FUNCTIONAL IMMUNOLIPOSOMES HARBORING A BIOSYNTHETICALLY LIPID-TAGGED SINGLE-CHAIN ANTIBODY

被引:36
作者
LAUKKANEN, ML
ALFTHAN, K
KEINANEN, K
机构
[1] VTT Biotechnology and Food Research, FIN-02044 VTT, Espoo
关键词
D O I
10.1021/bi00204a031
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
An anti-2-phenyloxazolone single-chain antibody was expressed in Escherichia coli as a lipoprotein fusion in order to generate a biosynthetically lipid-tagged molecule [Laukkanen et al. (1993) Protein Eng. 6, 449-454]. For purification, a hexahistidinyl tag was introduced to the C-terminus of the protein. The resulting antibody, termed Ox lpp-scFv-H6, was membrane-bound, displayed hapten-binding activity, and contained the lipoprotein-specific lipid modification as indicated by metabolic [H-3]palmitic acid labeling. The Ox lpp-scFv-H6 was purified by immobilized metal affinity chromatography followed by hapten-based affinity chromatography to essential homogeneity with a yield of 0.4-1.6 mg/L of culture. In detergent dialysis, the purified antibody partitioned quantitatively into phospholipid liposomes. The immunoliposome preparation consisting of a homogeneous population of unilamellar 100-200 nm vesicles displayed specific hapten-binding activity as measured by using ELISA and surface plasmon resonance (SPR)-based realtime biospecific interaction analysis. In SPR experiments, the immunoliposomes exhibited virtually irreversible binding to immobilized hapten compared to soluble antibody fragments, consistent with the predicted multivalent binding. Biosynthetic lipid-tagging of antibodies may prove useful for immunoliposome-based diagnostic and therapeutic applications.
引用
收藏
页码:11664 / 11670
页数:7
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