Materials and methods relating to the attachment and display...

Details Materials and methods relating to the attachment and display... Materials and methods relating to the attachment and display...

1998-03-06

2001-02-20

Navarro, Albert (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

C424S192100, C424S195110, C424S197110, C424S200100, C435S069300, C435S252300, C435S320100

Reexamination Certificate

active

06190662

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to materials and methods for the exportation, cell wall attachment and display of substances, in particular proteins and polypeptides, on the cell surface of Gram-positive bacteria such as
Lactococcus lactis.
Methods are disclosed providing for the surface expression of cell wall anchoring of proteins, including antigens, specific binding proteins and enzymes.
BACKGROUND TO THE INVENTION
The display of polypeptides on the surface of bacteria has been a subject of investigation for several years. This interest has arisen because recombinant DNA technology makes it possible to use bacterial cells as factories for the inexpensive production of a wide range of different proteins. Work has centred on the display of polypeptides on the surface of Gram-negative bacteria, see for example the review provided in U.S. Pat. No. 5,348,867. However, in Gram-negative bacteria it has proved difficult to find an adaptable method of displaying polypeptides on the surface of the cells so that they are securely attached to the surface at high density.
However, high density surface expression of recombinant products on the bacterial surface is a prerequisite for the use of these bacteria in certain industrial applications, and in the areas of vaccine development, the construction of peptide libraries and the production of whole bacterial cell adsorbents. There is no prior art known to the inventors which results in the consistent, stable, high density expression on the surface of bacteria, and more especially, on the cell surface of Gram-positive bacteria such as
Lactococcus lactis
and similar or related species.
Previous work on the bacterial surface presentation of polypeptides has focused mainly on the use of Gram-negative bacterial such as
E. coli
and Salmonella (Georgiou et al., 1993). In Gram-negative bacteria the peptidoglycan cell wall is bounded by two separate lipid membranes. The outer membrane is largely impermeable to molecules with a relative molecular mass greater than 500 (Mr) and as a result very few proteins are released into the growth medium. Owing to the presence of an outer membrane, surface exposed proteins of Gram-negative bacteria are either integral outer membrane proteins or protein appendages such as pili or flagella
The few proteins which are secreted by Gram-negative organisms are transported across the outer membrane by specialised transport systems which may involve up to 14 different gene products (eg pullulanase secretion; Kornacker and Pugsley 1990a).
The possibility of displaying foreign proteins by making C-terminal fusions to integral membrane proteins is complicated by the fact that the C-terminal regions of many integral membrane proteins appears to be necessary for targeting and correct assembly of the protein in the outer membrane. One way around this problem has been to use the N-terminal targeting sequence of the
E. coli
major lipoprotein (Lpp) to direct fusion proteins to the outer membrane. As Lpp does not transverse the outer membrane, the membrane spanning domain of an outer membrane protein (OMP; eg
E. coli
OmpA) is also required to localise a fusion protein to the cell surface. Nevertheless, the utility of these Lpp-OmpA fusions to display functional proteins such as &bgr;-lactamase, a single chain Fv antibody and a cellulose binding protein have been demonstrated (Georgiou et al., 1990).
Other approaches for the surface display of polypeptides have involved making amino acid insertions within outer membrane loops of integral membrane proteins such as Lam B or in the pilin and flagellin proteins which do not contain transmembrane domains (Charbit et al., 1991; Thiry et al., 1989, Steidler et al, 1993 a,b). The major problem with this approach has been that large amino acid insertions/substitutions disrupt the proper folding and/or localisation of the fusion protein in such a way that when used to immobilise the bacterial cells, the fusion proteins are extracted from the outer membrane (Steidler, PhD Thesis, University of Gent, Belgium).
Although significant progress has recently been made in the development of methods from the surface display of proteins in Gram-negative bacteria, the major drawback to the use of integral membrane proteins for such purposes is that the fusion proteins are not firmly attached to the cell surface. Furthermore, the high level expression of membrane proteins is known to be detrimental to the cell, a factor which may limit the possible applications for such systems in cases where high density surface expression is required.
In contrast, Gram-positive bacteria do not possess an outer membrane and proteins which are displayed on the cell surface are thought to be linked to the cell surface via their C-terminus (Schneewind et al., 1992). Many surface proteins of Gram-positive bacteria possess certain common features which are unique to these organisms. Each molecule has at its N-terminal end a secretion leader peptide which acts as an export signal for cell secretion, a LPXTG motif, a C-terminal hydrophobic domain and charged amino acids at the very C terminus. Most work on the characterisation and function of these has been carried out in
S. aureus
using the cell wall sorting and anchoring domains of
S. aureus
protein A (Navarre and Schneewind, 1994). It has been shown that protein A is covalently coupled to the cell wall of
S. aureus
by a mechanism which is yet to be elucidated.
SUMMARY OF THE INVENTION
The present invention demonstrates that the firm attachment of foreign proteins to the surface of
L. lactis
and other Gram-positive bacteria is made possible by constructing chimeric genes encoding a fusion protein which contains nucleic acid encoding (1) an N-terminal secretion signal, (2) the protein to be surface expressed and (3) the cell wall sorting and anchoring domains of
S. aureus
protein A.
By comparison with
E. coli,
as relatively few heterologous proteins have been expressed at high level in
L. lactis,
before the present work it was impossible to predict with certainty whether the expression of any particular protein would be possible or whether such expression would be toxic to the cell. Such toxicity is likely to occur when a secreted or membrane associated protein is expressed at high level, as protein export may adversely affect the integrity of the cell membrane or the function of the general secretory pathway.
Thus, the present invention shows for the first time that proteins or polypeptides can be firmly attached to the cell wall of Gram-positive bacteria when expressed as fusions with a cell wall anchoring domain. As is shown below, it is not possible to remove the protein by boiling the cells in the presence of an ionic detergent which would normally release non-covalently bound proteins. While not wishing to be bound by any particular theory, we believe that
L. lactis
appears to possess the necessary enzyme(s) required to link (covalently) the expressed protein A fusion to the cell wall peptidoglycan. This binding could not have been readily predicted from the possession by protein A of the LPXTG motif and C-terminal hydrophobic domains common to many surface proteins of Gram-positive bacteria, since these motifs are just necessary, but not sufficient conditions for the occurrence of covalent cell wall anchoring.
Accordingly, the present invention arises from the finding that it is possible to firmly attach proteins and polypeptides of interest (including but not restricted to adhesins, antigens, enzymes and ligands) to the outer face of the peptidoglycan cell wall of Gram-positive bacteria, such as
Lactococcus lactis,
using a chimeric gene comprising nucleic acid encoding a secretion signal sequence, a desired protein or polypeptide and a cell wall attachment domain, such as that derived from
Staphylococcus aureus
protein A.
Accordingly, in a first aspect, the present invention provides a recombinant vector including nucleic acid encoding a fusion protein comprising a secretion signal sequence, a desired protein or p

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