Temperature control of biotin binding and release with a streptavidin-poly(N-isopropylacrylamide) site-specific conjugate
Temperature control of biotin binding and release with a streptavidin-poly(N-isopropylacrylamide) site-specific conjugate
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DOI：
10.1021/bc980108s
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发表时间：
1999-05-01
影响因子：
4.7
通讯作者：
Stayton, PS
中科院分区：
文献类型：
--
作者：
Ding, ZL;Long, CJ;Stayton, PS
利用链霉亲和素作为分子适配器的许多实验室和诊断应用依赖于其高亲和力和与生物素本质上不可逆的相互作用。然而，在许多情况下，生物素化分子的回收是可取的。我们之前已经证明，聚（n -异丙基丙烯酰胺）（PNIPAAm）是一种温度敏感的聚合物，当聚合物的构象在较低的临界溶液温度（LCST）下发生变化时，它可以可逆地阻断生物素的结合。在这里，我们构建了一个链亲和素- pnipaam偶联物，它能够在漫游温度或更低的温度下结合生物素，并在37℃下释放结合的生物素。该偶联物可以在温度循环通过LCST时反复结合和释放生物素。基因工程链亲和素突变体E116C只有一个半胱氨酸残基，通过巯基与具有巯基反应性乙烯砜基团的PNIPAAm特异性偶联。偶联位点靠近色氨酸120残基，与生物素形成范德华接触，这对于产生大的结合自由能很重要。温度引起的聚合物位置116的构象变化可能导致色氨酸120区域的结构变化，该区域负责生物素和共轭链霉亲和素之间的可逆结合。
The many laboratory and diagnostic applications utilizing streptavidin as a molecular adaptor rely on its high affinity and essentially irreversible interaction with biotin. However, there are many situations where recovery of the biotinylated molecules is desirable. We have previously shown that poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, can reversibly block biotin association as the polymer's conformation changes at its lower critical solution temperature (LCST). Here, we have constructed a streptavidin-PNIPAAm conjugate which is able to bind biotin at roam temperature or lower and release bound biotin at 37 degrees C. The conjugate can repeatedly bind and release biotin as temperature is cycled through the LCST. A genetically engineered streptavidin mutant, E116C, which has only one cysteine residue, was conjugated site specifically via the sulfhydryl groups with a PNIPAAm that has pendent sulfhydryl-reactive vinyl sulfone groups. The conjugation site is near the tryptophan 120 residue, which forms a van der Waals contact with biotin that is important in generating the large binding free energy. The temperature-induced conformational change of the polymer at position 116 may lead to structural changes in the region of tryptophan 120 that are responsible for the reversible binding between biotin and the conjugated streptavidin.