Improving the copy numbers of antibody fragments expressed on the major coat protein of bacteriophage M13.
BACKGROUND
Antibody fragments have been expressed on the major coat protein of filamentous phage using a gene VIII expression system, but with low copy numbers (averaging 0.2 Fab/phage).


OBJECTIVE
As a general strategy to increase copy number, the phage vector was optimized by site directed mutagenesis.


METHODS
One mutation was the introduction of a random six amino acid tether between the heavy chain and pseudo gene VIII to form a phage library. The other mutation was the removal of two cysteine residues which form a disulfide bond between the heavy and light chains. An assay was developed to measure Fab concentrations and used to calculate the average number of copies displayed on phage.


RESULTS
Phage libraries containing random tethers were panned, and clones containing a proline rich motif were extracted. Removing the interchain disulfide had a greater effect on copy number and soluble Fab concentrations in the periplasmic space of the bacterial cultures.


CONCLUSIONS
A tenfold increase in the copy number was achieved using the optimized vector. Incorporation of these vector mutations may be a general strategy for optimizing Fab display on the major coat protein of bacteriophage M13.
Nakayama
G R
GR
Ixsys, Inc., San Diego, CA 92121, USA.
Valkirs
G
G
McGrath
D
D
Huse
W D
WD
D017104:Bacteriophage M13 / Q000235:genetics / Q000378:metabolism*; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D015870:Gene Expression; D005814:Genes, Viral; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D012995:Solubility
D004220:Disulfides; D007128:Immunoglobulin Fragments; D003545:Cysteine