Yılmaz Hilal, Özdemir Fatma İnci, Ergenekon Pınar, Özkan Melek
Gebze Technical University, Department of Environmental Engineering, 41400, Gebze Kocaeli, Turkey.
Gebze Technical University, Department of Molecular Biology and Genetics, 41400, Gebze Kocaeli, Turkey.
Protein Expr Purif. 2020 Sep;173:105664. doi: 10.1016/j.pep.2020.105664. Epub 2020 May 5.
In this study, effect of affinity tags, Histidine (His) and Glutathione-S-Transferase (GST), on the activity of halophilic aquaporin was analyzed. The gene coding for H. elongata aquaporin was cloned into pET28a vector and expressed in E. coli BL21 successfully. Stopped flow light scattering measurements showed that His-tagged aquaporin is functional. The difference in the filtration parameters caused by affinity tags were determined by using thin film composite nano-filtration (NFC) membranes prepared with the aquaporins. At 100 mM salt concentration, water permeability (L/m.h) and the % salt rejection of NFC membranes produced with the His-tagged aquaporin was found to be higher than that of the membrane with GST-tagged aquaporin. Salt rejection of His-tagged aquaporin-membrane was found to be 53% with a lower solute permeability value (B). Use of short affinity tag (His tag) for cloning resulted in higher solute rejection ability of TFC membranes prepared with H. elongata aquaporins.
在本研究中,分析了亲和标签组氨酸(His)和谷胱甘肽 - S - 转移酶(GST)对嗜盐水通道蛋白活性的影响。将编码嗜盐栖热放线菌水通道蛋白的基因克隆到pET28a载体中,并成功在大肠杆菌BL21中表达。停流光散射测量表明,带有His标签的水通道蛋白具有功能。通过使用用水通道蛋白制备的复合纳滤(NFC)膜来确定亲和标签引起的过滤参数差异。在100 mM盐浓度下,发现用带有His标签的水通道蛋白制备的NFC膜的水渗透率(L/m.h)和盐截留率高于带有GST标签的水通道蛋白的膜。带有His标签的水通道蛋白膜的盐截留率为53%,溶质渗透率值(B)较低。使用短亲和标签(His标签)进行克隆导致用嗜盐栖热放线菌水通道蛋白制备的TFC膜具有更高的溶质截留能力。