Kabir M Enamul, Krishnaswamy Senthilkumar, Miyamoto Masahiko, Furuichi Yasuhiro, Komiyama Tadazumi
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niigata 956-8603, Japan.
Protein Expr Purif. 2010 Jul;72(1):59-65. doi: 10.1016/j.pep.2010.01.002. Epub 2010 Jan 7.
Single-domain single-chain variable fragment (scFv) antibody is sometimes critical for purification using affinity tagging strategy. We failed in our initial effort to purify a prematurely developed Camelid-like E-tagged short scFv-K2 antibody that contained a complete variable region of the heavy chain and partial region of the light chain by using an anti-E-tag affinity column. To expedite the purification of this altered but interesting antimycotic agent, we replaced a long and large E-tag by a short and hydrophilic 6x-Histidine (His(6)) affinity tag by polymerase chain reaction. The short and compact His(6)-tag was placed on the previously constructed expression vector pCANTAB 5 E that contained the large affinity E-tag sequence (13 amino acids) by PCR-based mutagenesis and was expressed in Escherichia coli. The recombinant protein can then be purified by immobilized metal affinity chromatography (IMAC) and be used for biochemical and other functional characterization. This His(6)-tagged short scFv-K2 antibody (20 kDa) had strong cytocidal activity against Saccharomyces and Candida species with a IC(50) value of 0.44x10(-6)M and 1.10 x 10(-6)M, respectively. Tag replacement facilitates the purification of a Camelid-like single-domain scFv antibody and after that meets its different functional characteristics. The present study reflects that the V(H) domain of the scFv antibody is mainly responsible for its biological activity and single-domain scFv antibody may acts as a potent antimicrobial agent.
单域单链可变片段（scFv）抗体有时对于采用亲和标签策略进行纯化至关重要。我们最初尝试使用抗E标签亲和柱纯化一种过早开发的骆驼科动物样E标签短scFv-K2抗体，该抗体包含重链的完整可变区和轻链的部分区域，但未成功。为了加快这种经过改造但有趣的抗真菌剂的纯化，我们通过聚合酶链反应将长而大的E标签替换为短而亲水性的6x组氨酸（His(6)）亲和标签。通过基于聚合酶链反应的诱变将短而紧凑的His(6)标签置于先前构建的含有大亲和E标签序列（13个氨基酸）的表达载体pCANTAB 5 E上，并在大肠杆菌中表达。然后可以通过固定化金属亲和色谱（IMAC）纯化重组蛋白，并用于生化和其他功能表征。这种His(6)标签短scFv-K2抗体（20 kDa）对酿酒酵母和念珠菌具有很强的杀细胞活性，其IC(50)值分别为0.44×10(-6)M和1.10×10(-6)M。标签替换有助于纯化骆驼科动物样单域scFv抗体，并在此之后满足其不同的功能特性。本研究表明，scFv抗体的V(H)结构域主要负责其生物活性，单域scFv抗体可能作为一种有效的抗菌剂。
