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- W2023239354 abstract "Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers.Codon-optimized Opti-Pgp and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from T(m) ∼40 °C to 49 °C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids.The significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins." @default.
- W2023239354 created "2016-06-24" @default.
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- W2023239354 date "2011-08-03" @default.
- W2023239354 modified "2023-10-18" @default.
- W2023239354 title "A Gene Optimization Strategy that Enhances Production of Fully Functional P-Glycoprotein in Pichia pastoris" @default.
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- W2023239354 doi "https://doi.org/10.1371/journal.pone.0022577" @default.
- W2023239354 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3149604" @default.
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- W2023239354 hasPublicationYear "2011" @default.
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