Chaperonin assisted phage display of antibody fragments on filamentous bacteriophages.
We have used the GroE chaperonins to assist in the packing of a new phage display vector, pEXmide3. Titers of the packed phagemid increased almost 200-fold from approximately 4 x 10(11) cfu/ml, without coexpression of the GroE proteins, to approximately 7 x 10(13) cfu/ml with their coexpression. Equal titers of non-assisted and assisted phagestocks exhibited the same antigen specificity and ELISA reactivity, indicating the same frequency of displayed Fab-fragments. While the diversity of antibody libraries depends on the bacterial transformation efficiency, the copy number of each antibody is determined by subsequent amplification of the phage, thus chaperonin assisted phagemid packing in bacteriophage M13 can be used as a general and simple tool to increase the amplification level of expressed Fab fragments. pEXmide3 was developed for display of Fab and single chain Fv-fragments (scFv), using restriction enzymes that do not cut, or cut with low frequencies, in genes encoding immunoglobulin variable domains. The vector allows cloning of genes for the variable domains linking these to predetermined human constant domains or cloning of the entire light and heavy Fab chains. A modification of the pelB leader sequence, with a glutamine to alanine substitution at residue 18, was used for export of the light chain.
Söderlind
E
E
Department of Immunotechnology, Lund University, Sweden.
Lagerkvist
A C
AC
Dueñas
M
M
Malmborg
A C
AC
Ayala
M
M
Danielsson
L
L
Borrebaeck
C A
CA
D001426:Bacterial Proteins / Q000235:genetics / Q000494:pharmacology*; D001435:Bacteriophages / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D018835:Chaperonin 10; D018834:Chaperonin 60; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D015870:Gene Expression; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics / Q000494:pharmacology*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D000939:Epitopes; D006360:Heat-Shock Proteins; D007140:Immunoglobulin Fab Fragments