Production and Optimization of LTE, a Leishmania tarentolae Derived Cell-Free Protein Expression System for Recombinant Protein Production.

IF 1.2 4区 综合性期刊 Q3 MULTIDISCIPLINARY SCIENCES
Wayne A Johnston, Juan Alfaro Palma, Kirill Alexandrov
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引用次数: 0

Abstract

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate Leishmania tarentolae, referred to as Leishmania Translational Extract or LTE. Although this organism originally evolved as a parasite of geckos, it can be cultivated easily and inexpensively in flasks or bioreactors. Unlike Leishmania major, it is non-pathogenic to humans and does not require special laboratory precautions. Another advantage of using Leishmania for CFPS is that the addition of a single antisense oligonucleotide to the CFPS, targeting a conserved splice leader sequence on the 5'-end of all protein-coding RNAs, can suppress endogenous protein expression. We provide procedures for cell disruption and lysate processing, which have been simplified and improved compared to previous versions. These procedures start with simple flask cultures. Additionally, we explain how to introduce genetic information using vectors containing species-independent translation initiation sites (SITS) and how to perform straightforward batch optimization and quality control to ensure consistent protein expression quality.

生产和优化用于重组蛋白生产的无细胞利什曼原虫蛋白表达系统 LTE。
本方案概述了一种真核无细胞蛋白表达系统(CFPS)的生产和优化过程,该系统源自单细胞鞭毛虫--透明利什曼原虫(Leishmania tarentolae),被称为利什曼原虫转化提取物(Leishmania Translational Extract)或 LTE。虽然这种生物最初是作为壁虎的寄生虫进化而来,但它可以在烧瓶或生物反应器中以低廉的成本轻松培养。与大利什曼原虫不同,它对人类无致病性,不需要特殊的实验室预防措施。利用利什曼原虫制造 CFPS 的另一个优势是,在 CFPS 中加入单个反义寡核苷酸,以所有编码蛋白质 RNA 5'-end 上的保守剪接前导序列为目标,可以抑制内源性蛋白质的表达。我们提供的细胞破碎和裂解物处理程序与以前的版本相比有所简化和改进。这些程序从简单的烧瓶培养开始。此外,我们还解释了如何使用含有物种无关翻译起始位点(SITS)的载体引入遗传信息,以及如何进行直接的批次优化和质量控制,以确保一致的蛋白质表达质量。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Jove-Journal of Visualized Experiments
Jove-Journal of Visualized Experiments MULTIDISCIPLINARY SCIENCES-
CiteScore
2.10
自引率
0.00%
发文量
992
期刊介绍: JoVE, the Journal of Visualized Experiments, is the world''s first peer reviewed scientific video journal. Established in 2006, JoVE is devoted to publishing scientific research in a visual format to help researchers overcome two of the biggest challenges facing the scientific research community today; poor reproducibility and the time and labor intensive nature of learning new experimental techniques.
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