Laukkanen M L, Teeri T T, Keinänen K
VTT Biotechnical Laboratory, Espoo, Finland.
Protein Eng. 1993 Jun;6(4):449-54. doi: 10.1093/protein/6.4.449.
In order to achieve a stable and functional immobilization of antibodies, we investigated the possibility of adding hydrophobic membrane anchors to antibody fragments expressed in Escherichia coli. The DNA sequence encoding the signal peptide and the nine N-terminal amino acid residues of the major lipoprotein of E.coli was fused to the sequence of an anti-2-phenyloxazolone single-chain Fv antibody fragment [Takkinen et al. (1991) Protein Engng, 4,837-841]. The expression of the fusion construct in E.coli resulted in specific accumulation of an immunoreactive 28 kDa polypeptide. Unlike the unmodified single-chain Fv fragment, the fusion protein was cell-associated, labelled by [3H]palmitate which is indicative of the presence of N-terminal lipid modification, partitioned into the detergent phase upon Triton X-114 phase separation and was localized predominantly in the bacterial outer membrane. The fusion antibody displayed specific 2-phenyloxazolone-binding activity in the membrane-bound form and after solubilization with non-ionic detergents. Furthermore, upon removal of detergent the fusion antibody was incorporated into proteoliposomes which displayed specific hapten-binding activity. Our results show that antibodies can be converted to membrane-bound proteins with retention of antigen-binding properties by introduction of lipid anchors during biosynthesis. This approach may prove useful in the design of immunoliposomes and immunosensors.
为了实现抗体的稳定且功能性固定，我们研究了在大肠杆菌中表达的抗体片段添加疏水膜锚定物的可能性。将编码大肠杆菌主要脂蛋白信号肽和九个N端氨基酸残基的DNA序列与抗2-苯基恶唑酮单链Fv抗体片段的序列融合[Takkinen等人（1991年）《蛋白质工程》，4，837 - 841]。融合构建体在大肠杆菌中的表达导致一种免疫反应性28 kDa多肽的特异性积累。与未修饰的单链Fv片段不同，融合蛋白与细胞相关，被[3H]棕榈酸标记，这表明存在N端脂质修饰，在Triton X - 114相分离时分配到去污剂相中，并且主要定位于细菌外膜。融合抗体在膜结合形式以及用非离子去污剂溶解后均表现出特异性的2-苯基恶唑酮结合活性。此外，去除去污剂后，融合抗体被整合到具有特异性半抗原结合活性的蛋白脂质体中。我们的结果表明，通过在生物合成过程中引入脂质锚定物，抗体可以转化为具有抗原结合特性的膜结合蛋白。这种方法可能在免疫脂质体和免疫传感器的设计中证明是有用的。
