来自Pseudomonas sp.的II型L-阿斯巴拉金酶的分泌表达和优化 在PCH199199中使用
Subhash Kumar1, Virender Kumar2, Dharam Singh3
1Molecular and Microbial Genetics Lab, CSIR-Institute of Himalayan Bioresource Technology, Palampur 176 061, Himachal Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201 002, India; Department of Bio Medical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu 632 014, India.
概括
这项研究通过使用分泌表达系统来增强大肠杆菌中的L-阿斯巴拉金酶II (Pg-ASNase II) 生产. 优化的方法增加了细胞外酶产量,降低了治疗应用的下游加工成本.
科学领域:
- 生物技术是生物技术.
- 酶工程是什么? 酶工程是什么?
- 分子生物学分子生物学
背景情况:
- 再组合L-阿斯巴拉基酶 (L-ASNase) 对于治疗急性淋巴细胞白血病和减少食品中的烯胺至关重要.
- 目前L-ASNase的生产方法通常涉及大肠杆菌的细胞质表达,需要复杂的净化.
研究的目的:
- 开发一种高效的分泌表达系统,用于E. coli中的重组L-asparaginase II (Pg-ASNase II).
- 为了增强Pg-ASNase II的细胞外生产,用于潜在的治疗应用.
主要方法:
- 经过工程设计的大肠杆菌带有pelB信号,用于Pg-ASNase II的周等离子体表达.
- 优化培养条件,包括添加Tween 80,以增强蛋白质分泌.
- 使用单阶段的Q-色色谱,净化Pg-ASNase II.
主要成果:
- 成功地实现了Pg-ASNase II的分泌表达和分泌到大肠杆菌超体中.
- 通过将介质成分优化为0.2%的Tween 80,使蛋白质分泌量从0.33升至0.77毫克/毫升.
- 获得了8.0 mg/L的净化蛋白质,活性为60.0 U/mg,产量为75%,净化增加了15倍.
结论:
- 与原生水平相比,开发的分泌表达系统显著增强了细胞外Pg-ASNase II的产生.
- 这种方法降低了下游加工成本,并有助于周等离子体Pg-ASNase II的潜在治疗用途.
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