Enhancing phage display of antibody fragments using gIII-amber suppression.
The effect of utilizing Ex12 helper phage, a mutant M13K07 helper having two amber codons at the gIII (gIII-amber), in combination with Escherichia coli host strains belonging to the supE genotype on improving the phage display of antibody fragments was investigated. Because of an inefficient read-through of the UAG codons, Ex12 helper phage produced approximately 10% of the intracellular wt pIII in the supE host cells compared to M13K07. The phage progenies rescued from the supE XL-1 Blue MRF' strain carrying the recombinant phagemid, pCMTG-SP112, by Ex12 helper phage displayed both antibody-DeltapIII fusion and wt pIII at a ratio of 1:1.5, and achieved a 50-fold greater display of the antibody-DeltapIII compared to those obtained by a conventional phage rescue using M13K07. Additionally observed were a 100-fold increase in antigen-binding functionality and a drastic improvement on antigen-specific panning efficiency by the phage progenies. Our approach permits the display of at least one antibody fragment as well as more than one copy of wt pIII on the surface of recombinant phages, and this would make the phagemid-based phage display technology more practical and reliable.
Oh
Mi-Young
MY
IG Therapy Co., Rm. 112, Nong 3rd Building, Kangwon National University, Chuncheon, Gangwon-do 200-701, South Korea.
Joo
Hyun-Yoo
HY
Hur
Byung-Ung
BU
Jeong
Yeon-Ho
YH
Cha
Sang-Hoon
SH
D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000235:genetics; D000918:Antibody Specificity / Q000235:genetics; D017104:Bacteriophage M13 / Q000235:genetics*; D016153:Genes, Suppressor; D006378:Helper Viruses / Q000235:genetics*; D007128:Immunoglobulin Fragments / Q000235:genetics*; D008969:Molecular Sequence Data; D019151:Peptide Library
D007128:Immunoglobulin Fragments; D019151:Peptide Library