Modifying filamentous phage capsid: limits in the size of the major capsid protein.
Ff filamentous phages are long thin cylindrical structures that infect bacteria displaying the F pilus and replicate without lysing the host. These structures are exploited to display peptides by fusing them to the amino terminus of either the bacterial receptor protein (pIII) or the major coat protein (pVIII). We have analysed a vast collection of phage mutants containing substitutions and insertions in the amino terminus of pVIII to ask whether any chemical group of this solvent exposed region of the phage capsid has any key function in the phage life cycle. Any of the five amino-terminal residues can be substituted by most amino acids without affecting phage assembly suggesting that this region does not play any essential role in morphogenesis. However, a deletion of three residues delta (Gly3Asp4Asp5) results in a phage clone with an decreased ability to produce infective particles. By engineering phages designed to display peptides by fusion to the amino terminus of the major coat protein we have found that phage viability is affected by peptide length while peptide sequence plays a minor "tuning" role. Most peptides of six residues are tolerated irrespective of their sequence while only 40% of the phages carrying an amino-terminal extension of eight residues can form infective particles. This fraction drops to 20% and 1% when we attempt to insert peptides 10 and 16 amino acids long. We have used this information to build phage libraries where each phage displays approximately 2700 copies of a different octapeptide all over the phage surface.
Iannolo
G
G
Dipartimento di Biologia, Universitá di Roma Tor Vergata, Italia.
Minenkova
O
O
Petruzzelli
R
R
Cesareni
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry / Q000235:genetics*; D005814:Genes, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D017912:Inovirus / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion / Q000502:physiology; D015678:Viral Structural Proteins / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins