Establishment of a reliable dual-vector system for the phage display of antibody fragments.
To resolve some of the technical limitations in a phage-displayed Fab library, we have designed two dual-vector systems, DVS-I and DVS-II, composed of a set of replicon-compatible plasmid (pLA-1 or pLT-2) for producing soluble L chain fragments and phagemid (pHf1g3T-1 or pHf1g3A-2) for expressing Fd (V(H)+C(H1))-DeltapIII fusion molecules as well as a genotype-phenotype linkage. Compared to the DVS-I (pLA-1 and pHf1g3T-1), the DVS-II (pLT-2 and pHf1g3A-2) showed stable transformation efficiency regardless of the order of the vectors introduced into the host cells. In addition, expression of soluble Fab molecules with antigen-binding reactivity, recombinant phage titer and display level of functional Fab-DeltapIII on the phage progenies of the DVS-II were comparable with a conventional phage display system using a single phagemid vector. More importantly, the phage displaying target-specific Fab-DeltapIII molecules was successfully enriched by panning, which allows isolation of the pHf1g3A-2 phagemid encoding antigen-specific Fd molecules. We believe that the DVS-II may provide a valuable tool in the construction of a combinatorial phage-displayed Fab library with large diversity. Furthermore, it can be readily applied to isolation of desired antibody clones if L chain promiscuity of antibodies in determining antigen-binding specificity is considered, or in guided-selection or chain shuffling of mAbs of non-human origin.
Joo
Hyun-yoo
HY
Division of Molecular and Medical Biotechnology, College of Bioscience and Biotechnology, Kangwon National University, Chunchon, Gangwon 200-701, South Korea.
Hur
Byung-ung
BU
Lee
Kyung-woo
KW
Song
Suk-yoon
SY
Cha
Sang-hoon
SH
D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D018274:Electroporation / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics* / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction
D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D004247:DNA