Department of Biochemistry, Faculty of Medicine, Cumhuriyet University, 58140, Sivas, Turkey.
Protein J. 2010 Oct;29(7):501-8. doi: 10.1007/s10930-010-9280-2.
Heat shock protein 70 is a conserved protein among organisms. Hsp70 helps substrate proteins to fold correctly. Unfolded substrate proteins increase the probability of the aggregate formation. High level recombinant protein expression in biotechnology often leads insoluble inclusion bodies. To prevent aggregation and to obtain high levels of soluble proteins, Hsp co-expression with desired recombinant protein in yeast becomes a popular method. For this purpose, S. cerevesiae cytosolic Hsp70 (Ssa1) biochemical properties were characterized. Alteration of Ssa1 structure between ATP- and ADP-bound states regulates its function. Therefore, conformation-dependent Ssa1 hydrophobicity and as a result aggregation may also play a key role in Ssa1 function. Therefore, a combination of FTIR, acrylamide quenching, and ANS was used to investigate the effect of nucleotide binding on the structure of Ssa1. Ssa1 secondary structure alterations and hydrophobic properties in aqueous solutions with differing ionic strengths and temperature were also studied.
热休克蛋白 70 是生物体内保守的蛋白质。Hsp70 有助于底物蛋白正确折叠。未折叠的底物蛋白增加了聚集体形成的概率。生物技术中高水平的重组蛋白表达常常导致不溶性包涵体。为了防止聚集并获得高水平的可溶性蛋白，在酵母中与所需重组蛋白共表达 Hsp 成为一种流行的方法。为此，对酿酒酵母胞质 Hsp70（Ssa1）的生化特性进行了表征。在 ATP 和 ADP 结合状态之间改变 Ssa1 的结构调节其功能。因此，构象依赖性 Ssa1 疏水性以及由此产生的聚集也可能在 Ssa1 功能中发挥关键作用。因此，使用傅里叶变换红外光谱、丙烯酰胺猝灭和 ANS 来研究核苷酸结合对 Ssa1 结构的影响。还研究了在不同离子强度和温度的水溶液中 Ssa1 二级结构的变化和疏水性。
