Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-06-29
2004-07-06
Celsa, Bennett (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007320, C435S219000, C530S350000, C530S825000
Reexamination Certificate
active
06759205
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the construction and expression of a sublancin-derived Lantibody Display Peptide in a strain of
Bacillus subtilis
and the use thereof.
BACKGROUND OF THE INVENTION
Lantibodies were initially described by the inventors of this application (1). Lantibodies are derived from lantibiotics which are a family of natural peptides that have antimicrobial activity. Lantibiotics have unique chemical and biological properties that are conferred by the presence of unusual amino acid residues such as dehydroalanine, dehydrobutyrine, lanthionine, and 3-methyllanthionine. The dehydro residues are electrophilic, and are capable of reacting with nucleophilic groups on polypeptide surfaces (1). By constructing suitable polypeptide environments around the dehydro residues, it is possible to control the reactivity and specificity of the dehydro residues, to react in a highly specific way with particular nucleophilic groups on the polypeptide surface. This reaction can alter the biological activity of the polypeptide surface, and if it is on a pathogen such as a bacterium or a virus, the activity of the pathogen can be destroyed. If the polypeptide surface is part of an enzyme, the activity of the enzyme can be altered in some useful way.
In this disclosure, a novel process by which the lantibodies are designed and constructed is described, and the lantibodies thus produced are versatile in their use. Also disclosed is a novel means for the identification of lantibodies which bind specifically to desired target molecules.
The inspiration for this invention is the mammalian immune system, in which stem cells differentiate into B-cells. This differentiation involves random recombination events among the variable regions of antibody genes, so that the resulting B-cell becomes programmed for the production of a particular antibody whose antigen-combining regions have been determined by a random process. The antibody that any B-cell can make is then displayed on the surface of the respective B-cell, and this surface antibody can interact with circulating pathogenic antigens. In the event that an antigen binds tightly to one of the displayed antibodies, the binding triggers cell division and further maturation of the B-cell into a plasma cell, which then produces and secretes large quantities of the antibody, which then leads to the destruction of the antigen (2-3).
Essential features of this natural process include the random generation of a population of antibodies, each of which is produced by a cell that displays the antibody that it is genetically programmed to make. Then, there is a highly-efficient process for the selection and amplification of those antibodies that bind to a specific antigen. The amplification is achieved by stimulating the division of those B-cells that display the antigen-binding antibodies.
Using discoveries in the Inventor's laboratory, the lantibody that a given bacterial cell produces is displayed on the surface of the cell. It is demonstrated that a population of lantibody producing cells can be exposed to a target molecule, and that the cells whose surface lantibodies can bind to the target can be specifically recovered, so that the population of target-binding cells is enriched. This enriched population can be subjected to repeated selection and enrichment, so that a purified population of the specific antigen-binding cells can be obtained. A very important aspect of this process is that once the target-binding cells are obtained, the lantibody that is displayed on the surface of the cell can be further characterized or the corresponding lantibody peptide can be used to identify novel nucleophilic groups on target molecules or even novel target molecules.
Knowledge of the structure of the lantibody is very important, because it can provide the basis of understanding the fundamental principles that are responsible for causing a particular lantibody to bind to a particular antigen. This knowledge can be applied to the rational design of new lantibodies that are directed toward nucleophilic targets, so that the methods by which new lantibodies are made is not solely dependent on random chance.
SUMMARY OF THE INVENTION
An object of the invention is a lantibiotic-spacer-subtilin leader sequence in anchoring a lantibiotic peptide to the cell surface of a host cell. The construction of the lantibiotic-spacer-subtilin chimera comprises lantibiotic structural regions being fused at the C-terminus to a spacer which is fused to the N-terminus of the subtilin signal leader sequence.
Another object of the invention is a mutagenesis-vector for replacing the endogenous lantibiotic chromosomal gene with a mutagenized lantibiotic sequence in an expression host.
Another object of the invention is a
Bacillus subtilis
host strain engineered to contain a deletion of a portion of the sun A gene with the remaining portion of the sunA gene being flanked by an erm gene.
Another object of the invention is a method for selecting a Lantibody Display Peptide having the ability to bind to a nucleophilic group on a target molecule.
Another object of the invention is a kit containing a bacterial host cell expressing a Lantibody Display Peptide for use in the identification of a target molecule.
REFERENCES:
patent: 5218101 (1993-06-01), Hansen
patent: 5516682 (1996-05-01), Hansen
patent: 5576420 (1996-11-01), Hansen
patent: 5861275 (1999-01-01), Hansen
patent: 5885811 (1999-03-01), Hansen
patent: 6153405 (2000-11-01), Hansen
patent: WO 00/39152 (2000-07-01), None
J. Rudinger, Peptide Hormones (Ed. J.A. Parsons, Univ. Press; Jun. 1976) pp. 1-6.*
U.S. patent application Ser. No. 60/215,449, Hansen, filed Jun. 29, 2000.
Hansen, “Nisin and Related Antimicrobial Peptides”, Biotechnology of Antibiotics, Second Edition, pp. 437-467.
Arent & Fox PLLC
Celsa Bennett
University of Maryland
LandOfFree
Construction of a strain of Bacillus subtilis 168 that... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Construction of a strain of Bacillus subtilis 168 that..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Construction of a strain of Bacillus subtilis 168 that... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3243501