of the library and thus the new library cloning strategy. This simple and efficient strategy places larger libraries within reach for nonspecialist researchers to hopefully expand the possible targets of phage display applications.
AB - The success of phage display, used for developing target-specific binders based on peptides and proteins, depends on the size and diversity of the library screened, but generating large libraries of phage-encoded polypeptides remains challenging. New peptide phage display libraries developed in recent years rarely contained more than 1 billion clones, which appears to have become the upper size limit for libraries generated with reasonable effort. Here, we established a strategy based on whole-plasmid PCR and self-ligation to clone a library with more than 2 × 1010 members. The enormous library size could be obtained through amplifying the entire vector DNA by PCR, which omitted the step of vector isolation from bacterial cells, and through appending DNA coding for the peptide library via a PCR primer, which enabled efficient DNA circularization by end-ligation to facilitate the difficult step of vector-insertion of DNA fragments. Panning the peptide repertoires against a target yielded high-affinity ligands and validated the quality of the library and thus the new library cloning strategy. This simple and efficient strategy places larger libraries within reach for nonspecialist researchers to hopefully expand the possible targets of phage display applications.
UR - http://www.scopus.com/inward/record.url?scp=85095828648&partnerID=8YFLogxK
U2 - 10.1021/acschembio.0c00497
DO - 10.1021/acschembio.0c00497
M3 - Article
C2 - 33125222
AN - SCOPUS:85095828648
SN - 1554-8929
VL - 15
SP - 2907
EP - 2915
JO - ACS Chemical Biology
JF - ACS Chemical Biology
IS - 11
ER -