A General Method for Insertion of Functional Proteins within Proteins via Combinatorial Selection of Permissive Junctions.
A major goal of modern protein chemistry is to create new proteins with different functions. One approach is to amalgamate secondary and tertiary structures from different proteins. This is difficult for several reasons, not the least of which is the fact that the junctions between secondary and tertiary structures are not degenerate and usually affect the function and folding of the entire complex. Here, we offer a solution to this problem by coupling a large combinatorial library of about 10(7) different N- and C-terminal junctions to a powerful system that selects for function. Using this approach, the entire Leptin and follicle-stimulating hormone (FSH) were inserted into an antibody. Complexes with full retention of function in vivo and in vitro, although rare, were found easily by using an autocrine selection system to search for hormonal activity. Such large diversity systems, when coupled to robust selection systems, should enable construction of novel therapeutic proteins.
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Yingjie
Y
Peng
Laboratory of Molecular Genetics, Rockefeller University, New York, NY 10065, USA; Howard Hughes Medical Institute, Rockefeller University, New York, NY 10065, USA.
Wenwen
W
Zeng
School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, P. R. China.
Hui
H
Ye
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Kyung Ho
KH
Han
The Scripps Research Molecular Screening Center, The Scripps Research Institute, Scripps Florida, FL 33458, USA; Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA; Translational Research Institute, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA.
Venkatasubramanian
V
Dharmarajan
The Scripps Research Molecular Screening Center, The Scripps Research Institute, Scripps Florida, FL 33458, USA; Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA; Translational Research Institute, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA.
Scott
S
Novick
Department of Integrative Structural and Computational Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Ian A
IA
Wilson
The Scripps Research Molecular Screening Center, The Scripps Research Institute, Scripps Florida, FL 33458, USA; Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA; Translational Research Institute, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA.
Patrick R
PR
Griffin
Laboratory of Molecular Genetics, Rockefeller University, New York, NY 10065, USA; Howard Hughes Medical Institute, Rockefeller University, New York, NY 10065, USA.
Jeffrey M
JM
Friedman
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address: rlerner@scripps.edu.
Richard A
RA
Lerner
D020650:Combinatorial Chemistry Techniques / Q000379:methods; D005640:Follicle Stimulating Hormone / Q000097:blood / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000737:chemistry; D020738:Leptin / Q000097:blood / Q000737:chemistry; D008958:Models, Molecular; D019151:Peptide Library; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D013329:Structure-Activity Relationship
D007074:Immunoglobulin G; D020738:Leptin; D019151:Peptide Library; D011506:Proteins; D005640:Follicle Stimulating Hormone