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...

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Bibliographic Details
Published in:Journal of molecular biology 1998-09, Vol.282 (1), p.125-135
Main Authors: Santini, C, Brennan, D, Mennuni, C, Hoess, R H, Nicosia, A, Cortese, R, Luzzago, A
Format: Article
Language:English
Subjects:
Antibodies, Monoclonal
Antibodies, Viral
Antibody Specificity
Bacteriophage lambda - genetics
Bacteriophage M13 - genetics
Capsid Proteins
DNA, Complementary - genetics
DNA, Viral - genetics
DNA-Binding Proteins - genetics
Gene Expression
Gene Library
Hepacivirus - genetics
Hepatitis C - blood
Hepatitis C virus
Humans
Phage lambda
Phage M13
Recombinant Fusion Proteins - biosynthesis
Selection, Genetic
Sequence Analysis, DNA
Viral Fusion Proteins - genetics
Viral Proteins - genetics
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Summary: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.
ISSN:0022-2836
DOI:10.1006/jmbi.1998.1986