RT–PCR‐Based Cloning of the Human BETA‐Amyloid Precursor Protein Gene and the Construction of its Expression Plasmids

Abstract

Beta‐amyloid peptide (βA) is a major fibrillar component of neuritic plaques in Alzheimer's diseased brains and is related to the pathogenesis of the disease. βA generation depends on proteolytic cleavage of the amyloid precursor protein (APP). Obtaining full‐length APP is essential for the identification of biological activities that occur in the APP molecule; full‐length APP is also essential for the identification of proteases capable of creating βA as well as the factors that regulate the levels of βA. The present work offers a new approach that makes it easier for any laboratory interested in Alzheimer's disease research to obtain full‐length recombinant APP. Presented herein is the development of a new procedure for the cloning of human βA precursor protein gene (human APP gene) based on the reverse transcription (RT) and the polymerase chain reaction (PCR). This procedure for cloning human APP gene by means of RT–PCR reactions is cost‐effective, not time‐consuming, and is suited for any laboratory interested in research related to Alzheimer's disease. The present work also includes a new procedure for the construction of expression plasmids, (a) using the pFastBac™ HTb and the pBlueBacHis2 A transfer vectors for the purpose of obtaining human APP in insect cells; and (b) using the pET‐28a (+) transfer vector for the purpose of obtaining human APP in bacteria. The development of this new procedure is a significant contribution to the field in terms of augmenting the availability of full‐length APP critical for the exploration of therapeutic and preventative strategies toward the treatment of Alzheimer's disease.