The rational design of a 'type 88' genetically stable peptide display vector in the filamentous bacteriophage fd.
Filamentous bacteriophages are particularly efficient for the expression and display of combinatorial random peptides. Two phage proteins are often employed for peptide display: the infectivity protein, PIII, and the major coat protein, PVIII. The use of PVIII typically requires the expression of two pVIII genes: the wild-type and the recombinant pVIII gene, to generate mosaic phages. 'Type 88' vectors contain two pVIII genes in one phage genome. In this study a novel 'type 88' expression vector has been rationally designed and constructed. Two factors were taken into account: the insertion site and the genetic stability of the second pVIII gene. It was found that selective deletion of recombinant genes was encountered when inserts were cloned into either of the two non-coding regions of the phage genome. The deletions were independent of recA yet required a functional F-episome. Transcription was also found to be a positive factor for deletion. Taking the above into account led to the generation of a novel vector, designated fth1, which can be used to express recombinant peptides as pVIII chimeric proteins in mosaic bacteriophages. The fth1 vector is not only genetically stable but also of high copy number and produces high titers of recombinant phages.
Enshell-Seijffers
D
D
Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Smelyanski
L
L
Gershoni
J M
JM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D036022:Capsid Proteins; D003227:Conjugation, Genetic / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D000939:Epitopes / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D018628:Gene Dosage; D005814:Genes, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral; D017912:Inovirus / Q000235:genetics* / Q000254:growth & development; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D019151:Peptide Library; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D011995:Recombination, Genetic / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D013753:Tetracycline Resistance / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D000911:Antibodies, Monoclonal; D036022:Capsid Proteins; D004274:DNA, Recombinant; D000939:Epitopes; D019151:Peptide Library; D011994:Recombinant Proteins; C119217:coat protein pVIII, Enterobacteria phage fd; D011938:Rec A Recombinases