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在Pichia pastoris中开发人类护送BiP的高效表达系统:生产优化和功能验证

Eimantas Žitkus^1,2, Evaldas Čiplys^3,4, Mantas Žiaunys¹

¹Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio 7, Vilnius, LT-10257, Lithuania.

Microbial cell factories

|March 19, 2025

概括

优化的酵母发酵产生了高产的重组人类BiP (rhBiP),这是一种治疗应用至关重要的蛋白质. 这种可扩展的生物工艺使rhBiP的进一步研究成为可能.

科学领域:

生物化学生化学
分子生物学分子生物学
生物技术是生物技术.

背景情况:

热冲击蛋白BiP (GRP78) 是一种分子伴侣,主要存在于内质网膜中.
BiP与各种疾病有关,并显示出治疗潜力.
这项研究重点是使用Pichia pastoris生产复合人类BiP (rhBiP).

研究的目的:

优化高细胞密度发酵过程,以在Pichia pastoris.中生产rhBiP.
开发一种可扩展和具有成本效益的方法来获得生物活性rhBiP.
研究在矿物质介质中增强rhBiP分泌的条件.

主要方法:

使用Pichia pastoris用于重组蛋白质表达.
在摇瓶和生物反应器中优化发酵条件.
采用疏水相互作用和离子交换染色学来进行净化.
评估了rhBiP活性及其对体外蛋白质聚合的影响.

主要成果:

在经过优化条件的定义矿物基础盐介质 (BSM) 中实现了高产量的rhBiP生产.
确定了增加rhBiP分泌的重要添加剂 (DTT,碳源).
在生物反应器发酵中以氧气有限的策略达到大约70 mg/L的rhBiP.
纯化rhBiP达到90%的纯度,显示ATPase活性和体外抑制粉样β和α-synuclein聚合.

结论:

开发了一种可扩展的生物工艺,用于在P. pastoris.中产生高产量,生物活性的rhBiP.
该过程使用化学定义的矿物介质,促进进一步的治疗应用.
这项工作为加速与疾病相关的研究提供了可靠的rhBiP来源.

关键词:

这就是Pichia pastoris.这就是BiP BiP BiP.在 DTT 上,你会看到 DTT.发酵发酵是一个过程.矿物质介质是一种矿物质介质.混合养是一种混合养方式.密封的分泌密封的分泌

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