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Application of a protein domain as chaperone for enhancing biological activity and stability of other proteins.
Journal of Biotechnology ( IF 4.1 ) Pub Date : 2020-01-31 , DOI: 10.1016/j.jbiotec.2020.01.017
Rajender Jena 1 , Dushyant K Garg 2 , Mohan Murali V Achary 3 , Jasdeep Singh 1 , Rachana Tomar 1 , Lipsa Choudhury 4 , Ruby Bansal 5 , Bishwajit Kundu 1
Affiliation  

Chaperones are a diverse class of molecules known for increasing thermo-stability of proteins, preventing protein aggregation, favoring disaggregation, increasing solubility and in some cases imparting resistance to proteolysis. These functions can be employed for various biotechnological applications including point of care testing, nano-biotechnology, bio-process engineering, purification technologies and formulation development. Here we report that the N-terminal domain of Pyrococcus furiosusl-asparaginase, (NPfA, a protein chaperone lacking α-crystallin domain) can serve as an efficient, industrially relevant, protein additive. We tested the effect of NPfA on substrate proteins, ascorbate peroxidase (APX), IgG peroxidase antibodies (I-HAbs) and KOD DNA polymerase. Each protein not only displayed increased thermal stability but also increased activity in the presence of NPfA. This increase was either comparable or higher than those obtained by common osmolytes; glycine betaine, sorbitol and trehalose. Most dramatic activity enhancement was seen in the case of KOD polymerase (∼ 40 % increase). NPfA exerts its effect through transient binding to the substrate proteins as discerned through isothermal titration calorimetry, dynamic light scattering and size exclusion chromatography. Mechanistic insights obtained through simulations suggested a remodeled architecture and emergence of H-binding network between NPfA and substrate protein with an effective enhancement in the solvent accessibility at the active site pocket of the latter. Thus, the capability of NPfA to engage in specific manner with other proteins is demonstrated to reduce the concentration of substrate proteins/enzymes required per unit operation. The functional expansion obtained through our finding establishes NPfA as a novel class of ATP-independent molecular chaperone with immense future biotechnological applications.

中文翻译:

蛋白质结构域作为分子伴侣的应用,可增强其他蛋白质的生物活性和稳定性。

分子伴侣是一类多样的分子,可提高蛋白质的热稳定性,防止蛋白质聚集,促进分解,增加溶解度,并在某些情况下赋予对蛋白水解的抵抗力。这些功能可以用于各种生物技术应用,包括即时检验,纳米生物技术,生物工艺工程,纯化技术和配方开发。在这里,我们报告激烈热球菌(Pyrococcus furiosusl)-天冬酰胺酶(NPfA,缺乏α-晶状蛋白结构域的蛋白伴侣)的N末端结构域可以作为一种有效的,与工业相关的蛋白添加剂。我们测试了NPfA对底物蛋白,抗坏血酸过氧化物酶(APX),IgG过氧化物酶抗体(I-HAbs)和KOD DNA聚合酶的影响。在NPfA存在下,每种蛋白质不仅显示出增加的热稳定性,而且显示出增加的活性。这种增加与普通渗透压相当或更高。甘氨酸甜菜碱,山梨糖醇和海藻糖。在KOD聚合酶的情况下,可以看到最显着的活性增强(增加约40%)。NPfA通过与底物蛋白质的瞬时结合发挥作用,如通过等温滴定热分析,动态光散射和尺寸排阻色谱法所见。通过模拟获得的机械学见解表明,NPfA和底物蛋白之间的H-结合网络经过重新架构和出现,有效地增强了后者活性位点上的溶剂可及性。从而,NPfA以特定方式与其他蛋白质结合的能力被证明可以降低单位操作所需的底物蛋白质/酶的浓度。通过我们的发现获得的功能扩展将NPfA建立为一类新型的ATP独立分子伴侣,具有巨大的未来生物技术应用前景。
更新日期:2020-01-31
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