The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins
The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins
复制标题
DOI：
10.1016/j.pep.2003.08.014
复制
发表时间：
2004-01-01
影响因子：
1.6
通讯作者：
Erdmann, VA
中科院分区：
文献类型：
--
作者：
Lamla, T;Erdmann, VA
我们提出了一种新的链霉亲和素结合肽的纯化和重组蛋白的检测。该肽对链霉亲和素具有纳摩尔亲和力，因此被称为纳米标签。纳米标签（15）是15个氨基酸长并且以4 nM的解离常数结合链霉亲和素，并且纳米标签（9）是具有17 nM的解离常数的9-mer肽。我们证明了一步纯化纳米标记的蛋白质，即牛心脂肪酸结合蛋白（FABP），细菌氯霉素乙酰转移酶（CAT），和绿色荧光蛋白（GFP），从体外翻译系统，以及从大肠杆菌裂解物。未观察到Nano-tag 15和亲和层析过程中的条件对蛋白质的成熟或活性的显著影响，而Nano-tagg显示合成蛋白质的量和活性略有下降。Nano-tag的主要优点是用洗涤缓冲液加生物素或相关化合物进行温和和特异性洗脱，这使得结合的融合蛋白能够以天然状态从链霉亲和素柱洗脱。此外，纳米标签允许通过链霉亲和素-碱性磷酸酶缀合物在蛋白质印迹上检测重组蛋白。(C)2003年爱思唯尔公司All rights reserved.
We present a new streptavidin-binding peptide for both the purification and the detection of recombinant proteins. The peptide possesses nanomolar-affinity for streptavidin and therefore was termed Nano-tag. The Nano-tag(15) is 15 amino acids long and binds to streptavidin with a dissociation constant of 4nM and the Nano-tag(9) is a 9-mer peptide with a dissociation constant of 17 nM. We demonstrate the one-step purification of Nano-tagged proteins, namely bovine heart fatty acid-binding protein (FABP), bacterial chloramphenicol acetyltransferase (CAT), and green fluorescent protein (GFP), from an in vitro translation system as well as from an Escherichia coli lysate. No significant influence of the Nano-tag15 and of the conditions during affinity chromatography on maturation or activity of the proteins was observed whereas the Nano-tagg revealed a slight decline in the amount and activity of the synthesized proteins. The main advantage of the Nano-tag is the mild and specific elution with washing buffer plus biotin or related compounds, which enables the elution of the bound fusion protein from the streptavidin column in the native state. Additionally, the Nano-tag allowed the detection of recombinant proteins on Western blots by a streptavidin-alkaline phosphatase conjugate. (C) 2003 Elsevier Inc. All rights reserved.