A bacteriophage lambda vector for the cloning and expression of immunoglobulin Fab fragments on the surface of filamentous phage.
We have combined the high cloning efficiency of the lambda bacteriophage vectors with the surface expression screening method for the display of combinatorial antibody fragment (Fab) libraries on the surface of filamentous phage particles. The utility of the herein described ImmunoZAP 13 system for the isolation of Fabs that specifically bind antigen is demonstrated using two phagemid display libraries prepared from a previously characterized human combinatorial library. The percentage of clones that specifically bind antigen is maintained throughout the process of subcloning the LC and VH genes into ImmunoZAP 13, in vivo mass excision to convert the lambda library to a phagemid library, and preparation of phagemid particles displaying Fabs. Specific phagemid were isolated from libraries containing 0.6% and 0.03% tetanus toxoid (TT)-binding clones after two and three rounds of biopanning, respectively. Relative binding curves determined on a small sample of isolated clones indicate that several unique immunoglobulin Fab fragments have been isolated.
Stratacyte Corp., La Jolla, CA 92037.
H H
HH
Hogrefe
R L
RL
Mullinax
A E
AE
Lovejoy
B N
BN
Hay
J A
JA
Sorge
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D007140:Immunoglobulin Fab Fragments; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins