New display vector reduces biological bias for expression of antibodies in E. coli.
We report the development of a novel phagemid vector, pKM19, for display of recombinant antibodies in single-chain format (scFv) on the surface of filamentous phage. This new vector improves efficacy of selection and reduces the biological bias against antibodies that can be harmful to host bacteria. It is useful for generation of large new antibody libraries, and for the subsequent maturation of antibody fragments. In comparison with commonly used plasmids, this vector is designed to have relatively low expression levels of cloned scFv antibodies due to the amber codon positioned in a sequence encoding for the PhoA leader peptide. Moreover, fusion of antibodies to the carboxy terminal part only of the gene III protein improves display of scFv on bacteriophage surface in this system. Despite the lower antibody expression, the functional test performed with a new scFv library derived from human peripheral blood lymphocytes demonstrates that specific antibodies can be easily isolated from the library, even after the second selection round. The use of the pKM19 vector for maturation of an anti-CEA antibody significantly improves the final results. In our previous work, an analogous selection through the use of a phagemid vector, with antibody expression under the control of a lacP promoter, led to isolation of anti-CEA phage antibodies with improved affinities, which were not producible in soluble form. Probably due to the toxicity for E. coli of that particular anti-CEA antibody, 70% of maturated clones contained suppressed stop codons, acquired during various selection/amplification rounds. The pKM19 plasmid facilitates an efficient maturation process, resulting in selection of antibodies with improved affinity without any stop codons.
Pavoni
Emiliano
E
Kenton Labs, c/o Sigma-Tau, via Pontina km 30.400, Pomezia (RM), 00040 Italy.
Monteriù
Giorgia
G
Cianfriglia
Maurizio
M
Minenkova
Olga
O
D000906:Antibodies / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D002272:Carcinoembryonic Antigen / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D017912:Inovirus / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology
D000906:Antibodies; D002272:Carcinoembryonic Antigen; D007141:Immunoglobulin Fc Fragments; D011994:Recombinant Proteins