Beta-secretase enzyme compositions and methods
a technology of beta secretase and composition, applied in the field of beta secretase, can solve the problems of abnormal concentration of a peptide in amyloid plaques present in brains of people, and achieve the effect of maintaining or improving cognitive ability, reducing the amount of a deposition
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example 1
Isolation of Coding Sequences for Human β-Secretase
A. PCR Cloning
[0302] Poly A+ RNA from IMR human neuroblastoma cells was reverse transcribed using the Perkin-Elmer kit. Eight degenerate primer pools, each 8 fold degenerate, encoding the N and C terminal portions of the amino acid sequence obtained from the purified protein were designed (shown in Table 4; oligos 3407 through 3422)(SEQ ID NOS:3-21). PCR reactions were composed of cDNA from 10 ng of RNA, 1.5 mM MgCl2, 0.125 μl AmpliTaq® Gold, 160 μM each dNTP (plus 20 μM additional from the reverse transcriptase reaction), Perkin-Elmer TAQ buffer (from AmpliTaq® Gold kit, Perkin-Elmer, Foster City, Calif. ), in a 25 μl reaction volume. Each of oligonucleotide primers 3407 through 3414 was used in combination with each of oligos 3415 through 3422 for a total for 64 reactions. Reactions were run on the Perkin-Elmer 7700 Sequence Detection machine under the following conditions: 10 min at 95° C., 4 cycles of, 45° C. annealing for 15...
example 2
Screening of Human Fetal Brain cDNA Library
[0312] The Origene human fetal brain Rapid-Screen™ cDNA Library Panel is provided as a 96-well format array consisting of 5000 clones (plasmid DNA) per well from a human fetal brain library. Subplates are available for each well consisting of 96 wells of 50 clones each in E. coli. This is an oligo-dT primed library, size-selected and unidirectionally inserted into the vector pCMV-XL3.
[0313] 94 wells from the master plate were screened using PCR. The Reaction 1 Conditions described in Example 1, above, were followed, using only primers 3407 (SEQ ID NO:3) and 3416 (SEQ ID NO:12) with 30 ng of plasmid DNA from each well. Two pools showed the positive 70 bp band. The same primers and conditions were used to screen 1 μl E. coli from each well of one of the subplates. E. coli from the single positive well was then plated onto LB / amp plates and single colonies screened using the same PCR conditions. The positive clone, about 1 Kb in size, was la...
example 3
PCR Cloning Methods
[0314] 3′RACE was used in experiments carried out in support of the present invention to elucidate the polynucleotide encoding human β-secretase. Methods and conditions appropriate for replicating the experiments described herein and / or determining polynucleotide sequences encoding additional members of the novel family of aspartyl proteases described herein may be found, for example, in White, B. A., ed., PCR Cloning Protocols; Humana Press, Totowa, N.J., 1997, or Ausubel, supra, both of which are incorporated herein by reference.
RT-PCR
[0315] For reverse transcription polymerase chain reaction (RT-PCR), two partially degenerate primer sets used for RT-PCR amplification of a cDNA fragment encoding this peptide. Primer set 1 consisted of DNA's #3427-3434 (SEQ ID NOS:22-29 respectively), the sequences of which are shown in Table 5, below. Matrix RT-PCR using combinations of primers from this set with cDNA reverse transcribed from primary human neuronal cultures ...
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