Expression And Purification Of Human Death Receptor6in Pichia Pastoris And The Study Of The Interaction With Human Amyloid Precursor Protein In Vitro

Alzheimer’s disease (AD) is a kind of chronic disease of the central nervous system, is one of the most common types of senile dementia. Since the middle of the20th century, with the speeding up of aging and the rising incidence of AD, AD prevention has become a global attention hot spot. Recent researches have found out that amyloid precursor protein(APP) and Death receptor-6(DR6) participate in the self-destruction of nerve cells. APP is considered to be DR6’s ligand. The shortage of nutritional factors such as trophic factors can lead to neurodegeneration signaled by apoptosis receptor. DR6is expressed in most human tissues and organs. Some evidence suggests that the expression of DR6in the process of activation and differentiation can be adjusted. The interaction between NAPP and DR6is similar to that between antigen and antibody, whose effect on axons is affected by many factors. First of all, it is affected by neurotrophic factors, which plays an important role in the growth and survival of neurons. Nutrient deficiency will lead to atrophy degeneration of neurons. BF-CN is more sensitive to Nutrient deficiency, which accounts for the AD. The shortage of trophic factor triggers shedding of surface APP in a beta-secretase. A cleaved amino-terminal fragment of APP (N-APP) binds DR6and triggers neuronal cell apoptosis and axonal degeneration which depends on caspase-3and caspase-6signaling pathway. Because the NAPP-DR6interaction mechanism can explain the neuron axon degeneration in the process of AD development, which prove that DR6-NAPP interaction is the possible mechanisms of axonal degeneration of neurons. Therefore, to explore the function of DR6and study its interaction with NAPP has become one way of exploring the Alzheimer’s disease etiology, pathology, positioning the AD treatment drug target, and developing new AD drugs.In our study, we cloned cDNA encoding42-218amino acid and42-349amino acid fragments of human DR6into the expression vector pPIC9K, the recombinant plasmid transformed into pichia pastoris GS115after sequencing successfully. Transformers were obtained after screened by histidine nutrition deficient plate and antibiotic G418. PCR result showed the interested gene has been inserted into the host strain. Get two yeast strains of recombinants which could express DR6amino acid fragment respectively. Found exogenous proteins of26kD and43kD in supernatant by SDS assay after large-scale expressed,then DR6was purified and characterized by Western blot.In order to verify the interaction of DR6and NAPP, this research successfully constructs pGST-DR6(aa42-349). On the side, laboratory saved yeast strain expressing NAPP (aa18-285),we acquires NAPP after purification. The direct interaction between DR6and NAPP has been verified in vivo through GST pull-down assay.This experiment successfully constructed DR6(aa42-218/aa42-349) of pichia eukaryotic expression vector, and implements the efficient secretion expression, preliminarily proves the way for the further study of the biological functions of DR6as well as the action scope and interaction between APP and DR6.