Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13.
Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated. Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein. Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation. Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin. Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand. The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage.
Markland
W
W
Protein Engineering Corporation, Cambridge, MA 02138.
Roberts
B L
BL
Saxena
M J
MJ
Guterman
S K
SK
Ladner
R C
RC
D000595:Amino Acid Sequence; D007611:Aprotinin / Q000235:genetics*; D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D003090:Coliphages / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D007611:Aprotinin