Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-11-01
2004-02-03
Leffers, Gerry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S006120, C435S007400, C435S007800, C435S029000, C435S069100, C435S069700, C435S252300, C435S320100, C435S325000, C435S455000
Reexamination Certificate
active
06686168
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to methods for expressing recombinant proteins on the surface of host cells. In particular, the present invention relates to strategies for producing a fusion protein comprising a membrane anchor that allows extracellular attachment of the fusion protein in a type II orientation.
BACKGROUND OF THE INVENTION
The expression of foreign proteins on the surface of cells and virus particles provides a powerful tool for such diverse activities as obtaining specific antibodies, determining enzyme specificity, exploring protein-protein interactions, and introducing new functions into proteins. Surface display technology is also used for expression cloning, in which the biological function of a cloned gene product is used for selection.
A number of methods have been devised to display peptides and proteins on the surfaces of bacteria and bacteriophages. The surface display of heterologous protein in bacteria has been implemented for various purposes, such as the production of live bacterial vaccine delivery systems (see, for example, Georgiou et al., U.S. Pat. No. 5,348,867; Huang et al., U.S. Pat. No. 5,516,637; St{dot over (a)}hl and Uhlén,
Trends Biotechnol
. 15:185 (1995)). Bacterial surface display has been achieved using chimeric genes derived from bacterial outer membrane proteins, lipoproteins, fimbria proteins, and flagellar proteins. Bacteriophage display of foreign peptides and proteins has become a powerful tool for generating antigens, identifying peptide ligands, mapping enzyme substrate sites, isolation of high affinity antibodies, and the directed evolution of proteins (see, for example, Phizicky and Fields,
Microbiol. Rev
. 59:94 (1995); Kay et al.,
Phage Display of Peptides and Proteins
(Academic Press 1996); Lowman,
Annu. Rev. Biophys. Biomol. Struct
. 26:401 (1997)).
Either bacterial or bacteriophage surface display systems can be used for expression screening. Both approaches, however, share certain drawbacks for expressing eukaryotic proteins. Prokaryotic cells do not efficiently express functional eukaryotic proteins, and these cells lack the ability to introduce post-translational modifications, including glycosylation. Moreover, bacterial and bacteriophage display systems are limited by the small capacity of the display system, and as such, are more suited for the display of small peptides.
There are a limited number of reports on the eukaryotic cell surface display of heterologous proteins. Boder and Wittrup,
Nature Biotechnol
. 15:553 (1997), have described a library screening system using
Saccharomyces cerevisiae
as the displaying particle. This yeast surface display method uses the &agr;-agglutinin yeast adhesion receptor, which consists of two subunits, Aga1 and Aga2. The Aga1 subunit is anchored to the cell wall via a &bgr;-glucan covalent linkage, and Aga2 is linked to Aga1 by disulfide bonds. In this approach, recombinant yeast are produced that express Aga1 and an Aga2 fusion protein comprising a foreign polypeptide at the C-terminus of Aga2. Aga1 and the fusion protein associate within the secretory pathway of the yeast cell, and are expressed on the cell surface as a display scaffold.
Various approaches in eukaryotic systems achieve surface display by producing fusion proteins that contain the polypeptide of interest and a transmembrane domain from another protein to anchor the fusion protein to the cell membrane. In eukaryotic cells, the majority of secreted proteins and membrane-bound proteins are translocated across an endoplasmic reticulum membrane concurrently with translation (Wicker and Lodish,
Science
230:400 (1985); Verner and Schatz,
Science
241:1307 (1988); Hartmann et al.,
Proc. Nat'l Acad. Sci. USA
86:5786 (1989); Matlack et al.,
Cell
92:381 (1998)). In the first step of this co-translocational process, an N-terminal hydrophobic segment of the nascent polypeptide, called the “signal sequence,” is recognized by a signal recognition particle and targeted to the endoplasmic reticulum membrane by an interaction between the signal recognition particle and a membrane receptor. The signal sequence enters the endoplasmic reticulum membrane and the following nascent polypeptide chain begins to pass through the translocation apparatus in the endoplasmic reticulum membrane. The signal sequence of a secreted protein or a type I membrane protein is cleaved by a signal peptidase on the luminal side of the endoplasmic reticulum membrane and is excised from the translocating chain. The rest of the secreted protein chain is released into the lumen of the endoplasmic reticulum. A type I membrane protein is anchored in the membrane by a second hydrophobic segment, which is usually referred to as a “transmembrane domain.” The C-terminus of a type I membrane protein is located in the cytosol of the cell, while the N-teminus is displayed on the cell surface.
In contrast, certain proteins have a signal sequence that is not cleaved, a “signal anchor sequence,” which serves as a transmembrane segment. A signal anchor type I protein has a C-terminus that is located in the cytosol, which is similar to type I membrane proteins, whereas a signal anchor type II protein has an N-terminus that is located in the cytosol.
Several insect cell systems have been devised to express a fusion protein comprising a foreign amino acid sequence and a transmembrane domain. In one system, an expression vector was designed to allow fusion of a heterologous protein to the amino-terminus of the
Autographa californica
nuclear polyhedrosis virus major envelop glycoprotein, gp64 (Mottershead et al.,
Biochem. Biophys. Res. Commun
. 238:717 (1997)). Gp64, a type I integral membrane protein, functions as an anchor for the heterologous amino acid sequence, which is displayed on the surface of baculovirus particles (Monsma and Blissard,
J. Virol
. 69:2583 (1995)). More recently, Ernst et al.,
Nucl. Acids Res
. 26:1718 (1998), described a baculovirus surface display system for the production of an epitope library. In this case, a nucleotide sequence encoding a particular epitope was inserted into an
influenza virus hemagglutinin
gene. Influenza virus hemagglutinin, like gp64, is a type I integral membrane protein, which provides a membrane anchor for the foreign amino acid sequence (see, for example, Lamb and Krug, “
Orthomyxoviridae
: The Viruses and Their Replication,” in Fundamental Virology, 3
rd
Edition, pages 606-647 (Lippincott-Raven Publishers 1996)).
While both yeast and insect systems are useful for expressing eukaryotic polypeptides, post-translational modification of mammalian proteins in these systems does not necessarily produce proteins that are similar to those produced by mammalian cells. Accordingly, researchers are interested in developing display systems that use mammalian cells.
Cell surface display methods have been used to select molecules that encode proteins having a signal sequence or a transmembrane domain. For example, several techniques rely upon selection for nucleic acid fragments encoding a signal sequence to identify cDNA molecules that encode secreted proteins or type I membrane proteins (see, for example, Tashiro et al.,
Science
261:600 (1993); Yokoyama-Kobayashi et al.,
Gene
163:193 (1995)). According to these methods, a 5′-terminal fragment of the test cDNA is fused to a reporter gene, and the construct is introduced into cultured cells. If the fusion protein has a functional signal sequence, the product of the reporter gene will be detected in the cell membrane or in the culture medium. Similarly, Davis et al.,
Science
266:816 (1994), described an expression cloning method in which cDNA molecules encoding membrane-bound ligands were transfected into mammalian cells. Cells that expressed a membrane-bound ligand of interest were localized using detectably labeled soluble receptors, and cDNA encoding the ligand was rescued from the labeled cells.
In a related selection approach, Yokoyama-Kobayashi et al.,
Gene
228:161 (1999), described a method to test whether a
Leffers Gerry
Walker Shelby J.
ZymoGenetics Inc.
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