Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris
نویسندگان
چکیده
A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10(-8) M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.
منابع مشابه
Efficient display of active lipase LipB52 with a Pichia pastoris cell surface display system and comparison with the LipB52 displayed on Saccharomyces cerevisiae cell surface
BACKGROUND For industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes. RESULTS Two Pichia pastoris cell surface display vectors based on the flocculation functional domain of FLO with its own secretion signal...
متن کاملP-65: Effective Parameters on the Bovine Follicle Stimulating Hormone Expression in The Pichia Pastoris System
Background: Bovine follicle-stimulating hormone (bFSH) is a heterodimer hormone that consists of a common -subunit which noncovalently associated with the hormone-specific -subunit. During the past 15 years, the methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production because it is able to use methanol as a sole carbon and energy source. Th...
متن کاملImmunogenicity of heparin-binding hemagglutinin expressed by Pichia pastoris GS115 strain
Objective(s): Heparin-binding hemagglutinin (HBHA), a mycobacterial cell surface protein, mediates adhesion to nonphagocytic cells and the dissemination of Mycobacterium tuberculosis (M. tuberculosis) from the site of primary infection. Superior expression systems are required to obtain abundant M. tuberculosis proteins for the purpose of diagnosing M. tuberculosis infection or for the immuniza...
متن کاملMolecular cloning of cDNA for trehalase from the European honeybee, Apis mellifera L., and its heterologous expression in Pichia pastoris.
cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5' untranslated region (UTR) of 869 bp, and the 3' UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The Mr of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, w...
متن کاملEvaluation of Sorbitol-Methanol Co-Feeding Strategy on Production of Recombinant Human Growth Hormone in Pichia Pastoris
Recombinant protein production in Pichia pastoris is based on alcohol oxidase promoterswhich are regulated by methanol. However, the use of methanol has several disadvantages,which is why current trends in bioprocess development with Pichia pastoris (P. pastoris) arefocusing on methanol mixed feeding strategies. This work aimed to develop a new experimentalmethod and compare the effect of vario...
متن کامل