Phage Display Of Intact Domains At High Copy Number


Filamentous phage-based display systems have found widespread use in molecular biology, including many immunologic applications such as antigen presentation and the immuno-isolation of desired recombinants by "biopanning". The present invention relates to a phage display system in which the molecules to be displayed (i.e., molecules of interest) are covalently connected to dispensable capsid polypeptides such as SOC (small outer capsid) and HOC (highly antigenic outer capsid) polypeptides that are, in turn, bound to a surface lattice protein, such as those on the surface of a virion or polyhead. Polyheads are tubular capsid variants containing much longer numbers of the surface lattice protein. Molecules of interest may be displayed in various ways. For example, a chimeric polypeptide that includes a dispensable polypeptide and a polypeptide of interest can be expressed in Esherichia coli, purified, and then bound in vitro to separately isolated surface lattice proteins. The surface lattice proteins can be those on the surface of a capsid or polyhead from which the wild type dispensable polypeptides have been deleted. Similarly, a chimera that contains a dispensable polypeptide and a synthetic molecule of interest can be prepared in vitro and bound to surface lattice proteins. In another embodiment, a positive selection vector forces integration of a gene that encodes a dispensable polypeptide and a polypeptide of interest into the genome of a phage from which the wild type dispensable polypeptide is deleted. For example, a modified soc gene can be integrated into a soc-deleted T4 genome, leading to in vivo binding of the display molecule on progeny virions. More than one type of dispensable polypeptide can be used as part of the chimera for displaying one or more molecules of interest. For example, the surface lattice proteins of a phage may be bound to a chimera that contains SOC and a chimera that contains HOC.

The display system has been successfully demonstrated for three molecules of interest that vary in their length and character: (1) a tetrapeptide; (2) the 43 amino acid residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues).



Inventors:

Alasdair Steven (NIAMS)  ➽ more inventions...


Intellectual Property:
U.S. Pat: 7,041,441 issued 2006-05-09

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OTT Reference No: E-061-1997/0
Updated: Apr 1, 1998