Efficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda.
We describe the construction and characterization of a hepatitis C virus (HCV) cDNA expression library displayed as a fusion to the carboxy terminus of the capsid protein D of bacteriophage lambda. cDNA inserts were obtained by tagged random-priming of the HCV genome and cloned into a lambda vector from which chimeric phage bearing both wild-type D protein and D fusion products on the capsid surface were produced. The resulting library was affinity-selected with anti-HCV human monoclonal antibodies recognizing linear or conformational epitopes, and human sera from HCV-infected patients. Selection was monitored by immuno-screening experiments, ELISA, and sequence analysis of positive clones. The performance of this library was compared with two additional HCV cDNA display libraries generated as N-terminal fusions to the III and VIII capsid proteins of filamentous phage M13. The results obtained demonstrate the great potential of the lambda display system for constructing complex cDNA libraries for natural ligand discovery.
Santini
C
C
Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Pomezia, Italy.
Brennan
D
D
Mennuni
C
C
Hoess
R H
RH
Nicosia
A
A
Cortese
R
R
Luzzago
A
A
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000918:Antibody Specificity; D017104:Bacteriophage M13 / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D036022:Capsid Proteins; D018076:DNA, Complementary / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000097:blood; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D014760:Viral Fusion Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D036022:Capsid Proteins; C041745:D protein, Enterobacteria phage lambda; D018076:DNA, Complementary; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D014764:Viral Proteins