A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris.
A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris.
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DOI：
10.1371/journal.pone.0022577
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Urbatsch IL
中科院分区：
文献类型：
--
作者：
Bai J;Swartz DJ;Protasevich II;Brouillette CG;Harrell PM;Hildebrandt E;Gasser B;Mattanovich D;Ward A;Chang G;Urbatsch IL
哺乳动物膜蛋白的结构和生化研究仍然受到纯蛋白生产效率低下的阻碍。我们探索基于高表达毕赤酵母基因的密码子优化，以增强 P-糖蛋白 (Pgp) 的共翻译折叠和产生，Pgp 是一种参与癌症多药耐药性的 ATP 依赖性药物外排泵。密码子优化的“Opti-Pgp”和野生型 Pgp 的初级蛋白质序列相同，经过严格分析其功能或溶液结构的差异。 Opti-Pgp 的酵母表达水平和巴斯德毕赤酵母纯化蛋白的产量（约 130 mg/kg 细胞）比野生型蛋白高约三倍。 Opti-Pgp 对多种抗癌和杀菌药物表现出完全的体内耐药性。纯化的 Opti-Pgp 的 ATP 水解受到维拉帕米的强烈刺激～15 倍，并被环孢菌素 A 抑制，结合常数为 4.2±2.2 µM 和 1.1±0.26 µM，与野生型 Pgp 无法区分。最大周转数为 2.1±0.28 µmol/min/mg，比野生型 Pgp 提高了 1.2 倍，这可能是由于 Opti-Pgp 制剂的纯度更高。通过 CD、DSC 和有限的蛋白水解对纯化的野生型和 Opti-Pgp 进行分析表明具有相似的二级和三级结构。脂质的添加将热稳定性从 Tm ~40°C 提高到 49°C，并提高了总展开焓。折叠状态的增加可能是脂质存在下药物刺激的 ATP 酶活性增加的原因。天然折叠状态下蛋白质的产量显着提高，纯度更高，功能得到改善，确立了我们的基因优化方法的价值，并为提高其他膜蛋白的产量提供了基础。
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 Tm ∼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.
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影响因子：
4.8
作者：
Lee, JY;Urbatsch, IL;Wilkens, S
通讯作者：
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影响因子：
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DOI：
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发表时间：
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期刊：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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DOI：
10.1126/science.1168750
发表时间：
2009-03-27
期刊：
Science (New York, N.Y.)
影响因子：
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作者：
Aller SG;Yu J;Ward A;Weng Y;Chittaboina S;Zhuo R;Harrell PM;Trinh YT;Zhang Q;Urbatsch IL;Chang G
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