Secreted Expression Of Recombinant Human-amyloid Protein 1-42 In Pichia Pastoris

Alzheimer's disease, first announced by the German researcher Alosi Alzheimer in 1907, gains more and more concern now. Recently, studies on molecule biology and cytochemical involved senile plaques and neurofibrillary tangles have great progress.The latest results showed that the main pathologic characters of AD are extracellular senile plaques'formation and neurofibrillary tangles'accumulation in vivo. However, the main component of senile plaques isβ-amyloid peptide (Aβ_1-42 ) which is neuron toxicity. Due to its important functions on AD pathogenesis and immunity therapy, Aβ_1-42 needs to be examined clearly to find out the key factors of AD, and eliminate AD's pathological changes completely. In order to study and research the disease's mechanism and theory of immunity mentioned above, we need to get a great deal of Aβ_1-42. The existing acquiring manner is mainly by artificial synthesis, which has low biological activity with high cost. This study adopted gene engineering technology to generate Aβ_1-42, in order to both satisfy the need of research and also remarkably reduce the high cost from artificial synthesis.β-amyloid peptide is the main component of the brain senile plaques.Glenner and Wong found that the brain senile plaques were composed of 39-43 amino acids, which hadβ-collapse peptides, and were calledβ-amyloid peptides. Kang found them come from the hydrolyzation of membrane-sugar conformity proteins, which were called metabolize products ofβ-APP or APP. The research shows at least 3 proteolytic ferments'function (α,γ-andβ- secretase) related with Aβ's generation. The peptide bond between Met670 and Asp671 was hydrolyzed byβ- secretase, and the APP was incised into two products: soluble APP segment (APPsβ) with N terminal and C99 (99 amino acids adhered to membrane).γ- secretase hydrolyzes peptide bonds between 710 aa-712aa of C terminal in membrane intermediate zone, formingβstarch-like peptide 40 ,βstarch-like peptide 42 with different length ,βstarch-like peptide 40 has more fabric source thanβstarch-like peptide 42.αsecretase hydrolyzes peptide bonds in APP, produce APPsα(a soluble segment of 687 aa) and a C terminal residue C83 which adhered to membrane. The C terminal residue byαsecretase is hydrolysised byγ- secretase, and form protein (P3). The cleavage site ofαsecretase locates at Aβsection; therefore can block up the forming of Aβ.Although some papers revealed expression of Aβ_1-42 in E.coli, transient expression in MK cells cos-7, recombinant method to produce human in Aβ_1-42 Pachia pastoris is still unreported. The most common expression system with mature technology: the E.coli expression system contains some insurmountable flaws such as low productivity, incorrect folding and cleavage, difficult procedure and control, high cost in large scale production etc, which can better solved in Pachia pastoris expression system. Therefore, we consider that we should establish an expression system with high productivity and also can produce in large scale. Thus, to find a proper expression system to produce Aβ_1-42 has significant practical importance.1. Synthesis of Aβ_1-42 gene We changed the genetic codons of mature peptide to Pichia pastoris favorite codons by artificial synthesis . Cloning vector is pUC57, cloning site is XhoⅠ/ XbaⅠ.The primer inserted XhoⅠtarget site,Kex2 in 5'end,terminal codon taa and EcoRⅠt arger site in 3'end. Kex2 is essential to cutαsignal peptide efficiently. Using PCR we get the gene of Aβ_1-42 with XhoⅠ/EcoRⅠ.2. Aβ_1-42 stably and effectively1)Construction of eukaryotic expression vector pPICZα-Aβ_1-42Using PCR we get the gene of Aβ_1-42 with XhoⅠ/EcoRⅠ, after purification inserted into the pPICZαvector which was cut by the same enzymes. After confirmation by endonuclease digestation assay and sequencing, recombinant vector was linearized by SacI, then transformed into the X-33 via electroporation.2)Screening of recombinant Pichia pastorisTransformed yeasts were plated on YPD plates containing Zeocin to isolate-resistant clones. After clones were cultured for 72 hours at 28℃,16 single clones were picked into culture. Then the genomic DNA of the transformed yeasts were extracted to perform PCR using the primers.3)Expression and identification of Aβ_1-42Proliferate the PCR tested positively yeast clones, then induced the expression of Aβ_1-42 with 0.5% methanol. Perform SDS-PAGE, Western blot to identify Aβ_1-42. The results indicated that there was a 4 KDa protein in the supernatant.3. Studies on large-scale fermentation and purification process of Aβ_1-42:1)Studies on large-scale fermentation process of Aβ_1-42 Pichia pastoris has many advantages as a kind of expression host, and it's very suitable for large-scale expression of the extraneous proteins. We explored the large-scale fermentation process of Aβ_1-42 and found that the best pH was pH 4.0, DO between 20%～30% and the supply speed of methanol was 10ml/h/L initial fermentation volume. The concentration of Aβ_1-42 in the broth could reach 150mg/L.2)A new method to purify Aβ_1-42 at large-scaleThe Aβ_1-42 could be purified by SP Sepharose XL cation exchange and Source TM30 RPC hydrophobic chromatography, then the eluate was distilled to condense Aβ_1-42. The yield coefficient was higher than 75%.Results showed that our studies succeed in secreted expression of Aβ_1-42 in Pichia pastoris. Established initially expression system of Aβ_1-42. Via SDS-PAGE we discovered there was a 4000 D protein in the supernatant. The concentration of Aβ_1-42 in the broth could reach 150mg/L.