Methods of preparation of recombinant forms of human beta-amyloid protein and uses of these proteins
View Patent ↗The subject invention relates to the cloning, expression and isolation of recombinant forms of beta-amyloid protein containing a N-terminal methionine (or one or more amino acids) as well as to methods of using this recombinant protein in the production of therapeutic antibodies, in the identification of therapeutic small molecules, and in the performance of diagnostic assays.
1. A purified beta-amyloid protein consisting of an amino acid sequence at least 70% identical to the amino acid sequence of native beta-amyloid protein, and consists of a sequence represented by the formula [X+Y], wherein
“X” consists of one or more amino acids located at the amino terminus of the purified protein selected from the group consisting of methionine, valine, and leucine, and
“Y” comprises all or a portion of the amino acid sequence of beta amyloid protein, and contains a mutation selected from the group consisting of A2T, H6R, D7N, A21G, E22G, E22Q, E22K, D23N, A42T, and A42V, the numbers being relative to the N-terminus of the native beta-amyloid protein.
2. The purified beta-amyloid protein of claim 1 , wherein “X” is methionine.
3. A purified globulomer comprising at least two of the purified beta-amyloid proteins of claim 1 .
4. A vaccine comprising the purified beta-amyloid protein of claim 1 or the globulomer of claim 3 , and further comprising a pharmaceutically acceptable adjuvant.
5. A method of treating Alzheimer's Disease in a patient in need of treatment comprising the step of administering said vaccine of claim 4 to said patient in an amount sufficient to effect said treatment.
6. An isolated nucleic acid encoding the purified beta-amyloid protein of claim 1 .
7. A vector comprising said isolated nucleic acid molecule of claim 6 .
8. A host cell comprising said vector of claim 7 .
9. A method of producing the purified beta-amyloid protein of claim 1 , comprising the steps of:
(a) inserting a nucleic acid molecule encoding the purified beta-amyloid protein of claim 1 into a vector; and
(b) transforming the vector containing the nucleic acid molecule encoding the purified beta-amyloid protein of claim 1 into a host cell for a time and under conditions sufficient for expression of the beta-amyloid protein by the host cell.
10. The method of claim 9 wherein said host cell is prokaryotic or eukaryotic.
11. The method of claim 10 wherein said prokaryotic host cell is selected from the group consisting of Escherichia coli, Bacillus sp., Streptococcus sp, and Lactobacillus sp.
12. The method of claim 10 wherein said eukaryotic cell is selected from the group consisting of an insect cell, a yeast cell and a mammalian cell.
13. A method of diagnosing Alzheimer's Disease in a patient suspected of having this disease comprising the steps of:
(a) isolating a biological sample from said patient;
(b) contacting said biological sample with the globulomer of claim 3 for a time and under conditions sufficient for the formation of complexes comprising the globulomer and antibodies against the globulomer which may be present in the sample;
(c) adding a conjugate to the resulting antibody/globulomer complexes for a time and under conditions sufficient to allow said conjugate to bind to the complexes, wherein said conjugate comprises antibodies that bind to the anti-globulomer antibodies and comprises a compound capable of generating a detectable signal; and
(d) detecting the presence of antibodies that bind to the globulomers which may be present in the biological sample by detecting a signal generated by the compound, said signal indicating a diagnosis of Alzheimer's disease in said patient.
14. A method of diagnosing Alzheimer's Disease in a patient suspected of having Alzheimer's Disease comprising the steps of:
(a) isolating a biological sample from said patient;
(b) contacting said biological sample with the globulomer of claim 3 for a time and under conditions sufficient for the formation of complexes comprising the globulomer and antibodies against the globulomer which may be present in the sample;
(c) detecting the presence of said complexes in the sample, the presence of said complexes indicating a diagnosis of Alzheimer's Disease in said patient.